Extracellular pectin-RALF phase separation mediates FERONIA global signaling function.

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 in plant growth and survival.

Brassinosteroid recruits FERONIA to safeguard cell expansion in Arabidopsis.

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.

Using Hyper as a molecular probe to visualize hydrogen peroxide in living plant cells: An updated method.

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).

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.

Pollen-pistil interactions: It takes two to tangle but a molecular cast of many to deliver.

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.

RALF peptide signaling controls the polytubey block in Arabidopsis.

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.

Malectin/Malectin-like domain-containing proteins: A repertoire of cell surface molecules with broad functional potential.

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.

FERONIA receptor kinase-regulated reactive oxygen species mediate self-incompatibility in Brassica rapa.

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.

Pollen PCP-B peptides unlock a stigma peptide-receptor kinase gating mechanism for pollination.

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.

Programmed Cell Death in Stigmatic Papilla Cells Is Associated With Senescence-Induced Self-Incompatibility Breakdown in Chinese Cabbage and Radish.

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.

FERONIA controls pectin- and nitric oxide-mediated male-female interaction.

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 gametophyte1,2. The FERONIA receptor kinase has a dual role in ensuring sperm delivery and blocking polyspermy3. It has previously been reported that FERONIA generates a female gametophyte environment that is required for sperm release4. 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 LURE11, 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.

LLG2/3 Are Co-receptors in BUPS/ANX-RALF Signaling to Regulate Arabidopsis Pollen Tube Integrity.

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.

The FERONIA Receptor Kinase Maintains Cell-Wall Integrity during Salt Stress through Ca2+ Signaling.

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.

Arabidopsis pollen tube integrity and sperm release are regulated by RALF-mediated signaling.

In flowering plants, fertilization requires complex cell-to-cell communication events between the pollen tube and the female reproductive tissues, which are controlled by extracellular signaling molecules interacting with receptors at the pollen tube surface. We found that two such receptors in Arabidopsis, BUPS1 and BUPS2, and their peptide ligands, RALF4 and RALF19, are pollen tube-expressed and are required to maintain pollen tube integrity. BUPS1 and BUPS2 interact with receptors ANXUR1 and ANXUR2 via their ectodomains, and both sets of receptors bind RALF4 and RALF19. These receptor-ligand interactions are in competition with the female-derived ligand RALF34, which induces pollen tube bursting at nanomolar concentrations. We propose that RALF34 replaces RALF4 and RALF19 at the interface of pollen tube-female gametophyte contact, thereby deregulating BUPS-ANXUR signaling and in turn leading to pollen tube rupture and sperm release.

SIPP, a Novel Mitochondrial Phosphate Carrier, Mediates in Self-Incompatibility.

In Solanaceae, the S-specific interaction between the pistil S-RNase and the pollen S-Locus F-box protein controls self-incompatibility (SI). Although this interaction defines the specificity of the pollen rejection response, the identification of three pistil essential modifier genes unlinked to the S-locus (HT-B, 120K, and NaStEP) unveils a higher degree of complexity in the pollen rejection pathway. We showed previously that NaStEP, a stigma protein with homology with Kunitz-type protease inhibitors, is essential to SI in Nicotiana spp. During pollination, NaStEP is taken up by pollen tubes, where potential interactions with pollen tube proteins might underlie its function. Here, we identified NaSIPP, a mitochondrial protein with phosphate transporter activity, as a novel NaStEP-interacting protein. Coexpression of NaStEP and NaSIPP in pollen tubes showed interaction in the mitochondria, although when expressed alone, NaStEP remains mostly cytosolic, implicating NaSIPP-mediated translocation of NaStEP into the organelle. The NaSIPP transcript is detected specifically in mature pollen of Nicotiana spp.; however, in self-compatible plants, this gene has accumulated mutations, so its coding region is unlikely to produce a functional protein. RNA interference suppression of NaSIPP in Nicotiana spp. pollen grains disrupts the SI by preventing pollen tube inhibition. Taken together, our results are consistent with a model whereby the NaStEP and NaSIPP interaction, in incompatible pollen tubes, might destabilize the mitochondria and contribute to arrest pollen tube growth.

RopGEF1 Plays a Critical Role in Polar Auxin Transport in Early Development.

Polar auxin transport, facilitated by the combined activities of auxin influx and efflux carriers to maintain asymmetric auxin distribution, is essential for plant growth and development. Here, we show that Arabidopsis (Arabidopsis thaliana) RopGEF1, a guanine nucleotide exchange factor and activator of Rho GTPases of plants (ROPs), is critically involved in polar distribution of auxin influx carrier AUX1 and differential accumulation of efflux carriers PIN7 and PIN2 and is important for embryo and early seedling development when RopGEF1 is prevalently expressed. Knockdown or knockout of RopGEF1 induces embryo defects, cotyledon vein breaks, and delayed root gravity responses. Altered expression from the auxin response reporter DR5rev:GFP in the root pole of RopGEF1-deficient embryos and loss of asymmetric distribution of DR5rev:GFP in their gravistimulated root tips suggest that auxin distribution is affected in ropgef1 mutants. This is reflected by the polarity of AUX1 being altered in ropgef1 embryos and roots, shifting from the normal apical membrane location to a basal location in embryo central vascular and root protophloem cells and also reduced PIN7 accumulation at embryos and altered PIN2 distribution in gravistimulated roots of mutant seedlings. In establishing that RopGEF1 is critical for AUX1 localization and PIN differential accumulation, our results reveal a role for RopGEF1 in cell polarity and polar auxin transport whereby it imapcts auxin-mediated plant growth and development.

Stomatal Patterning: SERKs Put the Mouths in Their Right Place.

Plants have stomata, mouth-like pores on their surface, to adjust to environmental changes such as temperature and humidity to ensure optimum physiology and metabolism. A new study adds a key player, SERK, to the signal-sensing apparatus to inform where stomata are to be formed on the leaf.

Glycosylphosphatidylinositol-anchored proteins as chaperones and co-receptors for FERONIA receptor kinase signaling in Arabidopsis.

The Arabidopsis receptor kinase FERONIA (FER) is a multifunctional regulator for plant growth and reproduction. Here we report that the female gametophyte-expressed glycosylphosphatidylinositol-anchored protein (GPI-AP) LORELEI and the seedling-expressed LRE-like GPI-AP1 (LLG1) bind to the extracellular juxtamembrane region of FER and show that this interaction is pivotal for FER function. LLG1 interacts with FER in the endoplasmic reticulum and on the cell surface, and loss of LLG1 function induces cytoplasmic retention of FER, consistent with transport of FER from the endoplasmic reticulum to the plasma membrane in a complex with LLG1. We further demonstrate that LLG1 is a component of the FER-regulated RHO GTPase signaling complex and that fer and llg1 mutants display indistinguishable growth, developmental and signaling phenotypes, analogous to how lre and fer share similar reproductive defects. Together our results support LLG1/LRE acting as a chaperone and co-receptor for FER and elucidate a mechanism by which GPI-APs enable the signaling capacity of a cell surface receptor.