Recombinant Arabidopsis thaliana S- (+)-linalool synthase, chloroplastic (TPS14), partial

What is the primary function of TPS14 in Arabidopsis thaliana?

TPS14 functions as a terpene synthase that catalyzes the formation of the monoterpene S-(+)-linalool from geranyl diphosphate in chloroplasts. This enzyme plays a significant role in plant defense mechanisms by producing volatile compounds that can act as signaling molecules. Research indicates that linalool produced by TPS14 participates in early signaling transduction processes, including oxidative and calcium burst pathways, which are crucial for plant defense responses .

How does linalool produced by TPS14 contribute to plant defense responses?

Linalool produced by TPS14 contributes to plant defense by activating multiple signaling pathways. Studies show that linalool treatment induces defense responses against pests like Plutella xylostella in Arabidopsis by triggering hydrogen peroxide (H₂O₂) production and calcium signaling cascades . This activation leads to upregulation of jasmonic acid (JA)-related genes and defense genes, enhancing the plant's resistance capabilities. Experimental evidence demonstrates that linalool-treated Arabidopsis plants show reduced damage from larval feeding compared to untreated controls .

What are the key structural domains of the TPS14 enzyme?

The TPS14 enzyme contains characteristic structural domains common to plant terpene synthases, including the N-terminal transit peptide that targets the protein to chloroplasts (hence "chloroplastic"), and conserved catalytic domains responsible for substrate binding and conversion of geranyl diphosphate to linalool. The chloroplast localization is particularly important as it positions the enzyme where precursor molecules are synthesized via the methylerythritol phosphate (MEP) pathway.

How do H₂O₂ and calcium signaling interact downstream of linalool perception in Arabidopsis?

Research reveals a sophisticated signaling cascade where linalool triggers H₂O₂ production that subsequently leads to calcium release. When Arabidopsis mesophyll cells are treated with linalool, H₂O₂ levels rapidly increase and remain elevated for at least 15 minutes, showing approximately 50% increase above control levels . This H₂O₂ burst occurs upstream of calcium signaling, as demonstrated by experiments using NADPH oxidase inhibitor DPI and the NADPH oxidase mutant rbohd, which showed significantly suppressed calcium responses following linalool treatment . The data indicates that RBOHD is essential for linalool-induced H₂O₂ production, and this oxidative burst then triggers calcium release from internal stores through TPC1 channels. This sequence represents a specific "language" in the plant's defense signaling network .

What are the regulatory mechanisms controlling TPS14 expression in different tissues and developmental stages?

TPS14 expression follows tissue-specific and developmental patterns, with expression occurring primarily in floral tissues and under specific stress conditions. Regulatory elements including Up1 (which is almost identical to the site II motif) and Up2 have been identified in the promoter regions of genes induced during germination, including those involved in early signaling processes . TCP transcription factors, particularly AtTCP14, have been shown to regulate such genes. While not directly linked to TPS14 in the provided data, similar regulatory mechanisms likely control TPS14 expression during development and in response to environmental stimuli .

How does the interaction between CAM3 and ACA8 contribute to calcium homeostasis following linalool-induced calcium burst?

Following linalool-induced calcium burst, intracellular calcium levels must be restored to prevent cytotoxicity. Research indicates that calmodulin 3 (CAM3) interacts with calcium ATPase isoform 8 (ACA8) to facilitate calcium efflux and restore homeostasis . This interaction has been demonstrated through multiple experimental approaches, including yeast two-hybrid assays, firefly luciferase complementation imaging, and in vitro pulldown assays, which collectively show that CAM3 interacts with the N-terminus of ACA8 . Functionally, this interaction activates the calcium ATPase, promoting calcium transport out of the cytoplasm and allowing cells to return to their resting state following linalool-induced signaling events .

What are the optimal conditions for expressing recombinant TPS14 in heterologous systems?

For optimal expression of recombinant TPS14, researchers should consider using E. coli BL21(DE3) strain with pET expression vectors. Expression should be induced at OD₆₀₀ of 0.6-0.8 using 0.5-1.0 mM IPTG, with induction temperature at 18-20°C overnight to maximize soluble protein yield. For purification, use of a His-tag followed by immobilized metal affinity chromatography (IMAC) and subsequent size exclusion chromatography is recommended to obtain pure enzyme. Since TPS14 is normally chloroplast-targeted, expression constructs should exclude the transit peptide sequence to improve solubility. Additionally, supplementation with 5-10 mM MgCl₂ throughout purification helps maintain enzyme activity by preserving the proper folding of the catalytic domain.

How can researchers effectively measure TPS14 enzyme activity in vitro?

To effectively measure TPS14 enzyme activity in vitro, researchers should establish an assay system containing purified recombinant enzyme (10-50 μg/mL), buffer (typically 25 mM HEPES, pH 7.2-7.5), cofactors (5 mM MgCl₂ and 0.1 mM MnCl₂), reducing agent (5 mM DTT), and substrate (20-100 μM geranyl diphosphate). Reactions should be conducted in gas-tight vials with a glass insert containing organic solvent (hexane or pentane) to trap volatile products. After incubation at 30°C for 1-3 hours, the organic phase can be analyzed by gas chromatography-mass spectrometry (GC-MS) to quantify S-(+)-linalool production. Control reactions without enzyme or with heat-denatured enzyme are essential to confirm enzyme-specific activity. Kinetic parameters can be determined by varying substrate concentrations and analyzing the data using Michaelis-Menten or Lineweaver-Burk plots.

What experimental approaches are most effective for studying linalool-induced calcium signaling in planta?

For studying linalool-induced calcium signaling in planta, researchers should employ a multi-faceted approach combining genetic, molecular, and imaging techniques. Confocal laser scanning microscopy using calcium-sensitive fluorescent dyes such as Fluo3-AM is effective for visualizing calcium dynamics in mesophyll cells, as demonstrated in studies where intracellular calcium fluorescence rapidly increased by approximately 17% within 30 seconds of linalool treatment . This approach should be complemented with non-invasive micro-test technology (NMT) to measure calcium flux across cell membranes .

For genetic analysis, researchers should utilize mutant lines affecting calcium signaling components (such as tpc1) and upstream regulators (such as rbohd), along with pharmacological inhibitors like ruthenium red (a calcium channel blocker) and DPI (an NADPH oxidase inhibitor) . This combined approach allows researchers to dissect the signaling pathway by determining which components are essential for the linalool-induced calcium response. Additionally, transgenic plants expressing genetically encoded calcium indicators like GCaMP or Yellow Cameleon provide another valuable tool for real-time monitoring of calcium dynamics in intact tissues.

How can contradictory results between in vitro and in vivo studies of TPS14 function be reconciled?

Contradictions between in vitro and in vivo studies of TPS14 function can be reconciled through systematic analysis of the experimental conditions and biological context. In vitro studies typically use purified recombinant enzymes under controlled conditions, while in vivo studies examine the enzyme in its native cellular environment with complex regulatory networks.

To address these contradictions, researchers should:

• Examine substrate availability differences: In chloroplasts, geranyl diphosphate concentrations may differ from those used in vitro, affecting enzyme kinetics.

• Consider post-translational modifications: TPS14 may undergo modifications in vivo that aren't present in recombinant proteins.

• Evaluate protein-protein interactions: TPS14 may interact with other proteins in vivo that affect its function.

• Analyze subcellular compartmentalization: The chloroplastic environment provides specific conditions that may not be replicated in vitro.

• Implement complementary approaches: Combine in vitro biochemical studies with in vivo genetic approaches (knockout/knockdown lines, overexpression lines) and metabolomic analyses to build a comprehensive understanding of enzyme function.

To effectively reconcile contradictory results, researchers should systematically test each of these possibilities using appropriate controls and multiple experimental approaches.

What statistical methods are most appropriate for analyzing linalool-induced calcium and H₂O₂ dynamics data?

For analyzing linalool-induced calcium and H₂O₂ dynamics data, the following statistical methods are most appropriate:

• For time-course experiments: Repeated measures ANOVA should be used when analyzing fluorescence intensity changes over time, as demonstrated in studies tracking H₂O₂ levels every 3 minutes for 15 minutes and calcium levels every 30 seconds . This accounts for the non-independence of measurements from the same cells/tissues over time.

• For comparing treatment groups: When comparing multiple experimental groups (e.g., wild-type vs. various mutants or inhibitor treatments), Dunnett's C test is preferred when variance is not homogeneous across groups, as was used in the analysis of both H₂O₂ and calcium fluorescence data .

• For correlation analysis: Pearson's correlation coefficient helps determine relationships between H₂O₂ and calcium levels when measured simultaneously.

• For threshold determination: Change-point analysis can identify precise moments when significant increases in signaling molecules occur.

• For spatial analysis: For data from confocal microscopy images, spatial statistics and image analysis algorithms should be employed to quantify signal intensity, distribution, and cell-to-cell variation.

Data normalization is crucial, typically expressing results as percentage increase above control levels, as seen in studies reporting approximately 50% increases in H₂O₂ levels and 17% increases in calcium levels following linalool treatment .

How can researchers distinguish between direct and indirect effects of linalool in complex signaling networks?

To distinguish between direct and indirect effects of linalool in complex signaling networks, researchers should employ a systematic approach combining temporal, genetic, and pharmacological strategies:

For example, research has established that linalool-induced H₂O₂ production occurs upstream of calcium signaling by demonstrating that calcium response was inhibited in rbohd mutants and DPI-treated cells (blocking H₂O₂ production), while H₂O₂ production was relatively unaffected in tpc1 mutants and ruthenium-red-treated cells (blocking calcium release) . This hierarchical approach definitively established the sequence of events in the signaling pathway.

What are the most effective methods for analyzing linalool and its metabolites in plant tissues?

The most effective methods for analyzing linalool and its metabolites in plant tissues combine sample preparation techniques with advanced analytical instrumentation:

• Sample preparation:

  • Headspace solid-phase microextraction (HS-SPME) for volatile collection

  • Liquid-liquid extraction using pentane/hexane for tissue extraction

  • QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method for complex matrices

• Analytical techniques:

  • Gas chromatography-mass spectrometry (GC-MS) for volatile profiling

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for non-volatile metabolites

  • Chiral column chromatography to distinguish between S-(+) and R-(-) linalool enantiomers

• Quantification approaches:

  • Stable isotope dilution assays using deuterated internal standards

  • Standard addition method for complex matrices

  • Calibration curves using authentic standards

• Data analysis:

  • Targeted analysis for known metabolites

  • Untargeted metabolomics for discovery of novel derivatives

This comprehensive approach enables researchers to track linalool production, metabolism, and the formation of derivatives like linalool oxides, which research indicates may be modulated by cytochrome P450 enzymes in the CYP76 family, particularly CYP76C1 .

How can genome editing techniques be effectively applied to study TPS14 function in Arabidopsis?

Genome editing techniques, particularly CRISPR/Cas9, can be effectively applied to study TPS14 function in Arabidopsis through the following strategic approaches:

• Complete gene knockout: Design sgRNAs targeting the first exon of TPS14 to create frameshift mutations leading to complete loss of function. This allows assessment of the full phenotypic consequences of TPS14 absence, including effects on linalool production, defense responses, and signaling pathways.

• Domain-specific mutations: Create precise edits in catalytic domains to generate enzymes with altered activity rather than complete loss of function. This helps identify critical amino acid residues for substrate binding or catalysis.

• Promoter editing: Modify regulatory regions to alter expression patterns without changing the protein sequence, allowing study of TPS14 function in different developmental contexts.

• Base editing or prime editing: Make specific nucleotide changes to study the effects of naturally occurring TPS14 variants identified in different Arabidopsis ecotypes.

• Reporter gene integration: Insert reporter genes like GFP at the TPS14 locus to monitor expression patterns while maintaining native regulatory control.

For effective implementation, researchers should validate edited lines through sequencing, expression analysis, and enzyme activity assays. Phenotypic analysis should include metabolite profiling, response to biotic stressors, and detailed analysis of signaling pathways, including H₂O₂ and calcium dynamics, which have been demonstrated to be influenced by linalool in wild-type plants .

What are the best approaches for studying interactions between linalool signaling and other defense pathways in Arabidopsis?

The best approaches for studying interactions between linalool signaling and other defense pathways in Arabidopsis involve integrated multi-omics and systems biology strategies:

• Transcriptomics: RNA-seq analysis of wild-type and TPS14 mutant plants treated with linalool can identify differentially expressed genes involved in defense pathways. Research has already demonstrated that linalool treatment upregulates jasmonic acid (JA)-related genes and defense genes in Arabidopsis leaves .

• Proteomics: Quantitative proteomics using techniques like iTRAQ or TMT labeling can reveal changes in protein abundance and post-translational modifications following linalool treatment.

• Metabolomics: Comprehensive metabolite profiling using LC-MS/MS and GC-MS can track changes in defense-related compounds including jasmonates, salicylates, and specialized metabolites.

• Genetic interaction analysis: Creating double and triple mutants combining TPS14 mutations with mutations in key defense pathway genes helps determine epistatic relationships and pathway interactions.

• Calcium and ROS signaling: Using fluorescent probes like Fluo3-AM for calcium and H₂DCF-DA for H₂O₂, researchers can track real-time changes in these important second messengers following various treatments .

• Network analysis: Integrating multi-omics data through network analysis tools can reveal hub genes and regulatory nodes connecting linalool signaling to broader defense networks.

• Temporal resolution: Time-course experiments are crucial, as demonstrated by studies showing H₂O₂ burst occurs upstream of calcium signaling following linalool treatment , and gene expression changes occur in waves (JA-related genes at 5 min, 0.5h, and 2h; defense genes at 0.5h, 2h, and 8h) .

This integrated approach allows researchers to establish causal relationships and identify key nodes where linalool signaling interfaces with established defense pathways like JA, SA, and ethylene-mediated responses.