Prezygotic interspecific incompatibility prevents hybridization between species, which limits interbreeding strategies for crop improvement using wild relatives. The Brassica rapa female self-incompatibility determinant, S-locus receptor kinase (SRK), recognizes interspecific pollen. Here, we report the discovery of a pan-Brassicaceae SRK-interacting interspecific pollen signal (SIPS). On B. rapa stigmas, SIPSs from Arabidopsis and other Brassicaceae species target BrSRK and recruit the female fertility regulator FERONIA receptor kinase to increase stigmatic reactive oxygen species and reduce interspecific pollen viability. Arabidopsis thaliana sips mutant pollen failed to trigger interspecific incompatibility responses. Unlike self-incompatibility, which is controlled by the polymorphic S locus, different genetic variants of SRK interacted comparably with SIPS. This study establishes SIPS-SRK as a Brassicaceae-specific ligand-receptor pair that broadly maintains the stigmatic interspecific barrier in self-incompatible species.

Plant cell expansion is regulated by hormones and driven by turgor pressure,which stretches the cell wall and can potentially cause wall damage or rupture.How plant cells avoid cell wall rupture during hormone-induced rapid cell expansion remains poorly understood.Here,we show that the wall-sensing receptor kinase FERONIA(FER)plays an essential role in maintaining cell wall integrity during brassinosteroid(BR)-induced cell elongation.Compared with the wild type,the BR-treated fer mutants display an increased initial acceleration of cell elongation,increased cell wall damage and rupture,reduced production of reactive oxygen species(ROS),and enhanced cell wall acidification.Long-term treatments of fer with high concentrations of BR cause stress responses and reduce growth,whereas osmolytes,reducing turgor,alleviate the defects.These results show that BR-induced cell elongation causes damage to cell walls and the release of cell wall fragments that activate FER,which promotes ROS production,attenuates apoplastic acidification,and slows cell elongation,thereby preventing further cell wall damage and rupture.Furthermore,we show that BR signaling promotes FER accumulation at the plasma membrane(PM).When the BR level is low,the GSK3-like kinase BIN2 phosphorylates FER to reduce FER accumulation and translocation from the endoplasmic reticulum to PM.BR-induced inactivation of BIN2 leads to dephosphor-ylation and PM accumulation of FER.Thus,BR signaling enhances FER-mediated cell wall integrity surveil-lance while promoting cell expansion,whereas FER acts as a brake to maintain a safe cell elongation rate.Collectively,our study reveals a vital signaling circuit that coordinates hormone signaling with mechanical sensing to prevent cell rupture during hormone-induced cell expansion.

Auxin plays a critical role throughout plant development. We have established earlier that auxin activates the ROP (plant RHO GTPase) molecular switch, and that guanine nucleotide exchange factors ROPGEFs, which activate ROPs, are important regulators for myriad auxin-regulated processes. Here we show that auxin induces phosphorylation of RopGEF1 and that four receptor-like cytoplasmic kinases (RLCKs), named RopGEF1-activating kinases 1/2/3/4 (RAK1/2/3/4), mediate this process. We show that RAKs interact with RopGEFs and specifically phosphorylate S488 on RopGEF1, promoting its stability and recruitment to the cell membrane, and enhance ROP activation. Knockout of these RLCKs blocked auxin-stimulated RopGEF1 phosphorylation, reduced ROP activation and led to defects in PIN-FORMED (PIN)-mediated auxin distribution and multiple developmental processes. Phospho-mimic RopGEF1 (S488D) shows enhanced guanine nucleotide exchange activity in vitro, and its expression in rak quadruple mutants reverses their phenotypes. The RLCK–RopGEF linkage represents an important functional node and elucidates a critical missing link in ROP-meditated auxin signaling. Four receptor-like cytoplasmic kinases (RLCKs) phosphorylate guanine nucleotide exchange factors (RopGEFs), filling a critical gap in the signalling pathway connecting cell surface auxin and RHO GTPases (ROPs) in Arabidopsis.

Plants have evolved elaborate signaling networks, believed to be necessitated by the diversity and complexity of their morphology, developmental and reproductive strategies, and the need to cope with an ever-changing environment from which they are rooted and cannot escape. Their receptor-like kinase superfamilies, with members numbering in the hundreds to more than a thousand, exemplify how plants have evolved their signaling versatility. FERONIA (FER) receptor kinase from model Arabidopsis is a member of the Malectin-domain receptor kinase family conserved among plants. FER has a perplexingly broad functional range, impacting growth to reproduction throughout the plant life cycle, and survival when encountering biotic and abiotic stressors from the environment, such as pathogens and climatic adversity. Efforts to understand FER signaling have brought to light novel signaling strategies at the continuum of the plant cell wall and plasma membrane, and a network of cytoplasmic and nuclear pathways that together support its biological roles. The discussion here focuses on the cell surface mechanisms, including a sugar-peptide interaction-driven liquid-liquid phase separation process along the cell wall-plasma membrane interface and a plasma membrane-linked signaling node comprised of FER, a glycosylphosphatidylinositol-anchored protein, the RHO GTPase molecular switch and a generator for reactive oxygen species (ROS). The emerging recognition of how the broader FER-related receptor kinase family could impact plant wellness and agricultural productivity is also discussed.

The FERONIA (FER)-LLG1 co -receptor and its peptide ligand RALF regulate myriad processes for plant growth and survival. Focusing on signal -induced cell surface responses, we discovered that intrinsically disordered RALF triggers clustering and endocytosis of its cognate receptors and FER- and LLG1-dependent endocytosis of non -cognate regulators of diverse processes, thus capable of broadly impacting downstream responses. RALF, however, remains extracellular. We demonstrate that RALF binds the cell wall polysaccharide pectin. They phase separate and recruit FER and LLG1 into pectin-RALF-FER-LLG1 condensates to initiate RALF-triggered cell surface responses. We show further that two frequently encountered environmental challenges, elevated salt and temperature, trigger RALF-pectin phase separation, promiscuous receptor clustering and massive endocytosis, and that this process is crucial for recovery from stress -induced growth attenuation. Our results support that RALF-pectin phase separation mediates an exoskeletal mechanism to broadly activate FER-LLG1-dependent cell surface responses to mediate the global role of FER plant growth and survival.

Reactive oxygen species (ROS) are highly reactive reduced oxygen molecules that play a myriad of roles in animal and plant cells. In plant cells the production of ROS results from aerobic metabolism during respiration and photosynthesis. Therefore mitochondria, chloroplasts, and peroxisomes constitute an important source of ROS. However, ROS can also be produced in response to many physiological stimuli such as pathogen attack, hormone signaling, abiotic stresses or during cell wall organization and plant morphogenesis. The study of ROS in plant cells has been limited to biochemical assays and use of fluorescent probes, however, the irreversible oxidation of the fluorescent dyes prevents the visualization of dynamic changes. We have previously reported that Hyper 1 is a biosensor for H2O2 and consists of a circularly permutated YFP (cpYFP) inserted into the regulatory domain of the Escherichia coli hydrogen peroxide (H2O2) sensor protein OxyR rendering it an H2O2-specific quantitative probe (Bilan & Belousov, 2018; Hernandez-Barrera et al., 2015). Herein we describe an updated protocol for using the improved new version of Hyper 2 and Hyper 3 as a dynamic biosensor for H2O2 in Arabidopsis with virtually unlimited potential to detect H2O2 throughout the plant and under a broad range of developmental and environmental conditions (Bilan et al., 2013).

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.

Plant cell expansion is driven by turgor pressure and regulated by hormones. How plant cells avoid cell wall rupture during hormone-induced cell expansion remains a mystery. Here we show that brassinosteroid (BR), while stimulating cell elongation, promotes the plasma membrane (PM) accumulation of the receptor kinase FERONIA (FER), which monitors cell wall damage and in turn attenuates BR-induced cell elongation to prevent cell rupture. The GSK3-like kinase BIN2 phosphorylates FER, resulting in reduced FER accumulation and translocation from endoplasmic reticulum to PM. By inactivating BIN2, BR signaling promotes dephosphorylation and increases PM accumulation of FER, thereby enhancing the surveillance of cell wall integrity. Our study reveals a vital signaling circuit that coordinates hormone signaling with mechanical sensing to prevent cell bursting during hormone-induced cell expansion.

Explosive advances have been made in the molecular understanding of pollen-pistil interactions that underlie reproductive success in flowering plants in the past three decades. Among the most notable is the discovery of pollen tube attractants [1∗,2∗]. The roles these molecules play in facilitating conspecific precedence thus promoting interspecific genetic isolation are also emerging [3–5]. Male-female interactions during the prezygotic phase and contributions from the male and female gametophytes have been comprehensively reviewed recently. Here, we focus on key advances in understanding the mechanistic underpinnings of how these interactions overcome barriers at various pollen-pistil interfaces along the pollen tube growth pathway to facilitate fertilization by desirable mates.

Fertilization of an egg by multiple sperm (polyspermy) leads to lethal genome imbalance and chromosome segregation defects. In Arabidopsis thaliana, the block to polyspermy is facilitated by a mechanism that prevents polytubey (the arrival of multiple pollen tubes to one ovule). We show here that FERONIA, ANJEA, and HERCULES RECEPTOR KINASE 1 receptor-like kinases located at the septum interact with pollen tube-specific RALF6, 7,16, 36, and 37 peptide ligands to establish this polytubey block, The same combination of RALF (rapid alkalinization factor) peptides and receptor complexes controls pollen tube reception and rupture inside the targeted ovule. Pollen tube rupture releases the polytubey block at the septum, which allows the emergence of secondary pollen tubes upon fertilization failure. Thus, orchestrated steps in the fertilization process in Arabidopsis are coordinated by the same signaling components to guarantee and optimize reproductive success.

Most plants in the Brassicaceae evolve self-incompatibility (SI) to avoid inbreeding and generate hybrid vigor. Self-pollen is recognized by the S-haplotype-specific interaction of the pollen ligand S-locus protein 11 (SP11) (also known as S-locus cysteine-rich protein [SCR]) and its stigma-specific S-locus receptor kinase (SRK). However, mechanistically much remains unknown about the signaling events that culminate in self-pollen rejection. Here, we show that self-pollen triggers high levels of reactive oxygen species (ROS) in stigma papilla cells to mediate SI in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis). We found that stigmatic ROS increased after self-pollination but decreased after compatible(CP)- pollination. Reducing stigmatic ROS by scavengers or suppressing the expression of respiratory burst oxidase homologs (Rbohs), which encode plant NADPH oxidases that produce ROS, both broke down SI. On the other hand, increasing the level of ROS inhibited the germination and penetration of compatible pollen on the stigma, mimicking an incompatible response. Furthermore, suppressing a B. rapa FERONIA (FER) receptor kinase homolog or Rac/Rop guanosine triphosphatase (GTPase) signaling effectively reduced stigmatic ROS and interfered with SI. Our results suggest that FER-Rac/Rop signaling-regulated, NADPH oxidase-produced ROS is an essential SI response leading to self-pollen rejection.

Sexual reproduction in angiosperms relies on precise communications between the pollen and pistil. The molecular mechanisms underlying these communications remain elusive. We established that in Arabidopsis, a stigmatic gatekeeper, the ANJEA-FERONIA (ANJ-FER) receptor kinase complex, perceives the RAPID ALKALINIZATION FACTOR peptides RALF23 and RALF33 to induce reactive oxygen species (ROS) production in the stigma papillae, whereas pollination reduces stigmatic ROS, allowing pollen hydration. Upon pollination, the POLLEN COAT PROTEIN B-class peptides (PCP-Bs) compete with RALF23/33 for binding to the ANJ-FER complex, leading to a decline of stigmatic ROS that facilitates pollen hydration. Our results elucidate a molecular gating mechanism in which distinct peptide classes from pollen compete with stigma peptides for interaction with a stigmatic receptor kinase complex, allowing the pollen to hydrate and germinate.

Cell walls are at the front line of interactions between walled-organisms and their environment. They support cell expansion, ensure cell integrity and, for multicellular organisms such as plants, they provide cell adherence, support cell shape morphogenesis and mediate cell-cell communication. Wall-sensing, detecting perturbations in the wall and signaling the cell to respond accordingly, is crucial for growth and survival. In recent years, plant signaling research has suggested that a large family of receptor-like kinases (RLKs) could function as wall sensors partly because their extracellular domains show homology with malectin, a diglucose binding protein from the endoplasmic reticulum of animal cells. Studies of several malectin/malectin-like (M/ML) domain-containing RLKs (M/MLD-RLKs) from the model plant Arabidopsis thaliana have revealed an impressive array of biological roles, controlling growth, reproduction and stress responses, processes that in various ways rely on or affect the cell wall. Malectin homologous sequences are widespread across biological kingdoms, but plants have uniquely evolved a highly expanded family of proteins with ML domains embedded within various protein contexts. Here, we present an overview on proteins with malectin homologous sequences in different kingdoms, discuss the chromosomal organization of Arabidopsis M/MLD-RLKs and the phylogenetic relationship between these proteins from several model and crop species. We also discuss briefly the molecular networks that enable the diverse biological roles served by M/MLD-RLKs studied thus far.

Species that propagate by sexual reproduction actively guard against the fertilization of an egg by multiple sperm (polyspermy). Flowering plants rely on pollen tubes to transport their immotile sperm to fertilize the female gametophytes inside ovules. In Arabidopsis , pollen tubes are guided by cysteine-rich chemoattractants to target the female gametophyte 1 , 2 . The FERONIA receptor kinase has a dual role in ensuring sperm delivery and blocking polyspermy 3 . It has previously been reported that FERONIA generates a female gametophyte environment that is required for sperm release 4 . Here we show that FERONIA controls several functionally linked conditions to prevent the penetration of female gametophytes by multiple pollen tubes in Arabidopsis . We demonstrate that FERONIA is crucial for maintaining de-esterified pectin at the filiform apparatus, a region of the cell wall at the entrance to the female gametophyte. Pollen tube arrival at the ovule triggers the accumulation of nitric oxide at the filiform apparatus in a process that is dependent on FERONIA and mediated by de-esterified pectin. Nitric oxide nitrosates both precursor and mature forms of the chemoattractant LURE1 1 , respectively blocking its secretion and interaction with its receptor, to suppress pollen tube attraction. Our results elucidate a mechanism controlled by FERONIA in which the arrival of the first pollen tube alters ovular conditions to disengage pollen tube attraction and prevent the approach and penetration of the female gametophyte by late-arriving pollen tubes, thus averting polyspermy.

Self-incompatibility (SI) is a genetic mechanism flowering plants adopted to reject self-pollen and promote outcrossing. In the Brassicaceae family plants, the stigma tissue plays a key role in self-pollen recognition and rejection. We reported earlier in Chinese cabbage ( Brassica rapa ) that stigma tissue showed upregulated ethylene responses and programmed cell death (PCD) upon compatible pollination, but not in SI responses. Here, we show that SI is significantly compromised or completely lost in senescent flowers and young flowers of senescent plants. Senescence upregulates senescence-associated genes in B. rapa . Suppressing their expression in young stigmas by antisense oligodeoxyribonucleotide abolishes compatible pollination-triggered PCD and inhibits the growth of compatible pollen tubes. Furthermore, ethylene biosynthesis genes and response genes are upregulated in senescent stigmas, and increasing the level of ethylene or inhibiting its response increases or decreases the expression of senescence-associated genes, respectively. Our results show that senescence causes PCD in stigmatic papilla cells and is associated with the breakdown of SI in Chinese cabbage and in radish.

In angiosperms, two sperm cells are transported and delivered by the pollen tube to the ovule to achieve double fertilization. Extensive communication takes place between the pollen tube and the female tissues until the sperm cell cargo is ultimately released. During this process, a pollen tube surface-located receptor complex composed of ANXUR1/2 (ANX1/2) and Buddha’s Paper Seal 1/2 (BUPS1/2) was reported to control the maintenance of pollen tube integrity by perceiving the autocrine peptide ligands rapid alkalinization factor 4 and 19 (RALF4/19). It was further hypothesized that pollen-tube rupture to release sperm is caused by the paracrine RALF34 peptide from the ovule interfering with this signaling pathway. In this study, we identified two Arabidopsis pollen-tube-expressed glycosylphosphatidylinositol-anchored proteins (GPI-APs), LORELEI-like-GPI-anchored protein 2 (LLG2) and LLG3, as co-receptors in the BUPS-ANX receptor complex. llg2 llg3 double mutants exhibit severe fertility defects. Mutant pollen tubes rupture early during the pollination process. Furthermore, LLG2 and LLG3 interact with ectodomains of both BUPSs and ANXURs, and this interaction is remarkably enhanced by the presence of RALF4/19 peptides. We further demonstrate that the N terminus (including a YISY motif) of the RALF4 peptide ligand interacts strongly with BUPS-ANX receptors but weakly with LLGs and is essential for its biological function, and its C-terminal region is sufficient for LLG binding. In conclusion, we propose that LLG2/3 serve as co-receptors during BUPS/ANX-RALF signaling and thereby further establish the importance of GPI-APs as key regulators in plant reproduction processes.

In angiosperms, two sperm cells are transported and delivered by the pollen tube to the ovule to achieve double fertilization. Extensive communication takes place between the pollen tube and the female tissues until the sperm cell cargo is ultimately released. During this process, a pollen tube surface-located receptor complex composed of ANXUR1/2 (ANX1/2) and Buddha's Paper Seal 1/2 (BUPS1/2) was reported to control the maintenance of pollen tube integrity by perceiving the autocrine peptide ligands rapid alkalinization factor 4 and 19 (RALF4/19). It was further hypothesized that pollen-tube rupture to release sperm is caused by the paracrine RALF34 peptide from the ovule interfering with this signaling pathway. In this study, we identified two Arabidopsis pollen-tube-expressed glycosylphosphatidylinositol-anchored proteins (GPI-APs), LORELEI-like-GPI-anchored protein 2 (LLG2) and LLG3, as co-receptors in the BUPS-ANX receptor complex. llg2 llg3 double mutants exhibit severe fertility defects. Mutant pollen tubes rupture early during the pollination process. Furthermore, LLG2 and LLG3 interact with ectodomains of both BUPSs and ANXURs, and this interaction is remarkably enhanced by the presence of RALF4/19 peptides. We further demonstrate that the N terminus (including a YISY motif) of the RALF4 peptide ligand interacts strongly with BUPS-ANX receptors but weakly with LLGs and is essential for its biological function, and its C-terminal region is sufficient for LLG binding. In conclusion, we propose that LLG2/3 serve as co-receptors during BUPS/ANX-RALF signaling and thereby further establish the importance of GPI-APs as key regulators in plant reproduction processes.

Cells maintain integrity despite changes in their mechanical properties elicited during growth and environmental stress. How cells sense their physical state and compensate for cell-wall damage is poorly understood, particularly in plants. Here we report that FERONIA (FER), a plasma-membrane-localized receptor kinase from Arabidopsis, is necessary for the recovery of root growth after exposure to high salinity, a widespread soil stress. The extracellular domain of FER displays tandem regions of homology with malectin, an animal protein known to bind di-glucose in vitro and important for protein quality control in the endoplasmic reticulum. The presence of malectin-like domains in FER and related receptor kinases has led to widespread speculation that they interact with cell-wall polysaccharides and can potentially serve a wall-sensing function. Results reported here show that salinity causes softening of the cell wall and that FER is necessary to sense these defects. When this function is disrupted in the fer mutant, root cells explode dramatically during growth recovery. Similar defects are observed in the mur1 mutant, which disrupts pectin cross-linking. Furthermore, fer cell-wall integrity defects can be rescued by treatment with calcium and borate, which also facilitate pectin cross-linking. Sensing of these salinity-induced wall defects might therefore be a direct consequence of physical interaction between the extracellular domain of FER and pectin. FER-dependent signaling elicits cell-specific calcium transients that maintain cell-wall integrity during salt stress. These results reveal a novel extracellular toxicity of salinity, and identify FER as a sensor of damage to the pectin-associated wall.

AbstractThe sections in this article areIntroductionSexual Organ DevelopmentPollination and FertilizationFertilization and Seed DevelopmentFuture PerspectivesAcknowledgements