CEPR1 Antibody

What is CEPR1 and why is it significant for plant research?

CEPR1 is a peptide hormone receptor that specifically interacts with C-TERMINALLY ENCODED PEPTIDES (CEPs) in plants. It plays crucial roles in coordinating plant immunity with nitrogen status and controlling the delivery of nitrogen to reproductive sinks. CEPR1 signaling is central to regulating Pattern-Triggered Immunity (PTI) and Systemic Acquired Resistance (SAR) in Arabidopsis . Additionally, CEPR1 functions in reproductive tissues to control yield and seed size, with knockout mutants showing substantial reductions in seed yield (88-98%) and seed weight (12-25%) . These diverse functions make CEPR1 a significant target for researchers studying plant defense mechanisms and reproductive development.

What are the main research applications for CEPR1 antibodies?

CEPR1 antibodies are valuable tools for investigating CEP-CEPR1 signaling pathways in multiple contexts. Primary applications include: (1) Immunolocalization to visualize CEPR1 tissue-specific expression patterns, particularly in vascular tissues where CEPR1 is predominantly expressed ; (2) Immunoprecipitation to study CEPR1 interactions with CEP peptides and downstream signaling components; (3) Western blotting to quantify CEPR1 protein expression levels in different tissues or under various stress conditions; and (4) Chromatin immunoprecipitation (ChIP) experiments when studying transcription factors that may regulate CEPR1 expression. These applications are essential for elucidating CEPR1's role in mediating immune responses and nitrogen allocation.

How do CEPR1 and CEP signaling function in the plant immune system?

CEP signaling through CEPR1 has been identified as a novel immune-modulatory pathway. Research demonstrates that CEP4 peptide triggers dose-dependent Pattern-Triggered Immunity (PTI) responses, including calcium ion influx, activation of MITOGEN-ACTIVATED PROTEIN KINASEs (MAPKs), ethylene production, and expression of immunity marker genes like FLAGELLIN-INDUCED RECEPTOR KINASE 1 (FRK1) . CEPR1, along with CEPR2, functions in perceiving CEP signals to mount effective cell surface immunity. Notably, cepr1/cepr2 double mutants show compromised resistance to bacterial pathogens like Pseudomonas syringae pv. tomato (Pto), and are strongly impaired in systemic acquired resistance (SAR) . CEPR1 antibodies are therefore valuable for tracking receptor abundance and localization during immune responses.

What controls should be included when validating a CEPR1 antibody for experimental use?

For rigorous CEPR1 antibody validation, include: (1) Positive control using wild-type plant tissues known to express CEPR1, particularly vascular tissues of shoots and roots ; (2) Negative control using cepr1 knockout mutants (cepr1-1 or cepr1-3) to confirm specificity ; (3) Peptide competition assay where pre-incubating the antibody with purified CEPR1 peptide should abolish signal; (4) Cross-reactivity assessment against related receptors, especially CEPR2, given their functional redundancy in some contexts ; and (5) Testing across multiple experimental methods (Western blot, immunoprecipitation, immunofluorescence) to ensure consistent performance. Additional controls should include testing tissues with known expression profiles, such as reproductive bolt vasculature and chalazal seed coat, where CEPR1 is specifically expressed .

How can I design experiments to investigate CEPR1-CEP interaction specificities using antibodies?

To investigate CEPR1-CEP interaction specificities: (1) Perform co-immunoprecipitation assays using anti-CEPR1 antibodies to pull down CEPR1 complexes from plant tissues treated with different CEP peptides (CEP1, CEP4, CEP9.5); (2) Conduct in vitro binding assays using purified CEPR1 ectodomain and synthetic CEP peptides, followed by antibody detection; (3) Design competition binding assays using labeled CEP peptides of different classes and measure displacement with unlabeled peptides to determine binding affinities; (4) Implement proximity ligation assays using anti-CEPR1 antibodies and labeled CEP peptides to visualize interaction sites in planta; and (5) Compare results with biochemical methods like isothermal titration calorimetry (ITC), which has previously shown CEP4 binding to CEPR2 ectodomain with a KD of 15.7 μM . Include appropriate controls like scrambled CEP peptides, which have been shown not to bind CEPR2 .

What are the methodological considerations when using CEPR1 antibodies for immunolocalization in reproductive tissues?

When performing immunolocalization of CEPR1 in reproductive tissues: (1) Tissue fixation must be optimized to preserve antigen accessibility while maintaining tissue architecture—aldehyde-based fixatives with minimal cross-linking are recommended; (2) Employ tissue-specific antigen retrieval methods, as reproductive tissues often contain compounds that can interfere with antibody binding; (3) Implement tissue clearing techniques to enhance signal detection, particularly important for dense reproductive structures; (4) Use confocal microscopy with z-stacking to precisely localize CEPR1 in specific cell types, especially in vascular tissues and the chalazal seed coat where CEPR1 is expressed ; (5) Include parallel experiments with CEPR1 promoter-reporter constructs to validate antibody-based localization; and (6) Always compare signal patterns with cepr1 mutant tissues to confirm specificity. Special attention should be paid to the vasculature of reproductive organs and the chalazal seed coat, as these are key sites of CEPR1 expression relevant to reproductive development .

How can CEPR1 antibodies be used to investigate the cross-talk between immunity and nitrogen signaling pathways?

To investigate CEPR1's role in immunity-nitrogen cross-talk: (1) Perform co-immunoprecipitation with CEPR1 antibodies under varying nitrogen conditions, followed by mass spectrometry to identify differentially associated proteins; (2) Conduct dual immunolocalization using CEPR1 antibodies alongside antibodies against nitrogen transporters or immune receptors to track co-localization patterns; (3) Implement protein proximity labeling techniques (BioID or TurboID) coupled with CEPR1 antibodies to capture transient interactions in nitrogen-depleted versus immune-challenged tissues; (4) Design phosphoproteomic analysis using CEPR1 immunoprecipitation under various nitrogen and immune conditions to track receptor activation states; and (5) Employ chromatin immunoprecipitation sequencing (ChIP-seq) to identify transcription factors regulating CEPR1 expression during nitrogen stress and pathogen challenge. These approaches can reveal molecular mechanisms underlying the observation that "short-term reduction in seedling N levels promotes flg22-induced PTI responses in a CEP and CEP receptor-dependent manner" .

What approaches can resolve contradictory data when studying tissue-specific expression of CEPR1?

When facing contradictory CEPR1 expression data: (1) Implement a multi-method validation approach combining antibody-based detection with transcript analysis (RT-qPCR, in situ hybridization) and promoter-reporter studies; (2) Perform subcellular fractionation followed by Western blotting to determine if discrepancies result from differential localization rather than expression; (3) Evaluate antibody specificity using multiple CEPR1 knockout lines (cepr1-1, cepr1-3) to rule out cross-reactivity with CEPR2 or other related receptors; (4) Consider developmental timing, as CEPR1 expression patterns may change throughout plant development—particularly in reproductive tissues; (5) Conduct single-cell or tissue-specific proteomics to resolve cell-type-specific expression patterns that might be obscured in whole-tissue analyses; and (6) Use reciprocal bolt-grafting experiments with wild-type and cepr1 mutants, coupled with antibody detection, to distinguish local versus systemic effects on receptor expression . This multi-faceted approach helps reconcile apparent contradictions in CEPR1 expression patterns between vascular and reproductive tissues.

How can CEPR1 antibodies help elucidate the mechanism of CEPR1-mediated control of seed development?

To investigate CEPR1's role in seed development: (1) Perform immunohistochemistry with CEPR1 antibodies to track receptor localization during progressive stages of seed development, with particular focus on the chalazal seed coat where CEPR1 is specifically expressed ; (2) Conduct co-immunoprecipitation with CEPR1 antibodies from developing seeds to identify stage-specific interacting partners involved in nutrient transport; (3) Implement proximity-dependent labeling coupled with CEPR1 antibodies to map the local interactome in seed tissues; (4) Compare phosphorylation patterns of CEPR1 and downstream components between wild-type and cepr1 mutant seeds using phospho-specific antibodies; (5) Design pulse-chase experiments with labeled nitrogen compounds combined with CEPR1 immunolocalization to track changes in nutrient delivery correlating with receptor distribution; and (6) Analyze protein-metabolite interactions using CEPR1 antibodies to identify compounds that might regulate receptor function during seed filling. These approaches can help explain why cepr1 mutants show disproportionately large reductions in yield and seed size relative to their decreased vegetative growth .

What tissue preparation methods optimize CEPR1 antibody performance in different experimental contexts?

For optimal CEPR1 antibody performance: (1) For Western blotting, extract proteins using buffer systems containing non-ionic detergents (0.5-1% Triton X-100) to preserve membrane protein integrity, as CEPR1 is a transmembrane receptor; (2) For immunoprecipitation, use gentler solubilization with digitonin or CHAPS to maintain protein-protein interactions; (3) For immunohistochemistry, implement a progressive fixation protocol starting with 2-4% paraformaldehyde followed by careful permeabilization to maintain antigen recognition while preserving tissue architecture; (4) For reproductive tissues, which showed chlorosis and anthocyanin accumulation in cepr1 mutants , include antioxidants in extraction buffers to prevent interference from phenolic compounds; (5) When working with seed tissues, particularly the chalazal seed coat, optimize antigen retrieval using controlled proteolysis or heat-mediated procedures; and (6) For all applications, maintain consistent cold temperatures during extraction to minimize proteolysis of CEPR1, which may be particularly susceptible to degradation during sample preparation.

How can researchers distinguish between CEPR1 and CEPR2 in experimental systems using antibodies?

To differentiate between CEPR1 and CEPR2: (1) Generate and validate isoform-specific antibodies targeting unique epitopes in the less conserved regions of each receptor; (2) Implement antibody validation using cepr1 and cepr2 single mutants, as well as cepr1/cepr2 double mutants to confirm specificity ; (3) Perform epitope mapping using peptide arrays to identify antibody binding sites and potential cross-reactivity; (4) Conduct competition assays with recombinant CEPR1 and CEPR2 proteins to assess antibody preference; (5) Use genetic complementation models where one receptor is tagged to differentiate native from complemented protein when using antibodies; and (6) Combine antibody approaches with transcript analysis to correlate protein detection with known expression patterns—CEPR1 and CEPR2 show tissue-specific expression patterns, suggesting spatial cooperation in controlling plant immunity . This distinction is crucial because while some functions appear redundant, cepr1-3 was insensitive to CEP4-induced seedling growth inhibition, while cepr2 alleles showed varying levels of sensitivity .

What strategies can address non-specific binding issues when using CEPR1 antibodies in plant tissues with high phenolic content?

To overcome non-specific binding in phenolic-rich tissues: (1) Modify extraction buffers with polyvinylpolypyrrolidone (PVPP) and polyvinylpyrrolidone (PVP) to absorb phenolic compounds that may cross-react with antibodies; (2) Include higher concentrations (5-10%) of blocking agents like BSA or non-fat dry milk in incubation buffers; (3) Implement a pre-adsorption step where primary antibodies are incubated with extracts from cepr1 knockout plants to remove antibodies that bind non-specifically; (4) Add reducing agents like DTT or β-mercaptoethanol to prevent oxidation of phenolic compounds during sample preparation; (5) Consider using monovalent antibody fragments (Fab) rather than whole IgG to reduce non-specific binding mediated by Fc regions; and (6) Implement a dual-labeling approach using two different CEPR1 antibodies recognizing distinct epitopes—true signal should show co-localization. This is particularly important when examining reproductive tissues that accumulate anthocyanins, as observed in cepr1 mutants , which can contribute to high background signal.

How can CEPR1 antibodies be utilized to study the evolutionary conservation of CEP-CEPR signaling across plant species?

To investigate evolutionary conservation of CEP-CEPR signaling: (1) Test cross-reactivity of CEPR1 antibodies against orthologous receptors in diverse plant species, from model systems to crops; (2) Perform comparative immunolocalization studies across species to identify conserved expression domains, particularly in vascular and reproductive tissues; (3) Use antibodies for pull-down assays followed by mass spectrometry to compare CEPR1 interactomes across species; (4) Conduct heterologous expression studies where CEPR1 from different species is expressed in Arabidopsis cepr1 mutants, followed by antibody-based functional assays; (5) Implement co-immunoprecipitation studies to test if CEPs from one species can interact with CEPR1 from another; and (6) Design phylogenetic analyses combined with epitope conservation mapping to predict antibody utility across species. These approaches can expand our understanding beyond current knowledge of CEP-CEPR signaling in Arabidopsis and rice, where CEPs like OsCEP5 and OsCEP6 are specifically expressed at defined stages of reproductive development .

What methodological approaches can integrate CEPR1 antibody data with transcriptomic and metabolomic analyses?

To integrate multi-omics data with CEPR1 antibody studies: (1) Implement cell-type-specific immunoprecipitation of CEPR1-containing complexes followed by RNA-seq of associated transcripts to identify mRNAs regulated by CEPR1 signaling; (2) Conduct parallel analyses of CEPR1 protein levels (via quantitative immunoblotting) and transcriptomic changes during nitrogen stress and pathogen challenges; (3) Correlate CEPR1 tissue localization data from immunohistochemistry with spatial transcriptomics and metabolomics to create comprehensive tissue-specific regulatory maps; (4) Design integrative experiments where plants are subjected to nitrogen limitation followed by immune elicitation, with samples collected for antibody-based CEPR1 quantification, phosphoproteomics, transcriptomics, and metabolomics; (5) Develop computational models incorporating antibody-derived CEPR1 localization data with gene expression patterns to predict signaling network behavior under various conditions; and (6) Validate model predictions using CEPR1 antibodies to track receptor dynamics in real-time during environmental challenges. This integration is critical for understanding how "CEP-CEPR1 signaling curtails the expenditure of resources to control lateral root growth in response to elevated shoot-derived carbon" .

How can researchers use CEPR1 antibodies to investigate the mechanisms of nitrogen transport to reproductive tissues?

To study CEPR1's role in nitrogen transport: (1) Perform co-immunolocalization of CEPR1 with nitrogen transporters like USUALLY MULTIPLE ACIDS MOVE IN AND OUT TRANSPORTERS (UMAMITs) in reproductive tissues, particularly at the chalazal seed coat where CEPR1 is expressed ; (2) Conduct immunoprecipitation of CEPR1 complexes from reproductive tissues at different developmental stages to identify stage-specific interactions with transport machinery; (3) Implement in vivo crosslinking followed by CEPR1 antibody pull-down to capture transient interactions with nitrogen transport components; (4) Design pulse-chase experiments with labeled amino acids or nitrate combined with immunohistochemistry to correlate CEPR1 localization with nitrogen movement; (5) Compare the subcellular localization of CEPR1 and nitrogen transporters in wild-type versus nitrogen-deficient conditions; and (6) Analyze post-translational modifications of CEPR1 in response to nitrogen availability using modification-specific antibodies. These approaches can help explain why cepr1 mutants show phenotypic similarities with mutants impaired in nitrogen remobilization, which leads to reduced seed size and yield .

What is the comparative performance of monoclonal versus polyclonal CEPR1 antibodies across different experimental applications?

Notes: This comparative analysis is based on typical performance patterns. Optimal antibody choice depends on the specific research question. Polyclonal antibodies generally perform better for detecting CEPR1 in native conformation in plant tissues, particularly for vascular localization studies. Monoclonal antibodies offer higher specificity for distinguishing between CEPR1 and the related CEPR2 receptor, which show functional redundancy in some contexts .

What are the key differences between CEPR1 and CEPR2 signaling based on current research findings?

This comparative data suggests that while CEPR1 and CEPR2 have partially redundant functions in immunity, CEPR1 plays a more dominant role in reproductive development. These functional differences should be considered when designing antibody-based experiments targeting either receptor specifically.