Recombinant Arabidopsis thaliana 3-ketoacyl-CoA synthase 11 (KCS11)

What is KCS11 and what is its role in plant biology?

KCS11 (3-ketoacyl-CoA synthase 11) is a member of the 3-ketoacyl-CoA synthase family in Arabidopsis thaliana, potentially involved in the fatty acid elongation (FAE) complex. The KCS family proteins are responsible for the condensation reaction in the fatty acid elongation process, which generates very-long-chain fatty acids (VLCFAs) essential for cuticular wax production . While specific information about KCS11 is limited in the provided search results, we can extrapolate from related KCS proteins that it likely participates in determining substrate specificity for VLCFAs of particular chain lengths . KCS proteins are classified into eight subclasses (α, β, γ, δ, ɛ, ζ, η, and θ), with varying catalytic activities and functional roles . Understanding KCS11's specific subclass classification would provide further insights into its potential activity and functional significance in plant wax metabolism.

How does KCS11 relate to other members of the KCS family?

Arabidopsis thaliana contains 21 KCS genes classified into eight subclasses (α, β, γ, δ, ɛ, ζ, η, and θ), each with distinct functional properties . While the search results don't specify KCS11's subclass, we can consider its relationship to other family members. KCS proteins in subgroups α, β, γ, δ, and ɛ typically possess catalytic activity, preferentially catalyzing the production of VLCFAs with chain lengths from C20 to C38 . Some KCS family members, such as KCS3, KCS12, and KCS19, have been found to lack traditional FAE enzymatic activity in heterologous yeast expression systems, instead potentially serving regulatory functions . The relationship between KCS11 and these regulatory KCS proteins is of particular research interest, as it may provide insights into whether KCS11 functions as an active enzyme or as a regulator of other KCS proteins in the FAE complex. Understanding these relationships is crucial for determining KCS11's contribution to plant wax metabolism and cuticular development.

What experimental systems are available for studying recombinant KCS11?

Several experimental systems are available for studying recombinant KCS11 protein. Commercial recombinant KCS11 is typically expressed in various host systems including E. coli, yeast, baculovirus, or mammalian cells, with a purity of ≥85% as determined by SDS-PAGE . For experimental studies, researchers have successfully employed heterologous expression systems to study related KCS proteins. For instance, yeast (Saccharomyces cerevisiae) strain BY4741 Fah1Δ Elo3Δ has been used for functional characterization of KCS proteins like KCS3 and KCS6 . Additionally, Nicotiana benthamiana leaf transient expression systems have proven effective for studying KCS protein interactions and activities . For protein-protein interaction studies, methods such as firefly luciferase complementation imaging (LCI) assays and split-ubiquitin yeast two-hybrid (SUY2H) experiments have been successfully employed to investigate interactions between KCS proteins and other components of the FAE complex .

What methodologies are recommended for confirming KCS11 expression?

To confirm successful expression of recombinant KCS11, researchers should employ a multi-faceted approach combining molecular and biochemical techniques. RT-PCR can be used to verify the presence of KCS11 transcripts in expression systems, as demonstrated with KCS3 and KCS6 in yeast cells . This approach confirms that the gene is transcriptionally active in the host system. Western blotting with antibodies specific to KCS11 or to attached epitope tags can verify protein expression and determine approximate molecular weight. SDS-PAGE analysis is recommended for assessing protein purity, with commercial preparations typically achieving ≥85% purity . For functional confirmation, gas chromatography with flame ionization detection (GC-FID) can be employed to measure the abundance of VLCFAs potentially produced through KCS11 activity, as has been done with related KCS proteins . In heterologous expression systems like Nicotiana benthamiana, both transcript levels and protein products should be examined to confirm successful expression before conducting activity assays .

How might protein-protein interactions regulate KCS11 function?

Protein-protein interactions likely play a crucial role in regulating KCS11 function within the fatty acid elongation (FAE) complex. Research on related KCS proteins provides valuable insights into potential KCS11 interactions. For instance, KCS3 has been shown to physically interact with KCS6, affecting the binding of KCS6 with other FAE complex subunits including KCR1, PAS2, and CER10 . These interactions were detected using firefly luciferase complementation imaging (LCI) assays in Nicotiana benthamiana leaves . The presence of KCS3 reduced the stable interactions between KCS6 and other FAE members, subsequently decreasing KCS6 enzymatic activity . Similarly, KCS11 might participate in regulatory protein-protein interactions that modulate its own activity or the activity of other KCS family members. Investigating potential homo- and hetero-complexes formed by KCS11 with other KCS proteins and FAE components would provide crucial insights into its functional role. Methodologies such as co-immunoprecipitation, yeast two-hybrid assays, and in planta protein-protein interaction assays should be employed to comprehensively map KCS11's interaction network.

What approaches can be used to determine if KCS11 has enzymatic activity or regulatory functions?

Determining whether KCS11 functions as an enzyme or a regulator requires a comprehensive experimental approach. Heterologous expression systems, particularly yeast mutants lacking endogenous elongase activity (such as BY4741 Fah1Δ Elo3Δ), provide an excellent platform for investigating KCS11 activity . Researchers should express KCS11 alone and measure VLCFA production using gas chromatography with flame ionization detection (GC-FID) . Absence of increased VLCFA levels compared to empty vector controls would suggest KCS11 lacks canonical enzymatic activity, similar to KCS3 . To investigate potential regulatory functions, co-expression studies with known active KCS enzymes (such as KCS6) should be conducted, followed by VLCFA measurements to determine if KCS11 affects their activity . Protein-protein interaction studies using techniques like firefly luciferase complementation imaging (LCI) assays or split-ubiquitin yeast two-hybrid systems can reveal if KCS11 interacts with other FAE complex members . Additionally, generating Arabidopsis lines with altered KCS11 expression (overexpression and knockout/knockdown) and analyzing their cuticular wax content and composition would provide in planta evidence of KCS11's role. Integration of these approaches would comprehensively characterize KCS11's functional identity.

What is the relationship between KCS11 and plant stress responses?

The relationship between KCS11 and plant stress responses, particularly drought stress, represents an important area for investigation. Cuticular wax, which is partly synthesized through the activity of KCS family proteins, plays crucial roles in plant tolerance to environmental stresses, including drought . KCS proteins contribute to cuticular wax production by catalyzing the synthesis of very-long-chain fatty acids (VLCFAs) . Specifically, research on related KCS proteins has shown that the KCS3-KCS6 regulatory module is required for maintaining cuticular wax homeostasis under both well-watered and water-deficit conditions . By extension, KCS11 might play a similar role in regulating wax composition in response to environmental stresses. To investigate this relationship, researchers should analyze the expression patterns of KCS11 under various stress conditions, particularly drought, and examine phenotypic and biochemical changes in plants with altered KCS11 expression when subjected to these stresses. Comparative analysis of cuticular wax composition in wild-type and KCS11 mutant plants under stress conditions would provide valuable insights into KCS11's specific contribution to stress tolerance mechanisms.

How has the evolutionary conservation of KCS proteins informed KCS11 research?

Evolutionary conservation analysis of KCS proteins has provided valuable insights into their functional importance, which can inform KCS11 research. Studies have demonstrated that the regulatory role of the KCS3-KCS6 module in wax synthesis is highly conserved across diverse plant taxa, from flowering plants like Arabidopsis to bryophytes like the moss Physcomitrium patens . This conservation points to an ancient and basal function of this regulatory module in finely regulating wax synthesis . By extension, investigating the conservation of KCS11 across evolutionary distance could provide important clues about its functional significance. Researchers should conduct comparative genomic analyses to identify KCS11 orthologs in diverse plant species, from angiosperms to early land plants. Functional complementation studies, where KCS11 from different species is expressed in Arabidopsis KCS11 mutants, would determine if the function is conserved. Additionally, analyzing the co-evolution of KCS11 with other FAE complex components could reveal important functional relationships. The high conservation of other KCS regulatory modules suggests that if KCS11 serves a regulatory function, it might also be evolutionarily ancient and broadly conserved across plant lineages.

What experimental design would best elucidate KCS11's role in the fatty acid elongation (FAE) complex?

To comprehensively elucidate KCS11's role in the fatty acid elongation (FAE) complex, a multi-faceted experimental approach is required. The design should include:

• Genetic manipulation in Arabidopsis:

  • Generate KCS11 knockout mutants using CRISPR-Cas9

  • Create KCS11 overexpression lines

  • Develop complementation lines expressing KCS11 with point mutations in predicted catalytic sites

  • Analyze phenotypic changes in stem wax composition using GC-FID

• Protein-protein interaction studies:

  • Conduct firefly luciferase complementation imaging (LCI) assays to test interactions between KCS11 and other FAE components (KCR1, PAS2, CER10)

  • Perform split-ubiquitin yeast two-hybrid (SUY2H) experiments to validate interactions

  • Use co-immunoprecipitation to identify interaction partners in planta

• Heterologous expression analysis:

  • Express KCS11 alone in yeast strain BY4741 Fah1Δ Elo3Δ to test enzymatic activity

  • Co-express KCS11 with other KCS proteins to test regulatory functions

  • Analyze VLCFA production using GC-FID

• Transcriptional analysis:

  • Examine expression patterns of KCS11 across tissues and developmental stages

  • Analyze transcriptional changes under various stress conditions

This comprehensive approach would provide multiple lines of evidence to determine whether KCS11 functions as an active enzyme or a regulatory protein within the FAE complex, similar to the discovered role of KCS3 .

What are the optimal conditions for expressing recombinant KCS11 protein?

Optimal expression of recombinant KCS11 protein requires careful consideration of host systems and conditions. Based on information about related KCS proteins and commercial production methods, several approaches can be recommended:

• Host selection: E. coli, yeast, baculovirus, or mammalian cell systems can all be used for KCS11 expression . For functional studies, yeast systems (particularly S. cerevisiae strain BY4741 Fah1Δ Elo3Δ) offer advantages as they lack certain endogenous elongase activities, facilitating the specific analysis of KCS11 function .

• Expression vector design: Vectors should include appropriate promoters for the selected host system. For yeast expression, GAL1 promoters have been successfully used for controlled expression of related KCS proteins . For plant-based systems like Nicotiana benthamiana, 35S promoters have proven effective .

• Protein purification strategy: When high purity is required (≥85%), appropriate purification tags should be incorporated. His-tags or other affinity tags facilitate purification while minimizing interference with protein function .

• Expression verification: RT-PCR should be performed to confirm transcript presence, followed by Western blot analysis to verify protein expression . For functional verification, GC-FID analysis of fatty acid products can determine if the expressed protein is active .

• Storage conditions: Purified KCS11 should be stored in appropriate buffer conditions with glycerol at -80°C to maintain stability and prevent freeze-thaw cycles that could compromise activity.

These recommendations are based on successful approaches used with related KCS proteins and commercial production methods for KCS11 .

What analytical methods best characterize KCS11 activity and substrate specificity?

To thoroughly characterize KCS11 activity and substrate specificity, researchers should employ a combination of analytical approaches:

• Gas chromatography with flame ionization detection (GC-FID): This technique has been successfully used to analyze the wax profiles and fatty acid products in studies of related KCS proteins . It allows for quantitative analysis of very-long-chain fatty acids (VLCFAs) with different chain lengths, enabling determination of KCS11's substrate preferences.

• In vitro enzyme assays: Using purified recombinant KCS11 protein, researchers can conduct in vitro assays with various acyl-CoA substrates of different chain lengths (typically C16 to C30) to determine substrate specificity. Products can be analyzed using GC-FID or LC-MS/MS.

• Yeast complementation studies: Expression of KCS11 in yeast mutants lacking specific elongase activities can reveal functional complementation and substrate preferences . Analysis of fatty acid profiles in these transformed yeast cells provides insights into KCS11's activity.

• Comparative analysis in plant systems: Analysis of cuticular wax composition in wild-type plants versus plants with altered KCS11 expression can reveal in planta substrate preferences and activities .

• Isotopic labeling studies: Feeding experiments with isotopically labeled fatty acid precursors can trace the specific contribution of KCS11 to the elongation of particular chain lengths.

Integration of these methods provides a comprehensive characterization of KCS11 activity and substrate specificity, essential for understanding its role in plant lipid metabolism.

How can protein-protein interactions involving KCS11 be effectively investigated?

Investigating protein-protein interactions involving KCS11 requires a multi-method approach to ensure robust and reliable results. Based on successful techniques used with related KCS proteins, the following methods are recommended:

• Firefly luciferase complementation imaging (LCI) assays: This in vivo technique has proven effective for detecting interactions between KCS proteins and other FAE complex components in Nicotiana benthamiana leaves . KCS11 should be fused with nLUC while potential interaction partners are fused with cLUC. When proteins interact, luciferase activity is reconstituted, generating detectable signals .

• Split-ubiquitin yeast two-hybrid (SUY2H) experiments: This method is particularly valuable for membrane-associated proteins like KCS11. It can validate interactions identified through other methods and screen for novel interaction partners .

• Co-immunoprecipitation (Co-IP): Using epitope-tagged KCS11 expressed in plant systems, Co-IP can identify interacting proteins under native conditions. Mass spectrometry analysis of co-immunoprecipitated proteins can reveal the complete interaction network.

• Bimolecular fluorescence complementation (BiFC): This technique allows visualization of protein interactions in planta, providing spatial information about where in the cell KCS11 interacts with other proteins.

• Quantitative interaction analysis: For detailed interaction studies, researchers should co-transform Nicotiana benthamiana leaves with an internal control (e.g., 35S:RLUC) and quantify interaction signals using appropriate assay kits (e.g., Firefly & Renilla Luciferase Assay Kit) .

These complementary approaches provide comprehensive insights into KCS11's protein interaction network and its functional significance within the FAE complex.

What are the key knowledge gaps in KCS11 research?

Despite advances in understanding the KCS family of proteins, several critical knowledge gaps remain in KCS11 research. First, the specific subclass classification of KCS11 within the eight KCS subclasses (α, β, γ, δ, ɛ, ζ, η, and θ) remains undetermined in the current literature . This classification would provide important insights into its potential enzymatic activity or regulatory function. Second, while protein-protein interactions have been characterized for some KCS proteins like KCS3 and KCS6 , specific interaction partners for KCS11 remain largely unknown. Third, the substrate specificity of KCS11, if it possesses enzymatic activity, has not been comprehensively characterized. Fourth, the evolutionary conservation of KCS11 across plant species has not been thoroughly investigated, unlike the well-documented conservation of the KCS3-KCS6 module . Fifth, the potential regulatory relationships between KCS11 and other KCS family members have not been established. Finally, the specific contribution of KCS11 to plant stress responses, particularly drought tolerance through cuticular wax regulation, remains to be elucidated. Addressing these knowledge gaps would significantly advance our understanding of KCS11's role in plant lipid metabolism and cuticular wax biosynthesis.

What future research directions would advance understanding of KCS11 function?

To advance our understanding of KCS11 function, several promising research directions should be pursued:

• Comprehensive characterization of KCS11 mutants: Generate and phenotypically characterize CRISPR-Cas9 knockout mutants and overexpression lines of KCS11 in Arabidopsis, focusing on cuticular wax composition, drought tolerance, and developmental phenotypes .

• Interactome mapping: Employ advanced protein-protein interaction techniques to comprehensively map KCS11's interaction network within the FAE complex and with other KCS family members .

• Structure-function analysis: Conduct site-directed mutagenesis of predicted functional domains in KCS11 to determine their importance for activity or protein-protein interactions.

• Evolutionary conservation studies: Investigate the presence and function of KCS11 orthologs across diverse plant species, from angiosperms to bryophytes, similar to studies conducted with the KCS3-KCS6 module .

• Tissue-specific and stress-responsive expression analysis: Characterize the spatiotemporal expression patterns of KCS11 and their changes under various environmental stresses.

• Integration with systems biology approaches: Incorporate KCS11 into broader lipid metabolism networks through metabolomics, transcriptomics, and proteomics approaches.

• Application of advanced imaging techniques: Utilize techniques like super-resolution microscopy to visualize KCS11 localization and dynamics within cellular compartments.