Leucine-rich Repeat Extensins Research Articles

Identification, Classification, and Expression Analysis of Leucine-Rich Repeat Extensin Genes from Brassica rapa Reveals Salt and Osmosis Stress Response Genes

Identification, Classification, and Expression Analysis of Leucine-Rich Repeat Extensin Genes from Brassica rapa Reveals Salt and Osmosis Stress Response Genes

Rapid alkalinization factor 22 has a structural and signalling role in root hair cell wall assembly.

Pressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides, which bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSIN (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signalling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 and FERONIA, triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a feedback signalling molecule that regulates this process depending on its interaction partners. This mechanism may also underlie wall assembly and expansion in other plant cell types.

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.

High confidence QTLs and key genes identified using Meta-QTL analysis for enhancing heat tolerance in chickpea (Cicer arietinum L.).

The rising global temperatures seriously threaten sustainable crop production, particularly the productivity and production of heat-sensitive crops like chickpeas. Multiple QTLs have been identified to enhance the heat stress tolerance in chickpeas, but their successful use in breeding programs remains limited. Towards this direction, we constructed a high-density genetic map spanning 2233.5 cM with 1069 markers. Using 138 QTLs reported earlier, we identified six Meta-QTL regions for heat tolerance whose confidence interval was reduced by 2.7-folds compared to the reported QTLs. Meta-QTLs identified on CaLG01 and CaLG06 harbor QTLs for important traits, including days to 50% flowering, days to maturity, days to flower initiation, days to pod initiation, number of filled pods, visual score, seed yield per plant, biological yield per plant, chlorophyll content, and harvest index. In addition, key genes identified in Meta-QTL regions like Pollen receptor-like kinase 3 (CaPRK3), Flowering-promoting factor 1 (CaFPF1), Flowering Locus C (CaFLC), Heat stress transcription factor A-5 (CaHsfsA5), and Pollen-specific leucine-rich repeat extensins (CaLRXs) play an important role in regulating the flowering time, pollen germination, and growth. The consensus genomic regions, and the key genes reported in this study can be used in genomics-assisted breeding for enhancing heat tolerance and developing heat-resilient chickpea cultivars.

Exploring the extensin gene family: an updated genome-wide survey in plants and algae.

Extensins (EXTs), a class of hydroxyproline-rich glycoprotein with multiple Ser-Pro3-5 motifs, are known to play roles in cell wall reinforcement and environmental responses. EXTs with repetitive Tyr-X-Tyr (YXY) motifs for crosslinking are referred as crosslinking EXTs. Our comprehensive study spanned 194 algal and plant species, categorizing EXTs into seven subfamilies: classical extensins (EXT I and II), arabinogalactan-protein extensins (AGP-EXTs), proline-rich extensin-like receptor kinases (PERKs), leucine-rich repeat extensins (LRX I and II), formin homology (FH) domain-containing extensins (FH-EXTs), proline-rich, arabinogalactan proteins, conserved cysteines (PAC) domain-containing extensins (PAC I and II), and eight-cysteine motif (8CM)-containing extensins (8CM-EXTs). In the examined dataset, EXTs were detected ubiquitously in plants but infrequently in algae, except for one Coccomyxa and four Chlamydomonadales species. No crosslinking EXTs were found in Poales or certain Zingiberales species. Notably, the previously uncharacterized EXT II, PAC II, and liverwort-specific 8CM-EXTs were found to be crosslinking EXTs. EXT II, featuring repetitive YY motifs instead of the conventional YXY motif, was exclusively identified in Solanaceae. Furthermore, tandem genes encoding distinctive 8CM-EXTs specifically expressed in the germinating spores of Marchantia polymorpha. This updated classification of EXT types allows us to propose a plausible evolutionary history of EXT genes during the course of plant evolution.

Pseudomonas Cyclic Lipopeptide Medpeptin: Biosynthesis and Modulation of Plant Immunity

The multifunctional secondary metabolites known as cyclic lipopeptides (CLPs), which are produced by a large variety of bacteria, have become a key category of plant immunity elicitors. Pseudomonas-CLPs (Ps-CLPs) are extremely diverse in structure and biological activity. However, an understanding of CLP-plant structure–function interactions currently remains elusive. Here, we identify medpeptin, a novel CLP from Pseudomonas mediterranea that consists of 22 amino acids. Medpeptin is synthesized by a non-ribosomal peptide synthase (NRPS) gene cluster and regulated by a quorum-sensing system. Further research indicates that medpeptin does not exhibit antimicrobial activity; instead, it induces plant cell death immunity and confers resistance to bacterial infection. Comparative transcriptome analysis and virus-induced gene silencing (VIGS) reveal a set of immune signaling candidates involved in medpeptin perception. Silencing of a cell-wall leucine-rich repeat extensin protein (NbLRX3) or a receptor-like protein kinase (NbRLK25)—but not BAK1 or SGT1—compromises medpeptin-triggered cell death and resistance to pathogen infection in Nicotiana benthamiana. Our findings point to a noncanonical mechanism of CLP sensing and suggest perspectives for the development of plant disease resistance.

OsPEX1, an extensin-like protein, negatively regulates root growth in a gibberellin-mediated manner in rice

Leucine-rich repeat extensins (LRXs) are required for plant growth and development through affecting cell growth and cell wall formation. LRX gene family can be classified into two categories: predominantly vegetative-expressed LRX and reproductive-expressed PEX. In contrast to the tissue specificity of Arabidopsis PEX genes in reproductive organs, rice OsPEX1 is also highly expressed in roots in addition to reproductive tissue. However, whether and how OsPEX1 affects root growth is unclear. Here, we found that overexpression of OsPEX1 retarded root growth by reducing cell elongation likely caused by an increase of lignin deposition, whereas knockdown of OsPEX1 had an opposite effect on root growth, indicating that OsPEX1 negatively regulated root growth in rice. Further investigation uncovered the existence of a feedback loop between OsPEX1 expression level and GA biosynthesis for proper root growth. This was supported by the facts that exogenous GA3 application downregulated transcript levels of OsPEX1 and lignin-related genes and rescued the root developmental defects of the OsPEX1 overexpression mutant, whereas OsPEX1 overexpression reduced GA level and the expression of GA biosynthesis genes. Moreover, OsPEX1 and GA showed antagonistic action on the lignin biosynthesis in root. OsPEX1 overexpression upregulated transcript levels of lignin-related genes, whereas exogenous GA3 application downregulated their expression. Taken together, this study reveals a possible molecular pathway of OsPEX1mediated regulation of root growth through coordinate modulation of lignin deposition via a negative feedback regulation between OsPEX1 expression and GA biosynthesis.

Discovery of 2,6-Dihalopurines as Stomata Opening Inhibitors: Implication of an LRX-Mediated H+-ATPase Phosphorylation Pathway.

Stomata are pores in the leaf epidermis of plants and their opening and closing regulate gas exchange and water transpiration. Stomatal movements play key roles in both plant growth and stress responses. In recent years, small molecules regulating stomatal movements have been used as a powerful tool in mechanistic studies, as well as key players for agricultural applications. Therefore, the development of new molecules regulating stomatal movement and the elucidation of their mechanisms have attracted much attention. We herein describe the discovery of 2,6-dihalopurines, AUs, as a new stomatal opening inhibitor, and their mechanistic study. Based on biological assays, AUs may involve in the pathway related with plasma membrane H+-ATPase phosphorylation. In addition, we identified leucine-rich repeat extensin proteins (LRXs), LRX3, LRX4 and LRX5 as well as RALF, as target protein candidates of AUs by affinity based pull down assay and molecular dynamics simulation. The mechanism of stomatal movement related with the LRXs-RALF is an unexplored pathway, and therefore further studies may lead to the discovery of new signaling pathways and regulatory factors in the stomatal movement.

Self S-RNase restricts ABF-LRX signaling cascade to arrest pollen tube during pear self-incompatibility reaction.

Gametophytic self-incompatibility (GSI) has been widely studied in flowering plants, but the study on mechanism of arresting pollen tube growth by self S-RNase in the GSI species is limited. In this study, two leucine-rich repeat extensin genes, PbLRXA2.1 and PbLRXA2.2, were identified based on transcriptome and quantitative real-time PCR analyses. The expression levels of these two LRX genes were significantly higher in the pollen grains and pollen tubes of self-compatible cultivar 'Jinzhui' (a spontaneous bud mutation of the self-incompatible cultivar 'Yali') than in 'Yali'. Both PbLRXA2.1 and PbLRXA2.2 positively mediated pollen tube growth and attenuated self S-RNase toxicity on pollen tube growth by stabilizing actin cytoskeleton and cell wall integrity. This result indicates that the abnormal expression of PbLRXA2.1 and PbLRXA2.2 involve in the breakdown of self-incompatibility in 'Jinzhui'. The PbLRXA2.1 and PbLRXA2.2 promoters were directly bound by an ABRE-binding factor PbABF.D.2. Knockdown of PbABF.D.2 expression decreased PbLRXA2.1 and PbLRXA2.2 expression and inhibited pollen tube growth. Notably, the expressions of PbLRXA2.1, PbLRXA2.2, and PbABF.D.2 were repressed by self S-RNase, suggesting that self S-RNase can arrest pollen tube growth by restricting the PbABF.D.2-PbLRXA2.1/PbLRXA2.2 signal cascade. These results provide a novel insight of the pollen tube growth arrested by self S-RNase.

Regulation of immune receptor kinase plasma membrane nanoscale organization by a plant peptide hormone and its receptors.

Spatial partitioning is a propensity of biological systems orchestrating cell activities in space and time. The dynamic regulation of plasma membrane nano-environments has recently emerged as a key fundamental aspect of plant signaling, but the molecular components governing it are still mostly unclear. The receptor kinase FERONIA (FER) controls ligand-induced complex formation of the immune receptor kinase FLAGELLIN SENSING 2 (FLS2) with its co-receptor BRASSINOSTEROID-INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1), and perception of the endogenous peptide hormone RAPID ALKALANIZATION FACTOR 23 (RALF23) by FER inhibits immunity. Here, we show that FER regulates the plasma membrane nanoscale organization of FLS2 and BAK1. Our study demonstrates that akin to FER, leucine-rich repeat (LRR) extensin proteins (LRXs) contribute to RALF23 responsiveness and regulate BAK1 nanoscale organization and immune signaling. Furthermore, RALF23 perception leads to rapid modification of FLS2 and BAK1 nanoscale organization, and its inhibitory activity on immune signaling relies on FER kinase activity. Our results suggest that perception of RALF peptides by FER and LRXs actively modulates plasma membrane nanoscale organization to regulate cell surface signaling by other ligand-binding receptor kinases.

Malectin-like receptor kinases as protector deities in plant immunity.

Plant malectin-like receptor kinases (MLRs), also known as Catharanthus roseus receptor-like kinase-1-like proteins, are well known for their functions in pollen tube reception and tip growth, cell wall integrity sensing, and hormonal responses. Recently, mounting evidence has indicated a critical role for MLRs in plant immunity. Here we focus on the emerging functions of MLRs in modulating the two-tiered immune system mediated by cell-surface-resident pattern recognition receptors (PRRs) and intracellular nucleotide-binding leucine-rich repeat receptors (NLRs). MLRs complex with PRRs and NLRs and regulate immune receptor complex formation and stability. Rapid alkalinization factor peptide ligands, LORELEI-like glycosylphosphatidylinositol-anchored proteins and cell-wall-associated leucine-rich repeat extensins coordinate with MLRs to orchestrate PRR- and NLR-mediated immunity. We discuss the common theme and unique features of MLR complexes concatenating different branches of plant immune signalling.

OsPEX1, a leucine-rich repeat extensin protein, functions in the regulation of caryopsis development and quality in rice

Rice caryopses are enclosed by outer glumes. The size and dimension of the outer glume are the main determinants of caryopsis size. However, it is unclear whether caryopsis development is completely dependent on the size of the glume, or whether it can grow and expand autonomously despite the constraint of glume enclosure. We report the identification of a mutant line that produces normal-sized glumes with smaller mature caryopses that do not fill the entire glume cavity. The caryopsis phenotype in the pex1 mutant is caused by a reduction in cell size. OsPEX1, a leucine-rich repeat extensin gene, was highly expressed in the developing caryopsis. Overexpression of OsPEX1 driven by a constitutive promoter recapitulated the mutant phenotype, showing that the small-caryopsis phenotype is caused by overexpression of the OsPEX1 gene. Free amino acids, including several essential amino acids, and crude protein were increased in pex1 relative to the wild type, endowing pex1 with improved nutritional quality. Our results suggest that caryopsis development can be genetically uncoupled from maternally controlled glume development and that OsPEX1 might be a new resource for improving nutritional quality of rice cultivars.

Effect of extensin-like OsPEX1 on pollen fertility in rice.

LRXs (leucine-rich repeat extensins) are chimeric cell wall proteins containing an N-terminal leucine-rich repeat (LRR) and a C-terminal extensin domain. Increasing evidences suggest that LRXs family genes play important roles in pollen germination and pollen tube growth in Arabidopsis thaliana. However, the functions of rice (Oryza sativa L.) LRX genes in pollen development remain poorly understood. Bioinformatics analysis showed that the rice LRX gene family consist of eight members, namely OsPEX3, OsLRX3 and OsLRX5 located on chromosome 1, OsLRX1, OsLRX3, OsLRX2,OsPEX1 and OsPEX2 located on chromosome 2, 5, 6, 11 and 12, respectively. The OsPEX1 gene is preferentially expressed in rice anther, suggesting that it may be involved in the regulation of pollen development. Next, we further investigated the role of the OsPEX1 gene in rice by knockdown of its expression using an RNAi approach. The OsPEX1 RNAi transgenic lines showed a significant decrease in seed setting rate (10%~30%) due to pollen sterility. Further quantitative RT-PCR analysis indicated that the OsPEX1 gene was significantly down-regulated in the RNAi transgenic lines. The results indicate that the OsPEX1plays an important role in the regulation of rice pollen development. Further studies on this gene could provide insights on the molecular and genetic mechanisms in this developmental process.

Keeping up with the RALFs: how these small peptides control pollen-pistil interactions in Arabidopsis.

The pollen and pistil RALF peptides, along with multiple receptor-like kinases and leucine-rich repeat extensins, regulate pollen tube growth and the final burst within the ovule, where sperm cells are released for fertilisation to occur. This review introduces some new questions that arose about the regulation of this complex process.

The LRXs-RALFs-FER module controls plant growth and salt stress responses by modulating multiple plant hormones.

Salt stress is a major environmental factor limiting plant growth and productivity. We recently discovered an important new salt tolerance pathway, where the cell wall leucine-rich repeat extensins LRX3/4/5, the RAPID ALKALINIZATION FACTOR (RALF) peptides RALF22/23 and receptor-like kinase FERONIA (FER) function as a module to simultaneously regulate plant growth and salt stress tolerance. However, the intracellular signaling pathways that are regulated by the extracellular LRX3/4/5-RALF22/23-FER module to coordinate growth, cell wall integrity and salt stress responses are still unknown. Here, we report that the LRX3/4/5-RALF22/23-FER module negatively regulates the levels of jasmonic acid (JA), salicylic acid (SA) and abscisic acid (ABA). Blocking JA pathway rescues the dwarf phenotype of the lrx345 and fer-4 mutants, while disruption of ABA biosynthesis suppresses the salt-hypersensitivity of these mutants. Many salt stress-responsive genes display abnormal expression patterns in the lrx345 and fer-4 mutants, as well as in the wild type plants treated with epigallocatechin gallate (EGCG), an inhibitor of pectin methylesterases, suggesting cell wall integrity as a critical factor that determines the expression pattern of stress-responsive genes. Production of reactive oxygen species (ROS) is constitutively increased in the lrx345 and fer-4 mutants, and inhibition of ROS accumulation suppresses the salt-hypersensitivity of these mutants. Together, our work provides strong evidence that the LRX3/4/5-RALF22/23-FER module controls plant growth and salt stress responses by regulating hormonal homeostasis and ROS accumulation.

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.

RNA-Seq analysis of compatible and incompatible styles of Pyrus species at the beginning of pollination.

At the early stage of pollination, the difference in gene expression between compatibility and incompatibility is highly significant about the pollen-specific expression of the LRR gene, resistance, and defensin genes. In Rosaceae, incompatible pollen can penetrate into the style during the gametophytic self-incompatibility response. It is therefore considered a stylar event rather than a stigmatic event. In this study, we explored the differences in gene expression between compatibility and incompatibility in the early stage of pollination. The self-compatible pear variety "Jinzhuili" is a naturally occurring bud mutant from "Yali", a leading Chinese native cultivar exhibiting typical gametophytic self-incompatibility. We collected the styles of 'Yali' and 'Jinzhuili' at 0.5 and 2h after self-pollination and then performed high-throughput sequencing. According to the KEGG analysis of the differentially expressed genes, several metabolic pathways, such as "Plant hormone signal transduction", "Plant-pathogen interaction", are the main pathways was the most represented pathway. Quantitative PCR was used to validate these differential genes. The expression levels of genes related to pollen growth and disease inhibition, such as LRR (Leucine-rich repeat extensin), resistance, defensin, and auxin, differed significantly between compatible and incompatible pollination. Interestingly, at 0.5h, most of these genes were upregulated in the compatible pollination system compared with the incompatible pollination system. Calcium transport, which requires ATPase, also demonstrated upregulated expression. In summary, the self-incompatibility reaction was initiated when the pollen land on the stigma.

Leucine-Rich Repeat Extensin Proteins and Their Role in Cell Wall Sensing.

Plant cells are surrounded by a cell wall that provides shape and physically limits cell expansion. To sense the environment and status of cell wall structures, plants have evolved cell wall integrity-sensing mechanisms that involve a number of receptors at the plasma membrane. These receptors can bind cell wall components and/or hormones to coordinate processes in the cell wall and the cytoplasm. This review focuses on the role of leucine-rich repeat extensins (LRXs) during cell wall development. LRXs are chimeric proteins that insolubilize in the cell wall and form protein-protein interaction platforms. LRXs bind RALF peptide hormones that modify cell wall expansion and also directly interact with the transmembrane receptor FERONIA, which is involved in cell growth regulation. LRX proteins, therefore, also represent a link between the cell wall and plasma membrane, perceiving extracellular signals and indirectly relaying this information to the cytoplasm.

Extracellular matrix sensing by FERONIA and Leucine-Rich Repeat Extensins controls vacuolar expansion during cellular elongation in Arabidopsis thaliana.

Cellular elongation requires the defined coordination of intra‐ and extracellular processes, but the underlying mechanisms are largely unknown. The vacuole is the biggest plant organelle, and its dimensions play a role in defining plant cell expansion rates. Here, we show that the increase in vacuolar occupancy enables cellular elongation with relatively little enlargement of the cytosol in Arabidopsis thaliana. We demonstrate that cell wall properties are sensed and impact on the intracellular expansion of the vacuole. Using vacuolar morphology as a quantitative read‐out for intracellular growth processes, we reveal that the underlying cell wall sensing mechanism requires interaction of extracellular leucine‐rich repeat extensins (LRXs) with the receptor‐like kinase FERONIA (FER). Our data suggest that LRXs link plasma membrane‐localised FER with the cell wall, allowing this module to jointly sense and convey extracellular signals to the cell. This mechanism coordinates the onset of cell wall acidification and loosening with the increase in vacuolar size.

Mutations in the Arabidopsis ROL17/isopropylmalate synthase 1 locus alter amino acid content, modify the TOR network, and suppress the root hair cell development mutant lrx1.

The growth and development of organisms must be tightly controlled and adjusted to nutrient availability and metabolic activities. The Target of Rapamycin (TOR) network is a major control mechanism in eukaryotes and influences processes such as translation, mitochondrial activity, production of reactive oxygen species, and the cytoskeleton. In Arabidopsis thaliana, inhibition of the TOR kinase causes changes in cell wall architecture and suppression of phenotypic defects of the cell wall formation mutant lrx1 (leucine-rich repeat extensin 1). The rol17 (repressor of lrx1 17) mutant was identified as a new suppressor of lrx1 that induces also a short root phenotype. The ROL17 locus encodes isopropylmalate synthase 1, a protein involved in leucine biosynthesis. Dependent on growth conditions, mutations in ROL17 do not necessarily alter the level of leucine, but always cause development of the rol17 mutant phenotypes, suggesting that the mutation does not only influence leucine biosynthesis. Changes in the metabolome of rol17 mutants are also found in plants with inhibited TOR kinase activity. Furthermore, rol17 mutants show reduced sensitivity to the TOR kinase inhibitor AZD-8055, indicating a modified TOR network. Together, these data suggest that suppression of lrx1 by rol17 is the result of an alteration of the TOR network.