Recombinant Arabidopsis thaliana Glycerol-3-phosphate acyltransferase 5 (GPAT5)

What is GPAT5 and what is its primary function in Arabidopsis thaliana?

GPAT5 is an acyl-CoA:glycerol-3-phosphate acyltransferase that plays a crucial role in suberin biosynthesis in Arabidopsis thaliana. It belongs to a plant-specific family of GPATs that differs from those involved in membrane and storage lipid synthesis. GPAT5 is specifically required for the synthesis of suberin polymers that function as pathogen barriers and regulate water and solute transport across plant tissues . Unlike membrane/storage lipid GPATs with sn-1 regiospecificity, GPAT5 exhibits sn-2 acyltransferase activity without phosphatase activity, producing primarily sn-2 lysophosphatidic acids (LPAs) as products . This enzyme is particularly important for the accumulation of very long chain fatty acids and their ω-oxidized derivatives in root and seed coat suberin.

Where is GPAT5 expressed in Arabidopsis tissues?

GPAT5 expression has been characterized using RT-PCR and β-glucuronidase-promoter fusion analyses. The enzyme is predominantly expressed in seed coat, root, hypocotyl, and anther tissues . This expression pattern correlates with its functional role in suberin biosynthesis, as these tissues represent major sites of suberin deposition in plants. When examined at the transcript level using RT-PCR, GPAT5 mRNA was specifically detected in flowers, roots, and seeds, but notably absent in stem tissues . This tissue-specific expression pattern provides valuable information for researchers designing experiments to study GPAT5 function in different plant organs.

How does GPAT5 differ from other members of the Arabidopsis GPAT family?

Within the Arabidopsis genome, there are 10 genes annotated as GPATs, with GPAT5 belonging to a family of eight plant-specific GPATs (GPAT1-8). GPAT5 differs from other family members in several significant ways:

• Enzymatic activity: Unlike GPAT4, GPAT6, and GPAT8, which are bifunctional enzymes with both sn-2 acyltransferase and phosphatase activities, GPAT5 possesses only sn-2 acyltransferase activity without phosphatase function .

• Substrate specificity: GPAT5 exhibits a much broader chain length specificity compared to GPAT4, GPAT6, and GPAT8, accommodating substrates ranging from C16 to C24 with only 40-45% difference in activity .

• Functional role: While GPAT4, GPAT6, and GPAT8 are associated with cutin biosynthesis, GPAT5 is specifically involved in suberin formation .

• Evolutionary relationship: GPAT5 forms a distinct clade with GPAT7 (88% similar, 81% identical amino acid sequences), separate from the cutin-associated GPATs, suggesting specialized functional evolution .

What is the substrate specificity profile of GPAT5?

GPAT5 demonstrates a distinctive substrate specificity profile that differs significantly from other members of the GPAT family. When tested with ω-oxidized and unmodified acyl-CoAs with chain lengths from C16 to C24, GPAT5 exhibited the following characteristics:

GPAT5 shows highest activity with substrates of C22 chain length, but activity with other saturated substrates ranging from C16 to C24 differed by only 40-45% compared to C22 . Unlike cutin-associated GPATs, GPAT5 doesn't strongly discriminate between oxidized and non-oxidized acyl-CoA substrates of longer chain lengths, but it does show strong discrimination against unsaturated substrates, with activities with C18:1-FA, 18-OH C18:1-FA, and C18:1-DCA being among the lowest . This broad substrate specificity aligns with GPAT5's role in suberin synthesis, which incorporates a diverse range of very long chain fatty acids and their derivatives.

What is the regiospecificity of GPAT5 and how does it relate to its function?

The sn-2 regiospecificity of GPAT5 is particularly significant because it fundamentally differs from the GPATs involved in membrane or storage lipid synthesis, which typically exhibit sn-1 regiospecificity. This distinct regiospecificity suggests that GPAT5 participates in a specialized acylglycerol biosynthesis pathway dedicated to providing precursors for suberin biosynthesis rather than for membrane or storage lipids . The preference for sn-2 acylation appears to be a common feature among the plant-specific GPAT1-8 family, indicating evolutionary specialization for extracellular polyester synthesis.

How do the enzymatic properties of GPAT5 provide insight into the suberin biosynthetic pathway?

The enzymatic properties of GPAT5, particularly its substrate preferences, provide critical insights into the organization of the suberin biosynthetic pathway. A key question in polyester biosynthesis has been whether P450 oxidation reactions occur before or after glycerol-3-phosphate acylation. The substrate specificity study of GPAT5 helps clarify this sequence:

• GPAT5 efficiently utilizes ω-oxidized acyl-CoAs of long chain lengths (C22-C24), matching the composition of suberin in roots and seed coats.

• The observed reduction of C22:0- and C24:0-FA, ω-OHFA, and DCA monomers in gpat5 mutant seed coat suberin correlates directly with GPAT5's substrate preferences .

These findings strongly support a polyester biosynthetic pathway in which acyl transfer to glycerol occurs after oxidation of the acyl group . This pathway organization helps explain how the specificities of different GPATs can serve as major determinants of cutin and suberin composition in different plant tissues. For researchers, this understanding provides a framework for designing experiments that target specific steps in the suberin biosynthetic pathway.

How have GPAT5 knockout mutants been generated and characterized?

GPAT5 knockout mutants have been generated through T-DNA insertional mutagenesis and characterized through a combination of molecular and biochemical approaches:

• Mutant generation: Two independent T-DNA insertion lines, SALK_018117 and SALK_142456, were identified from the Salk Institute Genomic Analysis Laboratory collection . These lines were designated gpat5-1 and gpat5-2, respectively.

• Insertion confirmation: Homozygous mutants were identified using PCR-based screening. The gpat5-1 line has a T-DNA insertion in the first exon, while gpat5-2 has a T-DNA inserted in the only intron of the GPAT5 gene .

• Transcript analysis: RT-PCR analysis of flowers from homozygous gpat5-1 and gpat5-2 plants showed no detectable GPAT5 transcript, confirming complete knockout of the GPAT5 gene in both independent alleles .

• Phenotypic consistency: The observed phenotypes were consistent between both independent alleles (gpat5-1 and gpat5-2), confirming that the phenotypic effects could be attributed specifically to the disruption of the GPAT5 gene rather than to secondary mutations .

This methodological approach for generating and confirming GPAT5 mutants provides a robust foundation for researchers studying GPAT5 function through loss-of-function genetics.

What phenotypes are associated with GPAT5 knockout mutants?

GPAT5 knockout mutants exhibit specific phenotypes related to altered suberin composition in roots and seed coats:

• Root suberin: The gpat5 plants showed approximately 50% decrease in aliphatic suberin content in young roots compared to wild-type plants . This significant reduction demonstrates the critical role of GPAT5 in root suberin biosynthesis.

• Seed coat suberin: The mutants produced seed coats with a several-fold reduction in very long chain dicarboxylic acids and ω-hydroxy fatty acids, which are typical components of suberin . Specifically, reductions were observed in C22:0- and C24:0-FA, ω-OHFA, and DCA monomers in seed coat suberin .

• Specificity of effects: Importantly, the gpat5 mutations caused no change in the composition or content of membrane or storage glycerolipids or surface waxes . This specificity underscores GPAT5's dedicated role in suberin biosynthesis rather than in general lipid metabolism.

These phenotypes provide valuable insights into the specific contributions of GPAT5 to suberin composition and highlight the enzyme's importance in establishing proper barrier properties in roots and seed coats.

How can genetic complementation be used to confirm GPAT5 function?

Genetic complementation is a critical approach for confirming that observed mutant phenotypes are specifically due to the disruption of GPAT5. While the search results don't explicitly describe complementation experiments for GPAT5, a standard methodology would involve:

• Construct preparation: Cloning the full-length GPAT5 cDNA or genomic sequence (including its native promoter) into a plant transformation vector suitable for Arabidopsis.

• Transformation of mutants: Introducing the GPAT5 construct into homozygous gpat5-1 or gpat5-2 plants using Agrobacterium-mediated transformation.

• Selection of transformants: Identifying transformed plants using appropriate selection markers from the vector.

• Verification of GPAT5 expression: Confirming restoration of GPAT5 expression in complemented lines using RT-PCR or quantitative PCR.

• Phenotypic analysis: Examining whether the complemented lines show restoration of normal suberin content and composition in roots and seed coats compared to the mutant background.

Successful complementation would result in reversal of the mutant phenotypes, confirming that the observed defects in suberin biosynthesis are specifically due to the loss of GPAT5 function rather than to other genetic factors.

How can recombinant GPAT5 protein be expressed and purified for enzymatic studies?

Based on methodologies described in the search results, recombinant GPAT5 protein can be expressed and prepared using several approaches:

• Yeast expression system:

  • Clone the GPAT5 coding sequence into a suitable yeast expression vector

  • Transform into an appropriate yeast strain (e.g., Saccharomyces cerevisiae)

  • Induce expression under conditions optimized for GPAT5

  • Isolate microsomes containing the recombinant enzyme for activity assays

• Wheat germ in vitro translation:

  • Prepare GPAT5 construct for in vitro translation

  • Perform translation in the presence of liposomes

  • The resulting enzyme preparation can be used directly for activity assays

The choice between these systems depends on the specific requirements of the experiment. The yeast expression system produces both MAG and LPA products for enzymes with active phosphatase domains, while the wheat germ-derived enzyme produces almost exclusively MAG product with very little detectable LPA . Since GPAT5 lacks phosphatase activity and produces a simpler product profile (primarily LPA), the yeast expression system has been successfully used for GPAT5 enzymatic assays .

What assay methods are effective for measuring GPAT5 enzymatic activity?

Effective assay methods for measuring GPAT5 enzymatic activity include:

• Acyltransferase activity assay:

  • Prepare reaction mixtures containing recombinant GPAT5 (from yeast microsomes or wheat germ translation)

  • Add glycerol-3-phosphate as the acyl acceptor substrate

  • Add various acyl-CoA substrates (potentially including radiolabeled substrates for detection)

  • Incubate under optimized reaction conditions

  • Extract and analyze reaction products (LPAs for GPAT5) using thin-layer chromatography or liquid chromatography-mass spectrometry

• Substrate specificity profiling:

  • Conduct parallel reactions with different acyl-CoA substrates

  • Compare relative activities across substrates with varying chain lengths (C16-C24) and oxidation states (unmodified, ω-hydroxy, dicarboxylic)

  • Quantify products to establish a substrate preference profile

• Regiospecificity determination:

  • Analyze the stereochemistry of the acylated products to determine whether acylation occurs at the sn-1 or sn-2 position

  • This can be achieved through stereospecific enzymatic analysis or advanced analytical techniques such as nuclear magnetic resonance spectroscopy

These methods allow for comprehensive characterization of GPAT5's enzymatic properties, providing insights into its biological function in suberin biosynthesis.

How can GPAT5 expression patterns be analyzed in different plant tissues?

GPAT5 expression patterns can be analyzed using several complementary approaches:

• Transcript analysis:

  • RT-PCR can be used to detect GPAT5 mRNA in different tissues, as demonstrated in the search results where GPAT5 transcript was found in flowers, roots, and seeds but not in stems

  • Quantitative real-time PCR (qRT-PCR) provides more precise quantification of expression levels across tissues

  • RNA-seq analysis can offer genome-wide context for GPAT5 expression patterns

• Promoter-reporter fusion analysis:

  • β-glucuronidase (GUS) reporter gene fused to the GPAT5 promoter allows visualization of tissue-specific expression patterns through histochemical staining

  • This approach has successfully demonstrated GPAT5 expression in seed coat, root, hypocotyl, and anther tissues

• Protein localization:

  • Immunolocalization using specific antibodies against GPAT5

  • Fusion of GPAT5 with fluorescent proteins (e.g., GFP) for in vivo visualization

  • These approaches can reveal both tissue specificity and subcellular localization

These methodologies provide complementary information about GPAT5 expression at different levels (transcript, promoter activity, protein) and can help researchers understand the spatial and temporal regulation of GPAT5 in relation to suberin deposition.

How can GPAT5 be used for metabolic engineering of suberin properties in plants?

GPAT5 offers significant potential for metabolic engineering of suberin properties due to its key role in determining suberin composition:

• Altered expression strategies:

  • Overexpression of GPAT5 in native or heterologous plants could potentially increase suberin content, enhancing barrier properties

  • Tissue-specific expression using different promoters could direct suberin deposition to targeted tissues

  • Evidence from GPAT7 overexpression, which resulted in production of very-long-chain suberin-like monomers, suggests similar approaches could be effective with GPAT5

• Enzyme engineering approaches:

  • Modification of GPAT5's substrate specificity through protein engineering could alter suberin composition

  • Creating chimeric enzymes between GPAT5 and other GPATs could generate novel substrate preferences and product profiles

  • Site-directed mutagenesis of key residues could potentially introduce phosphatase activity, altering the balance between LPA and MAG products

• Potential applications:

  • Enhanced drought resistance through increased hydrophobic barriers

  • Improved pathogen resistance through strengthened suberin-based defense barriers

  • Targeted modification of root architecture and function through altered suberin composition

These approaches represent advanced research directions that could leverage GPAT5's properties for agricultural improvement and basic research into structure-function relationships in plant barrier polymers.

What is known about the evolutionary history of GPAT5 and its implications for research?

The evolutionary history of GPAT5 provides important context for understanding its function and for comparative studies:

• Phylogenetic analysis reveals that the plant-specific GPAT family has three distinct clades:

  • The cutin-associated clade (GPAT4/6/8) arose early in land plant evolution, appearing in bryophytes

  • The phosphatase-minus GPAT1-3 clade diverged later with the appearance of tracheophytes

  • The GPAT5/7 clade (suberin-associated) also emerged with tracheophytes

• Evolutionary implications:

  • The emergence of GPAT5/7 coincides with the evolution of vascular plants, suggesting a connection to specialized vascular tissue functions

  • The divergence of phosphatase activity across the family suggests functional specialization for different extracellular polymer synthesis pathways

  • The conservation of sn-2 acyltransferase activity across all subbranches of the Arabidopsis GPAT family indicates this is an ancient and fundamental activity

• Research applications:

  • Comparative studies of GPAT5 homologs across diverse plant species can reveal evolutionary adaptations in suberin biosynthesis

  • Understanding the evolutionary trajectory helps predict which plant species might have similar GPAT5 functions

  • Evolutionary analysis can guide the identification of conserved domains for structure-function studies

This evolutionary perspective provides a broader framework for GPAT5 research and connects it to fundamental questions about the adaptation of land plants to terrestrial environments.

How do GPAT5 and GPAT7 work together in suberin biosynthesis?

The relationship between GPAT5 and GPAT7 in suberin biosynthesis represents an area of ongoing research with important implications:

• Structural and functional similarity:

  • GPAT7 is most closely related to GPAT5 (88% similar and 81% identical amino acid sequences)

  • They constitute a distinct clade within the GPAT family tree

  • This high similarity suggests related but potentially specialized functions

• Evidence for GPAT7's role in suberin biosynthesis:

  • GPAT7 is induced by wounding

  • Overexpression of GPAT7 produces suberin-like monomers

  • These observations suggest GPAT7 likely functions in suberin biosynthesis, potentially in stress-responsive suberin formation

• Potential cooperation models:

  • Tissue-specific division of labor, with GPAT5 and GPAT7 predominating in different cell types

  • Condition-dependent roles, with GPAT5 functioning in developmental suberin formation and GPAT7 in stress-induced suberin synthesis

  • Substrate specialization, with each enzyme preferentially incorporating different acyl-CoA substrates

• Research questions:

  • Do GPAT5 and GPAT7 have different substrate preferences?

  • How do their expression patterns overlap or differ in response to development and stress?

  • What is the phenotype of gpat5/gpat7 double mutants compared to single mutants?

Understanding the coordinated functions of GPAT5 and GPAT7 represents an advanced research direction that could provide deeper insights into the regulation and specialization of suberin biosynthesis under different conditions.

What are common challenges in working with recombinant GPAT5 and how can they be addressed?

When working with recombinant GPAT5, researchers may encounter several challenges that require specific methodological approaches:

• Protein activity issues:

  • Challenge: Loss of enzymatic activity during purification

  • Solution: Using microsomal preparations from yeast rather than attempting complete purification can help maintain activity in a more native membrane environment

  • Challenge: Low expression levels

  • Solution: Optimization of expression conditions, codon optimization for the expression system, or trying alternative expression systems (yeast vs. wheat germ)

• Substrate availability:

  • Challenge: Limited commercial availability of specialized substrates (ω-oxidized acyl-CoAs)

  • Solution: Chemical or enzymatic synthesis of required substrates; collaboration with specialized lipid chemistry laboratories

• Assay sensitivity:

  • Challenge: Detecting low levels of enzymatic activity

  • Solution: Incorporation of radiolabeled substrates for increased detection sensitivity; development of more sensitive analytical methods like LC-MS/MS

• In vivo vs. in vitro discrepancies:

  • Challenge: Enzymatic behavior in vitro may not fully reflect in vivo activity

  • Solution: Complementary approaches combining in vitro enzymatic studies with in vivo genetic studies (mutant analysis, complementation)

These methodological considerations are important for researchers designing experiments to study GPAT5 function and can help address technical challenges in working with this enzyme.

How can researchers differentiate between the activities of different GPAT family members in experimental systems?

Differentiating between the activities of different GPAT family members, particularly the closely related GPAT5 and GPAT7, requires specific experimental approaches:

• Substrate specificity profiling:

  • Different GPAT enzymes show characteristic substrate preferences

  • GPAT5 exhibits broad chain length specificity (C16-C24) with highest activity toward C22 substrates

  • Cutin-associated GPATs (GPAT4, GPAT6, GPAT8) show strong preference for C16 and C18 ω-oxidized substrates

  • Comprehensive substrate panels can reveal distinctive enzymatic signatures

• Product analysis:

  • GPATs with active phosphatase domains (GPAT4, GPAT6, GPAT8) produce primarily MAGs

  • Phosphatase-minus GPATs (GPAT5) produce primarily LPAs

  • Analysis of reaction products can distinguish between these enzymatic activities

• Expression pattern analysis:

  • Tissue-specific and condition-dependent expression patterns differ between GPAT family members

  • GPAT5 is expressed in seed coat, root, hypocotyl, and anther

  • GPAT7 shows wound-inducible expression

  • Expression analysis can help identify which GPAT is likely active in a given context

• Genetic approaches:

  • Single and double mutant analysis

  • Complementation with specific GPAT genes

  • Overexpression studies with individual GPATs

These approaches allow researchers to distinguish the contributions of different GPAT family members to suberin and cutin biosynthesis, providing a more complete understanding of their specialized roles.