Defective pollen tube tip growth induces neo-polyploid infertility.

Genome duplication (generating polyploids) is an engine of novelty in eukaryotic evolution and a promising crop improvement tool. Yet newly formed polyploids often have low fertility. Here we report that a severe fertility-compromising defect in pollen tube tip growth arises in new polyploids of Arabidopsis arenosa. Pollen tubes of newly polyploid A. arenosa grow slowly, have aberrant anatomy and disrupted physiology, often burst prematurely, and have altered gene expression. These phenotypes recover in evolved polyploids. We also show that gametophytic (pollen tube) genotypes of two tip-growth genes under selection in natural tetraploid A. arenosa are strongly associated with pollen tube performance in the tetraploid. Our work establishes pollen tube tip growth as an important fertility challenge for neo-polyploid plants and provides insights into a naturally evolved multigenic solution.

Plant cell wall patterning and expansion mediated by protein-peptide-polysaccharide interaction.

Assembly of cell wall polysaccharides into specific patterns is required for plant growth. A complex of RAPID ALKALINIZATION FACTOR 4 (RALF4) and its cell wall-anchored LEUCINE-RICH REPEAT EXTENSIN 8 (LRX8)-interacting protein is crucial for cell wall integrity during pollen tube growth, but its molecular connection with the cell wall is unknown. Here, we show that LRX8-RALF4 complexes adopt a heterotetrametric configuration in vivo, displaying a dendritic distribution. The LRX8-RALF4 complex specifically interacts with demethylesterified pectins in a charge-dependent manner through RALF4's polycationic surface. The LRX8-RALF4-pectin interaction exerts a condensing effect, patterning the cell wall's polymers into a reticulated network essential for wall integrity and expansion. Our work uncovers a dual structural and signaling role for RALF4 in pollen tube growth and in the assembly of complex extracellular polymers.

Myosin XI-B is involved in the transport of vesicles and organelles in pollen tubes of Arabidopsis thaliana.

The movement of organelles and vesicles in pollen tubes depends on F-actin. However, the molecular mechanism through which plant myosin XI drives the movement of organelles is still controversial, and the relationship between myosin XI and vesicle movement in pollen tubes is also unclear. In this study, we found that the siliques of the myosin xi-b/e mutant were obviously shorter than those of the wild-type (WT) and that the seed set of the mutant was severely deficient. The pollen tube growth of myosin xi-b/e was significantly inhibited both in vitro and in vivo. Fluorescence recovery after photobleaching showed that the velocity of vesicle movement in the pollen tube tip of the myosin xi-b/e mutant was lower than that of the WT. It was also found that peroxisome movement was significantly inhibited in the pollen tubes of the myosin xi-b/e mutant, while the velocities of the Golgi stack and mitochondrial movement decreased relatively less in the pollen tubes of the mutant. The endoplasmic reticulum streaming in the pollen tube shanks was not significantly different between the WT and the myosin xi-b/e mutant. In addition, we found that myosin XI-B-GFP colocalized obviously with vesicles and peroxisomes in the pollen tubes of Arabidopsis. Taken together, these results indicate that myosin XI-B may bind mainly to vesicles and peroxisomes, and drive their movement in pollen tubes. These results also suggest that the mechanism by which myosin XI drives organelle movement in plant cells may be evolutionarily conserved compared with other eukaryotic cells.

LARP6C orchestrates posttranscriptional reprogramming of gene expression during hydration to promote pollen tube guidance.

Increasing evidence suggests that posttranscriptional regulation is a key player in the transition between mature pollen and the progamic phase (from pollination to fertilization). Nonetheless, the actors in this messenger RNA (mRNA)-based gene expression reprogramming are poorly understood. We demonstrate that the evolutionarily conserved RNA-binding protein LARP6C is necessary for the transition from dry pollen to pollen tubes and the guided growth of pollen tubes towards the ovule in Arabidopsis thaliana. In dry pollen, LARP6C binds to transcripts encoding proteins that function in lipid synthesis and homeostasis, vesicular trafficking, and polarized cell growth. LARP6C also forms cytoplasmic granules that contain the poly(A) binding protein and possibly represent storage sites for translationally silent mRNAs. In pollen tubes, the loss of LARP6C negatively affects the quantities and distribution of storage lipids, as well as vesicular trafficking. In Nicotiana benthamiana leaf cells and in planta, analysis of reporter mRNAs designed from the LARP6C target MGD2 provided evidence that LARP6C can shift from a repressor to an activator of translation when the pollen grain enters the progamic phase. We propose that LARP6C orchestrates the timely posttranscriptional regulation of a subset of mRNAs in pollen during the transition from the quiescent to active state and along the progamic phase to promote male fertilization in plants.

The role of diverse LURE-type cysteine-rich peptides as signaling molecules in plant reproduction.

In angiosperm sexual reproduction, the male pollen tube undergoes a series of interactions with female tissues. For efficient growth and precise guidance, the pollen tube perceives extracellular ligands. In recent decades, various types of secreted cysteine-rich peptides (CRPs) have been identified as peptide ligands that regulate diverse angiosperm reproduction processes, including pollen tube germination, growth, guidance, and rupture. Notably, in two distant core eudicot plants, multiple LURE-type CRPs were found to be secreted from egg-accompanying synergid cells, and these CRPs act as a cocktail of pollen tube attractants for the final step of pollen tube guidance. LURE-type CRPs have species-preferential activity, even among close relatives, and exhibit remarkably divergent molecular evolution with conserved cysteine frameworks, demonstrating that they play a key role in species recognition in pollen tube guidance. In this review, I focus on "reproductive CRPs," particularly LURE-type CRPs, which underlie common but species-specific mechanisms in angiosperm sexual reproduction, and discuss their action, functional regulation, receptors, and evolution.

Ethylene signaling modulates tomato pollen tube growth through modifications of cell wall remodeling and calcium gradient.

Ethylene modulates plant developmental processes including flower development. Previous studies have suggested ethylene participates in pollen tube (PT) elongation, and both ethylene production and perception seem critical at the time of fertilization. The full gene set regulated by ethylene during PT growth is unknown. To study this, we used various EThylene Receptor (ETR) tomato (Solanum lycopersicum) mutants: etr3-ko, a loss-of-function (LOF) mutant; and NR (NEVER RIPE), a gain-of-function (GOF) mutant. The etr3-ko PTs grew faster than wild-type (WT) PTs. Oppositely, NR PT elongation was slower than in WT, and PTs displayed larger diameters. ETR mutations result in feedback control of ethylene production. Furthermore, ethylene treatment of germinating pollen grains increased PT length in etr-ko mutants and WT, but not in NR. Treatment with the ethylene perception inhibitor 1-methylcyclopropene decreased PT length in etr-ko mutants and WT, but had no effect on NR. This confirmed that ethylene regulates PT growth. The comparison of PT transcriptomes in LOF and GOF mutants, etr3-ko and NR, both harboring mutations of the ETR3 gene, revealed that ethylene perception has major impacts on cell wall- and calcium-related genes as confirmed by microscopic observations showing a modified distribution of the methylesterified homogalacturonan pectic motif and of calcium load. Our results establish links between PT growth, ethylene, calcium, and cell wall metabolism, and also constitute a transcriptomic resource.

Quantitative cell biology of tip growth in moss.

KEY MESSAGE: Here we review, from a quantitative point of view, the cell biology of protonemal tip growth in the model moss Physcomitrium patens. We focus on the role of the cytoskeleton, vesicle trafficking, and cell wall mechanics, including reviewing some of the existing mathematical models of tip growth. We provide a primer for existing cell biological tools that can be applied to the future study of tip growth in moss. Polarized cell growth is a ubiquitous process throughout the plant kingdom in which the cell elongates in a self-similar manner. This process is important for nutrient uptake by root hairs, fertilization by pollen, and gametophyte development by the protonemata of bryophytes and ferns. In this review, we will focus on the tip growth of moss cells, emphasizing the role of cytoskeletal organization, cytoplasmic zonation, vesicle trafficking, cell wall composition, and dynamics. We compare some of the existing knowledge on tip growth in protonemata against what is known in pollen tubes and root hairs, which are better-studied tip growing cells. To fully understand how plant cells grow requires that we deepen our knowledge in a variety of forms of plant cell growth. We focus this review on the model plant Physcomitrium patens, which uses tip growth as the dominant form of growth at its protonemal stage. Because mosses and vascular plants shared a common ancestor more than 450 million years ago, we anticipate that both similarities and differences between tip growing plant cells will provide mechanistic information of tip growth as well as of plant cell growth in general. Towards this mechanistic understanding, we will also review some of the existing mathematical models of plant tip growth and their applicability to investigate protonemal morphogenesis. We attempt to integrate the conclusions and data across cell biology and physical modeling to our current state of knowledge of polarized cell growth in P. patens and highlight future directions in the field.

Membrane receptor-mediated mechano-transduction maintains cell integrity during pollen tube growth within the pistil.

Invasive or penetrative growth is critical for developmental and reproductive processes (e.g., pollen tube penetration of pistils) and disease progression (e.g., cancer metastasis and fungal hyphae invasion). The invading or penetrating cells experience drastic changes in mechanical pressure from the surroundings and must balance growth with cell integrity. Here, we show that Arabidopsis pollen tubes sense and/or respond to mechanical changes via a cell-surface receptor kinase Buddha's Paper Seal 1 (BUPS1) while emerging from compressing female tissues. BUPS1-defective pollen tubes fail to maintain cell integrity after emergence from these tissues. The mechano-transduction function of BUPS1 is established by using a microfluidic channel device mimicking the mechanical features of the in vivo growth path. BUPS1-based mechano-transduction activates Rho-like GTPase from Plant 1 (ROP1) GTPase to promote exocytosis that facilitates secretion of BUPS1's ligands for mechanical signal amplification and cell wall rigidification in pollen tubes. These findings uncover a membrane receptor-based mechano-transduction system for cells to cope with the physical challenges during invasive or penetrative growth.

Protein Analysis of Pollen Tubes after the Treatments of Membrane Trafficking Inhibitors Gains Insights on Molecular Mechanism Underlying Pollen Tube Polar Growth.

Pollen tube elongation is characterized by a highly-polarized tip growth process dependent on an efficient vesicular transport system and largely mobilized by actin cytoskeleton. Pollen tubes are an ideal model system to study exocytosis, endocytosis, membrane recycling, and signaling network coordinating cellular processes, structural organization and vesicular trafficking activities required for tip growth. Proteomic analysis was applied to identify Nicotiana tabacum Differentially Abundant Proteins (DAPs) after in vitro pollen tube treatment with membrane trafficking inhibitors Brefeldin A, Ikarugamycin and Wortmannin. Among roughly 360 proteins separated in two-dimensional gel electrophoresis, a total of 40 spots visibly changing between treated and control samples were identified by MALDI-TOF MS and LC-ESI-MS/MS analysis. The identified proteins were classified according to biological processes, and most proteins were related to pollen tube energy metabolism, including ammino acid synthesis and lipid metabolism, structural features of pollen tube growth as well modification and actin cytoskeleton organization, stress response, and protein degradation. In-depth analysis of proteins corresponding to energy-related pathways revealed the male gametophyte to be a reliable model of energy reservoir and dynamics.

Implications of ionizing radiation on pollen performance in comparison with diverse models of polar cell growth.

Research concerning the effects of ionizing radiation (IR) on plant systems is essential for numerous aspects of human society, as for instance, in terms of agriculture and plant breeding, but additionally for elucidating consequences of radioactive contamination of the ecosphere. This comprehensive survey analyses effects of x- and γ-irradiation on male gametophytes comprising primarily in vitro but also in vivo data of diverse plant species. The IR-dose range for pollen performance was compiled and 50% inhibition doses (ID50 ) for germination and tube growth were comparatively related to physiological characteristics of the microgametophyte. Factors influencing IR-susceptibility of mature pollen and polarized tube growth were evaluated, such as dose-rate, environmental conditions, or species-related variations. In addition, all available reports suggesting bio-positive IR-effects particularly on pollen performance were examined. Most importantly, for the first time influences of IR specifically on diverse phylogenetic models of polar cell growth were comparatively analysed, and thus demonstrated that the gametophytic system of pollen is extremely resistant to IR, more than plant sporophytes and especially much more than comparable animal cells. Beyond that, this study develops hypotheses regarding a molecular basis for the extreme IR-resistance of the plant microgametophyte and highlights its unique rank among organismal systems.

Organelle movement and apical accumulation of secretory vesicles in pollen tubes of Arabidopsis thaliana depend on class XI myosins.

F-actin and myosin XI play important roles in plant organelle movement. A few myosin XI genes in the genome of Arabidopsis are mainly expressed in mature pollen, which suggests that they may play a crucial role in pollen germination and pollen tube tip growth. In this study, a genetic complementation assay was conducted in a myosin xi-c (myo11c1) myosin xi-e (myo11c2) double mutant, and fluorescence labeling combined with microscopic observation was applied. We found that myosin XI-E (Myo11C2)-green fluorescent protein (GFP) restored the slow pollen tube growth and seed deficiency phenotypes of the myo11c1 myo11c2 double mutant and Myo11C2-GFP partially colocalized with mitochondria, peroxisomes and Golgi stacks. Furthermore, decreased mitochondrial movement and subapical accumulation were detected in myo11c1 myo11c2 double mutant pollen tubes. Fluorescence recovery after photobleaching experiments showed that the fluorescence recoveries of GFP-RabA4d and AtPRK1-GFP at the pollen tube tip of the myo11c1 myo11c2 double mutant were lower than those of the wild type were after photobleaching. These results suggest that Myo11C2 may be associated with mitochondria, peroxisomes and Golgi stacks, and play a crucial role in organelle movement and apical accumulation of secretory vesicles in pollen tubes of Arabidopsis thaliana.

AtPIG-S, a predicted Glycosylphosphatidylinositol Transamidase subunit, is critical for pollen tube growth in Arabidopsis.

BACKGROUND: Glycosylphosphatidylinositol (GPI) addition is one of the several post-translational modifications to proteins that increase their affinity for membranes. In eukaryotes, the GPI transamidase complex (GPI-T) catalyzes the attachment of pre-assembled GPI anchors to GPI-anchored proteins (GAPs) through a transamidation reaction. A mutation in AtGPI8 (gpi8-2), the putative catalytic subunit of GPI-T in Arabidopsis, is transmitted normally through the female gametophyte (FG), indicating the FG tolerates loss of GPI transamidation. In contrast, gpi8-2 almost completely abolishes male gametophyte (MG) function. Still, the unexpected finding that gpi8-2 FGs function normally requires further investigation. Additionally, specific developmental defects in the MG caused by loss of GPI transamidation remain poorly characterized. RESULTS: Here we investigated the effect of loss of AtPIG-S, another GPI-T subunit, in both gametophytes. Like gpi8-2, we showed that a mutation in AtPIG-S (pigs-1) disrupted synergid localization of LORELEI (LRE), a putative GAP critical for pollen tube reception by the FG. Still, pigs-1 is transmitted normally through the FG. Conversely, pigs-1 severely impaired male gametophyte (MG) function during pollen tube emergence and growth in the pistil. A pPIGS:GFP-PIGS transgene complemented these MG defects and enabled generation of pigs-1/pigs-1 seedlings. However, the pPIGS:GFP-PIGS transgene seemingly failed to rescue the function of AtPIG-S in the sporophyte, as pigs-1/pigs-1, pPIGS:GFP-PIGS seedlings died soon after germination. CONCLUSIONS: Characterization of pigs-1 provided further evidence that the FG tolerates loss of GPI transamidation more than the MG and that the MG compared to the FG may be a better haploid system to study the role of GPI-anchoring. Pigs-1 pollen develops normally and thus represent a tool in which GPI anchor biosynthesis and transamidation of GAPs have been uncoupled, offering a potential way to study free GPI in plant development. While previously reported male fertility defects of GPI biosynthesis mutants could have been due either to loss of GPI or GAPs lacking the GPI anchor, our results clarified that the loss of mature GAPs underlie male fertility defects of GPI-deficient pollen grains, as pigs-1 is defective only in the downstream transamidation step.

Plasma membrane H+ -ATPases-mediated cytosolic proton gradient regulates pollen tube growth.

The polar growth of pollen tubes is essential for the delivery of sperm cells during fertilization in angiosperms. How this polar growth is regulated has been a long-standing question. An in vitro pharmacological assay previously implicated proton flux in pollen tube growth, although genetic and cellular supporting evidence was lacking. Here, we report that protons form a gradient from the pollen tube tip to the shank region and this gradient is generated by three members of Arabidopsis H+ -ATPases (AHAs). Genetic analysis suggested that these AHAs are essential for pollen tube growth, thus providing new insight into the regulation of polar growth.

Plasma membrane H+-ATPases sustain pollen tube growth and fertilization.

Pollen tubes are highly polarized tip-growing cells that depend on cytosolic pH gradients for signaling and growth. Autoinhibited plasma membrane proton (H+) ATPases (AHAs) have been proposed to energize pollen tube growth and underlie cell polarity, however, mechanistic evidence for this is lacking. Here we report that the combined loss of AHA6, AHA8, and AHA9 in Arabidopsis thaliana delays pollen germination and causes pollen tube growth defects, leading to drastically reduced fertility. Pollen tubes of aha mutants had reduced extracellular proton (H+) and anion fluxes, reduced cytosolic pH, reduced tip-to-shank proton gradients, and defects in actin organization. Furthermore, mutant pollen tubes had less negative membrane potentials, substantiating a mechanistic role for AHAs in pollen tube growth through plasma membrane hyperpolarization. Our findings define AHAs as energy transducers that sustain the ionic circuit defining the spatial and temporal profiles of cytosolic pH, thereby controlling downstream pH-dependent mechanisms essential for pollen tube elongation, and thus plant fertility.

PRP8A and PRP8B spliceosome subunits act coordinately to control pollen tube attraction in Arabidopsis thaliana.

Precise guided pollen tube growth by the female gametophyte is a prerequisite for successful sexual reproduction in flowering plants. Cysteine-rich proteins (CRPs) secreted from the embryo sac are known pollen tube attractants perceived by pollen tube receptor-like kinases. How pre-mRNA splicing facilitates this cell-to-cell communication is not understood. Here, we report a novel function of Pre-mRNA PROCESSING factor 8 paralogs, PRP8A and PRP8B, as regulators of pollen tube attraction. Double mutant prp8a prp8b ovules cannot attract pollen tubes, and prp8a prp8b pollen tubes fail to sense the ovule's attraction signals. Only 3% of ovule-expressed genes were misregulated in prp8a prp8b Combination of RNA sequencing and the MYB98/LURE1.2-YFP reporter revealed that the expression of MYB98, LUREs and 49 other CRPs were downregulated, suggesting loss of synergid cell fate. Differential exon usage and intron retention analysis revealed autoregulation of PPR8A/PRP8B splicing. In vivo, PRP8A co-immunoprecipitates with splicing enhancer AtSF3A1, suggesting involvement of PRP8A in 3'-splice site selection. Our data hint that the PRP8A/PRP8B module exhibits spliceosome autoregulation to facilitate pollen tube attraction via transcriptional regulation of MYB98, CRPs and LURE pollen tube attractants.

Durotropic Growth of Pollen Tubes.

To reach the female gametophyte, growing pollen tubes must penetrate different tissues within the pistil, the female reproductive organ of a flower. Past research has identified various chemotropic cues that guide pollen tubes through the transmitting tract of the pistil, which represents the longest segment of its growth path. In addition, physical mechanisms also play a role in pollen tube guidance; however, these processes remain poorly understood. Here we show that pollen tubes from plants with solid transmitting tracts actively respond to the stiffness of the environment. We found that pollen tubes from Nicotiana tabacum and other plant species with a solid or semisolid transmitting tract increase their growth rate in response to an increasing matrix stiffness. By contrast, pollen tubes from Lilium longiflorum and other plant species with a hollow transmitting tract decrease their growth rate with increasing matrix stiffness, even though the forces needed to maintain a constant growth rate remain far below the maximum penetration force these pollen tubes are able to generate. Moreover, when confronted with a transition from a softer to a stiffer matrix, pollen tubes from N. tabacum display a greater ability to penetrate into a stiffer matrix compared with pollen tubes from L. longiflorum, even though the maximum force generated by pollen tubes from N. tabacum (11 µN) is smaller than the maximum force generated by pollen tubes from L. longiflorum (36 µN). These findings demonstrate a mechano-sensitive growth behavior, termed here durotropic growth, that is only expressed in pollen tubes from plants with a solid or semisolid transmitting tract and thus may contribute to an effective pollen tube guidance within the pistil.

TMT-Based Quantitative Proteomic Analysis Reveals the Crucial Biological Pathways Involved in Self-Incompatibility Responses in Camellia oleifera.

Camellia oleifera is a valuable woody oil plant belonging to the Theaceae, Camellia oil extracted from the seed is an excellent edible oil source. Self-incompatibility (SI) in C. oleifera results in low fruit set, and our knowledge about the mechanism remains limited. In the present study, the Tandem mass tag (TMT) based quantitative proteomics was employed to analyze the dynamic change of proteins response to self- and cross-pollinated in C. oleifera. A total of 6,616 quantified proteins were detected, and differentially abundant proteins (DAPs) analysis identified a large number of proteins. Combined analysis of differentially expressed genes (DEGs) and DAPs of self- and cross-pollinated pistils based on transcriptome and proteome data revealed that several candidate genes or proteins involved in SI of C. oleifera, including polygalacturonase inhibitor, UDP-glycosyltransferase 92A1-like, beta-D-galactosidase, S-adenosylmethionine synthetase, xyloglucan endotransglucosylase/hydrolase, ABC transporter G family member 36-like, and flavonol synthase. Venn diagram analysis identified 11 proteins that may participate in pollen tube growth in C. oleifera. Our data also revealed that the abundance of proteins related to peroxisome was altered in responses to SI in C. oleifera. Moreover, the pathway of lipid metabolism-related, flavonoid biosynthesis and splicesome were reduced in self-pollinated pistils by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. In summary, the results of the present study lay the foundation for learning the regulatory mechanism underlying SI responses as well as provides valuable protein resources for the construction of self-compatibility C. oleifera through genetic engineering in the future.

Genome-wide analysis of RopGEF gene family to identify genes contributing to pollen tube growth in rice (Oryza sativa).

BACKGROUND: In plants, the key roles played by RopGEF-mediated ROP signaling in diverse processes, including polar tip growth, have been identified. Despite their important roles in reproduction, a comprehensive analysis of RopGEF members has not yet been performed in rice (Oryza sativa). To determine whether RopGEF regulators are involved in rice pollen tube growth, we performed genome-wide analysis of this family in rice. RESULTS: Phylogenomic and meta-expression analysis of eleven RopGEFs in rice showed that four genes were preferentially expressed in mature pollen. These four genes contain the plant-specific Rop nucleotide exchanger (PRONE) domain and possible phosphorylated residues, suggesting a conserved role in polar tip growth with Arabidopsis thaliana. In subcellular localization analysis of the four RopGEFs through tobacco (Nicotiana benthamiana) infiltration, four proteins were predominantly identified in plasma membrane. Moreover, double mutants of RopGEF2/8 exhibited reduced pollen germination, causing partial male sterility. These genes possess unique cis-acting elements in their promoters compared with the other RopGEF genes. CONCLUSIONS: In this study, four RopGEF genes were identified as pollen-specific gene in eleven members of rice, and the expression pattern, promoter analysis, and evolutionary relationship of the RopGEF family were studied compared with Arabidopsis. Our study indicated that four RopGEF genes might function during pollen germination in distinct subcellular localization. Our study could provide valuable information on the functional study of RopGEF in rice.

Structural basis for recognition of RALF peptides by LRX proteins during pollen tube growth.

Plant reproduction relies on the highly regulated growth of the pollen tube for sperm delivery. This process is controlled by secreted RALF signaling peptides, which have previously been shown to be perceived by Catharanthus roseus RLK1-like (CrRLK1Ls) membrane receptor-kinases/LORELEI-like GLYCOLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEINS (LLG) complexes, or by leucine-rich repeat (LRR) extensin proteins (LRXs). Here, we demonstrate that RALF peptides fold into bioactive, disulfide bond-stabilized proteins that bind the LRR domain of LRX proteins with low nanomolar affinity. Crystal structures of LRX2-RALF4 and LRX8-RALF4 complexes at 3.2- and 3.9-Å resolution, respectively, reveal a dimeric arrangement of LRX proteins, with each monomer binding one folded RALF peptide. Structure-based mutations targeting the LRX-RALF4 complex interface, or the RALF4 fold, reduce RALF4 binding to LRX8 in vitro and RALF4 function in growing pollen tubes. Mutants targeting the disulfide-bond stabilized LRX dimer interface fail to rescue lrx infertility phenotypes. Quantitative biochemical assays reveal that RALF4 binds LLGs and LRX cell-wall modules with drastically different binding affinities, and with distinct and mutually exclusive binding modes. Our biochemical, structural, and genetic analyses reveal a complex signaling network by which RALF ligands instruct different signaling proteins using distinct targeting mechanisms.