SARD4 Antibody
Description
Introduction to SARD4 and Its Functional Context
SARD4 (SAR-DEFICIENT4) is a critical enzyme in Arabidopsis thaliana involved in the biosynthesis of pipecolic acid (Pip), a key regulator of systemic acquired resistance (SAR) against pathogens . SARD4 catalyzes the final step of Pip production by reducing the precursor Δ¹-piperideine-2-carboxylic acid (P2C) to Pip, working in tandem with the aminotransferase ALD1 . Pip accumulation is essential for amplifying salicylic acid (SA)-dependent defense signaling and establishing SAR .
SARD4 Antibody: Purpose and Development
While the provided sources do not explicitly describe the generation or commercial availability of a SARD4-specific antibody, its utility can be inferred from studies characterizing SARD4 mutants. For example:
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Mutant validation: T-DNA insertion mutants (e.g., sard4-5) were used to confirm the loss of SARD4 protein function . Antibodies against SARD4 would enable detection of protein expression levels in wild-type versus mutant plants.
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Protein localization: Immunolocalization studies could clarify SARD4’s tissue-specific expression during pathogen infection.
Key Research Findings Involving SARD4
The following table summarizes critical insights into SARD4’s role, which would require antibody-based validation:
Experimental Applications of a SARD4 Antibody
Hypothetical applications based on SARD4’s characterized roles:
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Western blotting: Quantify SARD4 protein levels in local vs. systemic tissues post-pathogen inoculation.
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Mutant screening: Confirm protein absence in sard4 alleles (e.g., sard4-3, sard4-5) .
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Subcellular localization: Determine if SARD4 is cytosolic or organelle-associated during immune responses.
Challenges and Research Gaps
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Redundancy in Pip biosynthesis: SARD4-independent pathways partially compensate for Pip production, complicating antibody-based phenotyping .
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Antibody specificity: No existing data confirm cross-reactivity with related enzymes (e.g., ornithine cyclodeaminase homologs).
Future Directions
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Develop monoclonal antibodies to distinguish SARD4 isoforms or post-translational modifications.
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Use immunofluorescence to map SARD4 expression dynamics during SAR.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- The Arabidopsis OCD-like protein (AtOCD), unlike its bacterial counterparts, may not catalyze the conversion of ornithine to proline. This observation aligns with the fact that three residues essential for the activity of bacterial OCDs are not conserved in AtOCD. [AtOCD] PMID: 24237637
Q&A
Basic Research Questions
How to validate SARD4 antibody specificity in Arabidopsis mutants?
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Perform immunoblotting with protein extracts from wild-type and sard4 knockout mutants (e.g., sard4-5 T-DNA line). Validate using:
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Quantify pathogen-induced Pip levels via LC-MS to confirm functional absence of SARD4 (Pip should be undetectable in systemic leaves of sard4 mutants) .
What protocols are recommended for detecting SARD4 expression in systemic acquired resistance (SAR) studies?
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Tissue-specific sampling: Collect local (infected) and systemic (uninfected) leaves separately at 24–48 h post-inoculation .
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RNA/protein extraction: Use TRIzol for qRT-PCR (primers targeting At5g52810) and RIPA buffer for protein isolation .
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Induction validation: Confirm SARD4 upregulation with pathogen treatment (e.g., Pseudomonas syringae ES4326) via RNA-seq or immunoblotting .
Advanced Research Questions
How to design experiments distinguishing SARD4’s role in local vs. systemic Pip biosynthesis?
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Local analysis: Measure Pip and Δ¹-piperideine-2-carboxylic acid (P2C) in sard4 mutants after localized pathogen infection. Expect partial Pip reduction in local leaves (due to ALD1 redundancy) .
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Systemic analysis: Compare Pip levels in distal leaves of wild-type and sard4 mutants. Pip should be absent in sard4 systemic tissues, confirming SARD4’s necessity for systemic Pip synthesis .
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Method: Use isotopic labeling (e.g., ¹⁵N-Lysine) to track Pip flux between tissues .
How to reconcile conflicting data on SARD4’s contribution to pathogen resistance?
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Contradiction: sard4 mutants show intact local resistance but compromised SAR .
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Resolution:
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Test multiple pathogens (e.g., Hyaloperonospora arabidopsidis vs. Pseudomonas syringae) to assess context-dependent roles .
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Quantify SA and reactive oxygen species (ROS) in systemic leaves; SARD4 deficiency reduces systemic SA/ROS priming .
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Use double mutants (e.g., sard4 fmo1) to dissect Pip/N-hydroxypipecolic acid (NHP) interplay .
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What methodologies confirm SARD4 enzymatic activity in vitro?
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Recombinant protein assay: Express SARD4 in E. coli with ALD1. Detect Pip via LC-MS after incubating lysates with L-lysine and cofactors (NADH/NADPH) .
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Kinetic parameters: Measure substrate affinity (Km for P2C) and catalytic efficiency (kcat) using purified SARD4 .
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Inhibitor screening: Test cyclodeaminase inhibitors (e.g., 3-mercaptopicolinic acid) to block SARD4 activity .
Mechanistic & Epistatic Analysis
How to determine epistatic relationships between SARD4 and SAR regulators like FMO1 or NPR1?
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Genetic crosses: Generate sard4 npr1 or sard4 fmo1 double mutants. Assess SAR via bacterial growth assays in systemic leaves .
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Key metrics:
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Transcriptomics: Compare SA/NHP-responsive genes (e.g., PR1, AZI1) in single/double mutants .
Data Table: Critical Findings on SARD4 Function
Best Practices for Contradictory Results
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Contextual variables: Control for growth conditions (light, humidity) and pathogen strain virulence .
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Multi-omics integration: Combine transcriptomics (SARD4 co-expressed genes) with metabolomics (Pip/NHP quantification) .
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Independent validation: Replicate key findings in sard4 alleles from different backgrounds (e.g., sard4-1 vs. sard4-5) .
