Recombinant Arabidopsis thaliana Acyl-CoA-binding domain-containing protein 1 (ACBP1)
Description
Introduction to Recombinant Arabidopsis thaliana Acyl-CoA-binding Domain-Containing Protein 1 (ACBP1)
Recombinant Arabidopsis thaliana Acyl-CoA-binding domain-containing protein 1 (ACBP1) is a protein derived from the model plant Arabidopsis thaliana. It belongs to a family of acyl-CoA-binding proteins (ACBPs) that play crucial roles in lipid metabolism and stress responses in plants. ACBP1 is particularly notable for its membrane-associated localization, which includes the plasma membrane and endoplasmic reticulum, and its ability to bind acyl-CoA esters and phosphatidic acid (PA) .
Structure and Localization of ACBP1
ACBP1 contains an amino-terminal transmembrane domain that targets it to the plasma membrane and the endoplasmic reticulum. This localization is critical for its function in lipid metabolism and stress responses. Additionally, ACBP1 possesses C-terminal ankyrin repeats, which may facilitate interactions with other proteins .
Functions of ACBP1
ACBP1 is involved in several key biological processes:
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Lipid Metabolism: ACBP1 can bind to acyl-CoA esters and phosphatidic acid, influencing the composition of membrane lipids. It regulates the expression of phospholipase Dα1 (PLDα1) and phospholipase Dδ (PLDδ), enzymes involved in the hydrolysis of phosphatidylcholine (PC) to PA .
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Stress Responses: ACBP1 plays a role in freezing tolerance. Mutant plants lacking ACBP1 show enhanced freezing tolerance due to reduced PA levels and increased PC levels, which contribute to membrane stability. Conversely, overexpression of ACBP1 leads to increased sensitivity to freezing .
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Cuticle Formation: ACBP1 is also involved in stem cuticle formation by trafficking very-long-chain acyl-CoA esters, which are precursors for wax biosynthesis. Mutants lacking ACBP1 exhibit reduced cuticular wax and altered cutin composition .
Effects on Lipid Composition and Stress Tolerance
| Genotype | PA Levels | PC Levels | Freezing Tolerance | PLDα1 Expression | PLDδ Expression |
|---|---|---|---|---|---|
| ACBP1 Overexpressors | Increased | Decreased | Reduced | Up-regulated | Down-regulated |
| acbp1 Mutants | Decreased | Increased | Enhanced | Down-regulated | Up-regulated |
Role in Cuticle Formation
| Genotype | Cuticular Wax Composition | Cutin Monomers | Susceptibility to Pathogens |
|---|---|---|---|
| Wild Type | Normal | Normal | Normal |
| acbp1 Mutants | Reduced | Altered | Increased |
References Li, X., & Chye, M. L. (2010). Membrane-Associated Acyl-CoA-Binding Protein 1 (ACBP1) Regulates Phospholipase D Activity and Freezing Tolerance in Arabidopsis. Plant Physiology, 152(2), 829–841. Xiao, S., & Chye, M. L. (2008). Arabidopsis acyl-CoA-binding proteins ACBP1 and ACBP2 show different roles in freezing stress. Plant Physiology, 148(2), 849–858. Shi, J., et al. (2014). Arabidopsis membrane-associated acyl-CoA-binding protein ACBP1 regulates stem cuticle formation. Journal of Experimental Botany, 65(11), 2929–2941.
Product Specs
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Target Background
- ACBP1 modulates the metabolism of fatty acids and sterols, impacting cellular signaling. PMID: 28500265
- ACBP1 is involved in ABA-mediated seed germination and seedling development. PMID: 23448237
- ACBP1 overexpression alters phosphatidylcholine molecular species, while knockouts enhance freezing tolerance. PMID: 20107029
- Arabidopsis lines overexpressing ACBP1 exhibit increased tolerance to Pb(II)-induced stress compared to wild-type lines. PMID: 18182029
Q&A
What structural features define recombinant AtACBP1 and its acyl-CoA-binding capability?
Recombinant AtACBP1 (10.4 kDa) contains a conserved acyl-CoA-binding (ACB) domain critical for interacting with long-chain (LC) and very-long-chain (VLC) acyl-CoA esters. Isothermal titration calorimetry (ITC) studies confirm its binding affinity for 18:1-, 18:2-, 18:3-, 24:0-, 25:0-, and 26:0-CoAs . The protein’s membrane-anchoring domain enables localization to cellular membranes, facilitating lipid transfer processes .
Key Data Table
| Acyl-CoA Type | Binding Affinity (Kd) | Functional Implication | Source |
|---|---|---|---|
| 18:1-CoA | High | Phospholipid remodelling | |
| 24:0-CoA | High | Cuticular wax biosynthesis | |
| 18:0-CoA | Moderate | Galactolipid synthesis |
How is ACBP1 expression regulated in plant tissues?
ACBP1 is differentially expressed across tissues:
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High levels (143 μg/g FW) in developing Brassica napus seeds during triacylglycerol accumulation .
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Stem-specific GUS expression in Arabidopsis transgenic lines, correlating with cuticle development .
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ABA-induced expression in seedlings, linking ACBP1 to stress responses .
Western blotting and qRT-PCR are standard methods for quantifying expression levels .
What contradictions emerge in ACBP1’s role in lipid metabolism?
Resolution: ACBP1 dual roles—binding acyl-CoAs for wax/cutin biosynthesis vs. interacting with PLDα1 to regulate PA production —suggest context-dependent functions. Mutant phenotypes highlight lipid class-specific dependencies.
How to validate ACBP1’s interaction with phospholipase Dα1 (PLDα1)?
Methodological Approaches:
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Bimolecular Fluorescence Complementation (BiFC):
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Yeast Two-Hybrid Assays:
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Test for protein-protein interactions using bait (ACBP1) and prey (PLDα1) constructs.
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Lipid Profiling:
What challenges exist in analyzing ACBP1’s role in embryogenesis?
Key Issues:
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Embryo lethality: acbp1acbp2 double mutants cannot survive beyond early stages, limiting adult-stage analysis .
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Compensation mechanisms: Single mutants (e.g., acbp1) show normal growth, complicating functional studies .
Solutions:
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Conditional knockouts: Use tissue-specific promoters to bypass embryonic lethality.
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Lipidomics: Compare acyl-CoA and phospholipid profiles in wild-type vs. mutant embryos using LC-MS .
What techniques are employed to study ACBP1’s subcellular localization?
| Method | Application | Advantage | Limitation |
|---|---|---|---|
| GUS reporter assays | Track expression in transgenic lines | High spatial resolution | Limited to promoter activity |
| Immunoelectron microscopy | Visualize membrane association | Ultrastructural detail | Requires specialized equipment |
| Subcellular fractionation | Isolate membrane vs. cytosolic fractions | Quantitative analysis | Risk of cross-contamination |
Best Practice: Combine GUS assays with immunogold labeling to confirm tissue-specific and subcellular localization .
Why does ACBP1 overexpression enhance ABA sensitivity but not drought tolerance?
Hypothesis: ACBP1 amplifies PLDα1-mediated PA production, which is critical for ABA signaling but not directly linked to osmotic stress responses .
Experimental Test:
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Phenotype comparison: Assess ABA germination inhibition vs. drought survival in ACBP1-OE lines.
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Gene expression profiling: Monitor RD29A, AREB1, and MYC2 (ABA-responsive) vs. P5CS1 (proline synthesis) under stress .
What is ACBP1’s role in pathogen defense?
Evidence: acbp1 mutant leaves show increased susceptibility to Botrytis cinerea, suggesting ACBP1 modulates cuticle integrity or defense-related lipid signaling .
Future Work:
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Lipid profiling: Compare VLC fatty acid profiles in infected vs. non-infected tissues.
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Gene co-expression networks: Identify ACBP1’s regulatory targets in pathogen response pathways.
How to reconcile lipidomic and transcriptomic data in ACBP1 studies?
Case Study: acbp1 mutants show reduced VLC acyl-CoA binding but unchanged expression of wax biosynthetic genes like CER1 or CER3.
Approach:
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Metabolite flux analysis: Track acyl-CoA ester turnover rates using isotopic labeling.
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ChIP-seq: Determine if ACBP1 regulates transcription factors (e.g., WIN1/SHN1) that control wax-related genes.
What controls are essential for ACBP1 binding assays?
Critical Controls:
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Negative control: Recombinant ACBP1 incubated with palmitoyl-CoA (C16:0-CoA) to confirm chain-length specificity .
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Competition assay: Add excess unlabeled acyl-CoA esters to confirm binding specificity.
Data Validation: Use ITC to measure binding thermodynamics (ΔH, ΔS) and confirm stoichiometry .
