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.

The single Marchantia polymorpha FERONIA homolog reveals an ancestral role in regulating cellular expansion and integrity.

Plant cells are surrounded by a cell wall, a rigid structure that is not only important for cell and organ shape, but is also crucial for intercellular communication and interactions with the environment. In the flowering plant Arabidopsis thaliana, the 17 members of the Catharanthus roseus RLK1-like (CrRLK1L) receptor kinase family are involved in a multitude of physiological and developmental processes, making it difficult to assess their primary or ancestral function. To reduce genetic complexity, we characterized the single CrRLK1L gene of Marchantia polymorpha, MpFERONIA (MpFER). Plants with reduced MpFER levels show defects in vegetative development, i.e. rhizoid formation and cell expansion, and have reduced male fertility. In contrast, cell integrity and morphogenesis of the gametophyte are severely affected in Mpfer null mutants and MpFER overexpression lines. Thus, we conclude that the CrRLK1L gene family originated from a single gene with an ancestral function in cell expansion and the maintenance of cellular integrity. During land plant evolution, this ancestral gene diversified to fulfill a multitude of specialized physiological and developmental roles in the formation of both gametophytic and sporophytic structures essential to the life cycle of flowering plants.

Genome-Wide Re-Identification and Analysis of CrRLK1Ls in Tomato.

The Catharanthus roseus receptor-like kinase 1-like (CrRLK1L), which is a vital member of the plant receptor-like kinase family, plays versatile roles in plant growth, development, and stress response. Although the primary screening of tomato CrRLK1Ls has been reported previously, our knowledge of these proteins is still scarce. Using the latest genomic data annotations, a genome-wide re-identification and analysis of the CrRLK1Ls in tomatoes were conducted. In this study, 24 CrRLK1L members were identified in tomatoes and researched further. Subsequent gene structures, protein domains, Western blot analyses, and subcellular localization analyses all confirmed the accuracy of the newly identified SlCrRLK1L members. Phylogenetic analyses showed that the identified SlCrRLK1L proteins had homologs in Arabidopsis. Evolutionary analysis indicated that two pairs of the SlCrRLK1L genes had predicted segmental duplication events. Expression profiling analyses demonstrated that the SlCrRLK1L genes were expressed in various tissues, and most of them were up- or down-regulated by bacteria and PAMP treatments. Together, these results will lay the foundation for elaborating the biological roles of SlCrRLK1Ls in tomato growth, development, and stress response.

Rice pollen-specific OsRALF17 and OsRALF19 are essential for pollen tube growth.

Pollen tube growth is essential for successful double fertilization, which is critical for grain yield in crop plants. Rapid alkalinization factors (RALFs) function as ligands for signal transduction during fertilization. However, functional studies on RALF in monocot plants are lacking. Herein, we functionally characterized two pollen-specific RALFs in rice (Oryza sativa) using multiple clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-induced loss-of-function mutants, peptide treatment, expression analyses, and tag reporter lines. Among the 41 RALF members in rice, OsRALF17 was specifically expressed at the highest level in pollen and pollen tubes. Exogenously applied OsRALF17 or OsRALF19 peptide inhibited pollen tube germination and elongation at high concentrations but enhanced tube elongation at low concentrations, indicating growth regulation. Double mutants of OsRALF17 and OsRALF19 (ralf17/19) exhibited almost full male sterility with defects in pollen hydration, germination, and tube elongation, which was partially recovered by exogenous treatment with OsRALF17 peptide. This study revealed that two partially functionally redundant OsRALF17 and OsRALF19 bind to Oryza sativa male-gene transfer defective 2 (OsMTD2) and transmit reactive oxygen species signals for pollen tube germination and integrity maintenance in rice. Transcriptomic analysis confirmed their common downstream genes, in osmtd2 and ralf17/19. This study provides new insights into the role of RALF, expanding our knowledge of the biological role of RALF in regulating rice fertilization.

OsMTD2-mediated reactive oxygen species (ROS) balance is essential for intact pollen-tube elongation in rice.

The highly specialized haploid male gametophyte-pollen consist of two sperm cells and a large vegetative cell. Successful fertilization requires proper growth timing and rupture of the pollen tube until it delivers sperm cells, which occur immediately after a pollen grain hydrates. Although a tight regulation on polar cell-wall expansion of the pollen tube is fundamentally important, the underlying molecular mechanism remains largely unknown, especially in crop plants. Here, we characterized the function of male-gene transfer defective 2 (OsMTD2) gene in rice (Oryza sativa), which belongs to the plant-specific receptor-like kinase, the CrRLK1L family. We demonstrated that OsMTD2 is an essential male factor participating in pollen-tube elongation based on genetic evidence and physiological observations. Because of unavailability of homozygous mutant via conventional methods, we used CRISPR-Cas9 system to obtain homozygous knockout mutant of OsMTD2. We were able to identify phenotypic changes including male sterility due to early pollen-tube rupture in the mutant. We observed that the production of reactive oxygen species (ROS) was dramatically reduced in mutants of OsMTD2 pollen grain and tubes with defective pectin distribution. Transcriptome analysis of osmtd2-2 versus wild-type anthers revealed that genes involved in defense responses, metabolic alteration, transcriptional and protein modification were highly upregulated in the osmtd2-2 mutant. Through yeast-two-hybrid screening, we found that OsMTD2 kinase interacts with E3 ligase SPL11. Taken together, we propose that OsMTD2 has crucial functions in promoting pollen-tube elongation through cell-wall modification, possibly by modulating ROS homeostasis during pollen-tube growth.

CrRLK1L receptor-like kinases HERK1 and ANJEA are female determinants of pollen tube reception.

Communication between the gametophytes is vital for angiosperm fertilisation. Multiple CrRLK1L-type receptor kinases prevent premature pollen tube burst, while another CrRLK1L protein, FERONIA (FER), is required for pollen tube reception in the female gametophyte. We report here the identification of two additional CrRLK1L homologues, HERCULES RECEPTOR KINASE 1 (HERK1) and ANJEA (ANJ), which act redundantly to promote pollen tube growth arrest at the synergid cells. HERK1 and ANJ localise to the filiform apparatus of the synergid cells in unfertilised ovules, and in herk1 anj mutants, a majority of ovules remain unfertilised due to pollen tube overgrowth, together indicating that HERK1 and ANJ act as female determinants for fertilisation. As in fer mutants, the synergid cell-specific, endomembrane protein NORTIA (NTA) is not relocalised after pollen tube reception; however, unlike fer mutants, reactive oxygen species levels are unaffected in herk1 anj double mutants. Both ANJ and HERK1 associate with FER and its proposed co-receptor LORELEI (LRE) in planta. Together, our data indicate that HERK1 and ANJ act with FER to mediate female-male gametophyte interactions during plant fertilisation.

New paradigms in cell adaptation: decades of discoveries on the CrRLK1L receptor kinase signalling network.

Receptor-like kinases (RLKs), which constitute the largest receptor family in plants, are essential for perceiving and relaying information about various environmental stimuli. Tremendous progress has been made in the past few decades towards elucidating the mechanisms of action of several RLKs, with emerging paradigms pointing to their roles in cell adaptations. Among these paradigms, Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) proteins and their rapid alkalinization factor (RALF) peptide ligands have attracted much interest. In particular, FERONIA (FER) is a CrRLK1L protein that participates in a wide array of physiological processes associated with RALF signalling, including cell growth and monitoring cell wall integrity, RNA and energy metabolism, and phytohormone and stress responses. Here, we analyse FER in the context of CrRLK1L members and their ligands in multiple species. The FER working model raises many questions about the role of CrRLK1L signalling networks during cell adaptation. For example, how do CrRLK1Ls recognize various RALF peptides from different organisms to initiate specific phosphorylation signal cascades? How do RALF-FER complexes achieve their specific, sometimes opposite, functions in different cell types? Here, we summarize recent major findings and highlight future perspectives in the field of CrRLK1L signalling networks.

Versatile Roles of the Receptor-Like Kinase Feronia in Plant Growth, Development and Host-Pathogen Interaction.

As a member of the Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) protein kinase subfamily, FERONIA (FER) has emerged as a versatile player regulating multifaceted functions in growth and development, as well as responses to environmental factors and pathogens. With the concerted efforts of researchers, the molecular mechanism underlying FER-dependent signaling has been gradually elucidated. A number of cellular processes regulated by FER-ligand interactions have been extensively reported, implying cell type-specific mechanisms for FER. Here, we provide a review on the roles of FER in male-female gametophyte recognition, cell elongation, hormonal signaling, stress responses, responses to fungi and bacteria, and present a brief outlook for future efforts.

Pollen tube integrity regulation in flowering plants: insights from molecular assemblies on the pollen tube surface.

Contents Summary 687 I. Introduction 687 II. Pollen tube membrane-localized receptors coordinate cell integrity and sperm release 689 III. RALF peptides mediate autocrine and paracrine signaling 689 IV. ROS and ion channel signaling mediate intracellular response 690 V. Involvements from pollen tube cell wall components 690 VI. Concluding remarks 691 Acknowledgements 692 Author contributions 692 References 692 SUMMARY: Unlike in animals, sperm in flowering plants are immotile and they are embraced as passive cargoes by a pollen tube which embarks on a long journey in the pistil to deliver them to the female gametophyte for fertilization. How the pollen tube switches from a rapid polarized growth towards its target to an abrupt disintegration for sperm cell release inside the female gametophyte is puzzling. Recent studies have shown that members of the Catharanthus roseus RLK1-like (CrRLK1L) receptor kinase family and their ligands, 5-kDa cysteine-rich peptide rapid alkalinization factors (RALFs), engage in an intricate balancing act involving autocrine and paracrine signaling to maintain pollen tube growth and induce timely tube rupture at the spatially confined pollen tube-female gametophyte interface. Here, we review recent progress related to pollen tube integrity control, mainly focusing on the molecular understanding of signaling as well as intracellular signaling nodes in Arabidopsis. Some missing links and future perspectives are also discussed.

T-DNA alleles of the receptor kinase THESEUS1 with opposing effects on cell wall integrity signaling.

Perturbation of cellulose synthesis in plants triggers stress responses, including growth retardation, mediated by the cell wall integrity-sensing receptor-like kinase (RLK) THESEUS1 (THE1). The analysis of two alleles carrying T-DNA insertions at comparable positions has led to conflicting conclusions concerning the impact of THE1 signaling on growth. Here we confirm that, unlike the1-3 and other the1 alleles in which cellular responses to genetic or pharmacological inhibition of cellulose synthesis are attenuated, the1-4 showed enhanced responses, including growth inhibition, ectopic lignification, and stress gene expression. Both the1-3 and the1-4 express a transcript encoding a predicted membrane-associated truncated protein lacking the kinase domain. However, the1-3, in contrast to the1-4, strongly expresses antisense transcripts, which are expected to prevent the expression of the truncated protein as suggested by the genetic interactions between the two alleles. Seedlings overexpressing such a truncated protein react to isoxaben treatment similarly to the1-4 and the full-length THE overexpressor. We conclude that the1-4 is a hypermorphic allele; that THE1 signaling upon cell wall damage has a negative impact on cell expansion; and that caution is required when interpreting the phenotypic effects of T-DNA insertions in RLK genes.

A Receptor-Like Kinase, Related to Cell Wall Sensor of Higher Plants, is Required for Sexual Reproduction in the Unicellular Charophycean Alga, Closterium peracerosum-strigosum-littorale Complex.

Here, we cloned the CpRLK1 gene, which encodes a receptor-like protein kinase expressed during sexual reproduction, from the heterothallic Closterium peracerosum-strigosum-littorale complex, one of the closest unicellular alga to land plants. Mating-type plus (mt(+)) cells with knockdown of CpRLK1 showed reduced competence for sexual reproduction and formed an abnormally enlarged conjugation papilla after pairing with mt(-) cells. The knockdown cells were unable to release a naked gamete, which is indispensable for zygote formation. We suggest that the CpRLK1 protein is an ancient cell wall sensor that now functions to regulate osmotic pressure in the cell to allow proper gamete release.

The receptor kinase OsANX limits precocious flowering and inflorescence over-branching and maintains pollen tube integrity in rice.

CrRLK1L subfamily members are involved in diverse growth- and development-related processes in Arabidopsis. However, the functions of their counterparts in rice are unknown. Here, OsANX expression was detected in developing inflorescences, mature pollen grains, and growing pollen tubes, and it was localized to the plasma membrane in pollen grains and tobacco epidermal cells. Homozygous osanx progeny could not be segregated from the CRISPR/Cas9-edited mutants osanx-c1+/- and osanx-c2+/-, and such progeny were segregated only occasionally from osanx-c3+/-. Further, all three alleles showed osanx male but not female gamete transmission defects, in line with premature pollen tube rupture in osanx-c3. Additionally, osanx-c3 exhibited precocious flowering, excessively branched inflorescences, and an extremely low seed setting rate of 1.4 %, while osanx-c2+/- and osanx-c3+/- had no obvious defects in inflorescence development or the seed setting rate compared to wild-type Nipponbare (Nip). Consistent with this, the complemented line pPS1:OsANX-GFP/osanx-c2 (PSC), in which the lack of OsANX expression was inflorescence-specific, showed slightly earlier flowering and overly-branched panicles. Multiple inflorescence meristem transition-related and inflorescence architecture-related genes were expressed at higher levels in osanx-c3 than in Nip; thus, they may partially account for the aforementioned mutant phenotypes. Our findings broaden our understanding of the biological functions of OsANX in rice.

Genome wide characterization and expression analysis of CrRLK1L gene family in wheat unravels their roles in development and stress-specific responses.

Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) genes encode a subfamily of receptor-like kinases (RLK) that regulate diverse processes during plant growth, development, and stress responses. The first CrRLK1L was identified from the Catharanthus roseus, commonly known as Madagascar periwinkle. Subsequently, CrRLK1L gene families have been characterized in many plants. The genome of T. aestivum encodes 15 CrRLK1L genes with 43 paralogous copies, with three homeologs each, except for -2-D and -7-A, which are absent. Chromosomal localization analysis revealed a markedly uneven distribution of CrRLK1L genes across seven different chromosomes, with chromosome 4 housing the highest number of genes, while chromosome 6 lacked any CrRLK1L genes. Tissue-specific gene expression analysis revealed distinct expression patterns among the gene family members, with certain members exhibiting increased expression in reproductive tissues. Gene expression analysis in response to various abiotic and biotic stress conditions unveiled differential regulation of gene family members. Cold stress induces CrRLK1Ls -4-B and -15-A while downregulating -3-A and -7B. Drought stress upregulates -9D, contrasting with the downregulation of -7D. CrRLK1L-15-B and -15-D were highly induced in response to 1 hr of heat, and combined drought and heat stress, whereas -10-B is downregulated. Similarly, in response to NaCl stress, only CrRLK1L1 homeologs were induced. Fusarium graminearum and Claviceps purpurea inoculation induces homeologs of CrRLK1L-6 and -7. The analysis of cis-acting elements in the promoter regions identified elements crucial for plant growth and developmental processes. This comprehensive genome-wide analysis and expression study provides valuable insights into the essential functions of CrRLK1L members in wheat.

PbrROP1/2-elicited imbalance of cellulose deposition is mediated by a CrRLK1L-ROPGEF module in the pollen tube of Pyrus.

Pollen tube growth is critical for the sexual reproduction of flowering plants. Catharanthus roseus receptor-like kinases (CrRLK1L) play an important role in plant sexual reproduction, pollen tube growth, and male and female gametophyte recognition. Here, we identified a CrRLK1L protein in pear (Pyrus bretschneideri), PbrCrRLK1L13, which is necessary for normal tip growth of pollen tube. When PbrCrRLK1L13 was knocked down, the pollen tube grew faster. Interaction analysis showed that the kinase domain of PbrCrRLK1L13 interacted with the C-terminal region of PbrGEF8, and PbrCrRLK1L13 activated the phosphorylation of PbrGEF8 in vitro. Furthermore, PbrROP1 and PbrROP2 were the downstream targets of PbrCrRLK1L13-PbrGEF8. When we knocked down the expression of PbrCrRLK1L13, PbrGEF8 or PbrROP1/2, the balance of cellulose deposition in the pollen tube wall was disrupted. Considering these factors, we proposed a model for a signaling event regulating pear pollen tube growth. During pear pollen tube elongation, PbrCrRLK1L13 acted as a surface regulator of the PbrROP1 and PbrROP2 signaling pathway via PbrGEF8 to affect the balance of cellulose deposition and regulate pear pollen tube growth.

Systematic Analysis of Tobacco CrRLK1L Family Genes and Functional Identification of NtCrRLK1L47 in Environmental Stresses.

The Catharanthus roseus RLK1-like (CrRLK1L) family is involved in the regulation of plant reproduction, growth and development, cell wall integrity sensing, as well as responses to both biotic and abiotic stress conditions. Extraordinary progress has been made in elucidating the CrRLK1L family receptor kinases-mediated signaling pathway, while limited research addressed the functions of CrRLK1L proteins in tobacco. In this study, we identified and analyzed 48 NtCrRLK1L members from the tobacco genome. The newly identified NtCrRLK1L members were divided into seven groups together with the Arabidopsis CrRLK1L members. The syntenic analysis revealed that four pairs of NtCrRLK1L genes were predicted to have arisen from segmental duplication events. Expression profiling showed that the NtCrRLK1L genes were expressed in various tissues, and most NtCrRLK1L genes were induced by salt and drought stress conditions. Notably, NtCrRLK1L47 was upregulated under drought and salinity stresses, and the NtCrRLK1L47-GFP fusion protein was located in the cell membrane. Furthermore, overexpression of the NtCrRLK1L47 gene enhanced the salt tolerance in tobacco seedlings.

Genome-Wide Analysis of the Catharanthus roseus RLK1-Like in Soybean and GmCrRLK1L20 Responds to Drought and Salt Stresses.

Abiotic stresses, such as drought and salinity, severely affects the growth, development and productivity of the plants. The Catharanthus roseus RLK1-like (CrRLK1L) protein kinase family is involved in several processes in the plant life cycle. However, there have been few studies addressing the functions of CrRLK1L proteins in soybean. In this study, 38 CrRLK1L genes were identified in the soybean genome (Glycine max Wm82.a2.v1). Phylogenetic analysis demonstrated that soybean CrRLK1L genes were grouped into clusters, cluster I, II, III. The chromosomal mapping demonstrated that 38 CrRLK1L genes were located in 14 of 20 soybean chromosomes. None were discovered on chromosomes 1, 4, 6, 7, 11, and 14. Gene structure analysis indicated that 73.6% soybean CrRLK1L genes were characterized by a lack of introns.15.7% soybean CrRLK1L genes only had one intron and 10.5% soybean CrRLK1L genes had more than one intron. Five genes were obtained from soybean drought- and salt-induced transcriptome databases and were found to be highly up-regulated. GmCrRLK1L20 was notably up-regulated under drought and salinity stresses, and was therefore studied further. Subcellular localization analysis revealed that the GmCrRLK1L20 protein was located in the cell membrane. The overexpression of the GmCrRLK1L20 gene in soybean hairy roots improved both drought tolerance and salt stresses and enhanced the expression of the stress-responsive genes GmMYB84, GmWRKY40, GmDREB-like, GmGST15, GmNAC29, and GmbZIP78. These results indicated that GmCrRLK1L20 could play a vital role in defending against drought and salinity stresses in soybean.

The CrRLK1L subfamily: One of the keys to versatility in plants.

Catharanthus roseous kinase 1L receptors (CrRLK1Ls) are a subfamily of membrane receptors unique to plant cells that perceive internal and external signals, integrate metabolic, physiological, and molecular processes, and regulate plant development. Recent genomic studies have suggested that this receptor subfamily arose during the emergence of terrestrial plants and has since diversified, preserving its essential functions. Participation of some of these CrRLK1Ls in different processes is presented and discussed herein, as well as the increasing number of interactors necessary for their function. At least five different responses have been detected after activating these receptors, such as physiological changes, formation or disassembly of protein complexes, metabolic responses, modification of gene expression, and modulation of phytohormone activity. To date, a common response mechanism for all processes involving CrRLK1Ls has not been described. In this review, the information available on the different functions of CrRLK1Ls was compiled. Additionally, the physiological and/or molecular mechanisms involved in the signaling processes triggered by these receptors are also discussed. In this review, we propose a possible common signaling mechanism for all processes regulated by CrRLK1Ls and pose questions to be answered in the future.

Stigmatic ROS: regulator of compatible pollen tube perception?

Accurate communication at the stigma surface is required to promote plants' own pollen and reject foreign pollen. Liu et al. have now discovered an autocrine signaling pathway at the surface of arabidopsis stigmatic papillae, accumulating ROS. Downregulation of ROS production via an antagonistic peptide from the pollen coat promotes pollen hydration and germination.

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.

Genome-Wide Identification of the CrRLK1L Subfamily and Comparative Analysis of Its Role in the Legume-Rhizobia Symbiosis.

The plant receptor-like-kinase subfamily CrRLK1L has been widely studied, and CrRLK1Ls have been described as crucial regulators in many processes in Arabidopsis thaliana (L.), Heynh. Little is known, however, about the functions of these proteins in other plant species, including potential roles in symbiotic nodulation. We performed a phylogenetic analysis of CrRLK1L subfamily receptors of 57 different plant species and identified 1050 CrRLK1L proteins, clustered into 11 clades. This analysis revealed that the CrRLK1L subfamily probably arose in plants during the transition from chlorophytes to embryophytes and has undergone several duplication events during its evolution. Among the CrRLK1Ls of legumes and A. thaliana, protein structure, gene structure, and expression patterns were highly conserved. Some legume CrRLK1L genes were active in nodules. A detailed analysis of eight nodule-expressed genes in Phaseolus vulgaris L. showed that these genes were differentially expressed in roots at different stages of the symbiotic process. These data suggest that CrRLK1Ls are both conserved and underwent diversification in a wide group of plants, and shed light on the roles of these genes in legume-rhizobia symbiosis.