Recombinant Arabidopsis thaliana Glycerol-3-phosphate acyltransferase 7 (GPAT7)

What is GPAT7 and how is it related to other GPATs in Arabidopsis?

GPAT7 (AT5G06090) is one of eight members of a land plant-specific glycerol-3-phosphate acyltransferase family in Arabidopsis thaliana. It is most closely related to GPAT5, with 88% similar and 81% identical amino acid sequences, and together they constitute a distinct clade within the GPAT gene family tree . The Arabidopsis GPAT family has three distinct clades with different functions, and GPAT7 belongs to the suberin-associated clade alongside GPAT5 . Unlike the GPAT4/6/8 clade that possesses both acyltransferase and phosphatase activities, GPAT7 lacks an active phosphatase domain .

What is the evolutionary history of GPAT7?

Phylogenetic analyses of the GPAT family indicate that while the GPAT4/6/8 clade arose early in land-plant evolution (appearing in bryophytes), the phosphatase-minus GPATs including GPAT7 likely emerged later in plant evolution . Comprehensive genome-wide analysis across 39 plant species including algae and land plants has tracked the distribution and evolution of the GPAT family members, showing how GPAT7 fits within the broader evolutionary context of plant lipid metabolism enzymes .

What is known about the structure of the GPAT7 gene and protein?

The GPAT7 gene (AT5G06090) contains information about its exon/intron organization that has been comparatively analyzed with other GPAT genes. The protein contains the characteristic acyltransferase domain but lacks an active phosphatase domain, distinguishing it from the GPAT4/6/8 clade . GPAT7 is localized to the endoplasmic reticulum (ER), like GPAT4-8, whereas GPAT1-3 are located in mitochondria and soluble GPAT is found in plastids .

What are the temporal and spatial expression patterns of GPAT7?

GPAT7 expression is strongly induced by wounding of leaf tissue, as demonstrated through promoter-GPAT7-GUS lines . The expression patterns of GPAT genes, including GPAT7, have been analyzed across different plant tissues using public microarray expression data from eudicots including A. thaliana . In the context of root suberization, GPAT7 works alongside GPAT5, with both being mainly required for abscisic acid (ABA)-regulated suberization processes .

How is GPAT7 expression regulated during stress responses?

GPAT7 expression is strongly induced by wounding, suggesting it participates in wound response mechanisms, particularly in suberin production in aerial tissues which is a characteristic wounding response . In root tissues, exogenous ABA application further induces GPAT7 expression, indicating its responsiveness to this stress hormone . Interestingly, in gpat4 gpat6 gpat8 triple mutants, GPAT7 expression increased 8-fold compared to wild type, suggesting compensatory regulation mechanisms within the GPAT family .

How can I design experiments to study GPAT7 expression?

To study GPAT7 expression, researchers can use:

• Promoter-GUS fusion lines to visualize tissue-specific expression patterns, particularly useful for studying wounding responses

• qRT-PCR analysis to quantify expression levels under different conditions, such as ABA treatment or in various mutant backgrounds

• Microarray or RNA-seq data analysis to compare GPAT7 expression with other GPATs across tissues and developmental stages

For effective expression studies, include appropriate controls and time-course analyses, especially when studying wound responses or hormone treatments.

What is the specific function of GPAT7 in plant metabolism?

GPAT7 likely functions in suberin biosynthesis, similar to its close homolog GPAT5 . Overexpression of GPAT7 in Arabidopsis results in the production of very-long-chain sn-1/3- and sn-2 monoacylglycerols (MAGs) together with C22:0 and C24:0 free fatty acids in seed and stem waxes, a phenotype similar to that found with GPAT5 ectopic expression . This suggests that GPAT7 plays a role in the biosynthetic pathway for suberin, particularly in response to wounding .

How does GPAT7 contribute to suberin formation?

The GPAT5/7 clade is crucial for the formation of the typical lamellated suberin ultrastructure observed by transmission electron microscopy . Unlike GPATs with phosphatase activity that produce 2-monoacylglycerol products, GPAT7 lacks an active phosphatase domain and synthesizes lysophosphatidic acids (LPAs) that appear to play a role in forming the lamellated structure of suberin . The gpat5 gpat7 double mutant exhibits distinct amorphous globular polyester structures deposited in the apoplast instead of the typical lamellated suberin .

What is the substrate specificity of GPAT7?

GPAT7 acyl substrate specificity appears similar to GPAT5, which can accommodate a broad chain length range of ω-oxidized and unsubstituted acyl-CoAs . While specific enzymatic assays for GPAT7 alone aren't detailed in the search results, the phenotypic analysis of GPAT7 overexpression lines suggests it can utilize very-long-chain fatty acyl-CoAs (C22:0 and C24:0) . In contrast to GPAT4/6/8 which strongly prefer C16:0 and C18:1 ω-oxidized acyl-CoAs, GPAT5 (and likely GPAT7 by extension) has broader substrate specificity .

How do GPAT7 and GPAT5 cooperate in suberin biosynthesis?

GPAT7 and GPAT5 constitute a distinct clade within the GPAT family and appear to have partially redundant functions in suberin biosynthesis . In the gpat5 gpat7 double mutant, there's an approximately 50% drop in the amount of aliphatic suberin monomers, with particularly strong decreases in C22 monomers (80-90%) without changes in C16 monomers . This suggests an additive effect when both genes are knocked out, with each having some specific roles in the synthesis of particular suberin monomers .

How can I express recombinant GPAT7 for biochemical studies?

For recombinant GPAT7 expression, two systems have been demonstrated effective for GPAT family members:

• Yeast expression system: For enzymes with an active phosphatase domain, this system produces both MAG and LPA products, allowing assessment of both activities

• Wheat germ cell-free expression system: This has been successfully used for expressing recombinant GPATs, including GPAT6 with similar structural features

When designing expression constructs, consider including epitope tags for purification and detection, and optimize codon usage if necessary for the expression system.

What methods are available for analyzing GPAT7 enzymatic activity?

To analyze GPAT7 enzymatic activity, researchers can:

• Measure acyltransferase activity using radiolabeled or fluorescently labeled glycerol-3-phosphate and various acyl-CoA substrates

• Analyze reaction products by thin-layer chromatography (TLC) or liquid chromatography-mass spectrometry (LC-MS) to detect lysophosphatidic acid (LPA) formation

• Compare substrate specificities using a range of acyl-CoA substrates with different chain lengths and oxidation states, similar to assays performed with GPAT5 and GPAT6

The choice of assay conditions should consider the lack of phosphatase activity in GPAT7, focusing on LPA rather than MAG products.

How can I generate and characterize GPAT7 mutants?

For generating and characterizing GPAT7 mutants:

• T-DNA insertion mutants can be obtained from Arabidopsis stock centers

• CRISPR/Cas9 technology can be used to create targeted mutations

• For functional characterization, analyze:

  • Suberin composition by gas chromatography-mass spectrometry (GC-MS) of aliphatic monomers after depolymerization

  • Suberin ultrastructure by transmission electron microscopy (TEM)

  • Wound responses using histochemical stains for suberin deposition

  • Expression of other GPAT genes to detect compensatory changes

Consider creating double mutants (e.g., gpat5 gpat7) to overcome potential functional redundancy between closely related GPATs .

How do the phosphatase-minus GPATs (like GPAT7) and phosphatase-active GPATs coordinate in suberin formation?

The coordination between phosphatase-minus GPATs (GPAT5, GPAT7) and phosphatase-active GPATs (GPAT4, GPAT6, GPAT8) appears crucial for proper suberin formation. The amorphous globular polyester structure observed in the gpat5 gpat7 double mutant was partially reverted by treatment with a phosphatase inhibitor or the expression of phosphatase-dead variants of GPAT4/6/8 . This suggests that the balance between LPA (produced by phosphatase-minus GPATs) and MAG (produced by phosphatase-active GPATs) is important for the correct lamellated structure of suberin .

Research approaches to further investigate this coordination include:

• Creating various combinations of mutants

• Expressing phosphatase-dead variants in different genetic backgrounds

• Developing in vitro reconstitution assays with defined ratios of both types of enzymes

What is the role of GPAT7 in response to different stresses beyond wounding?

While GPAT7 is known to be induced by wounding, its role in other stress responses deserves further investigation. GPAT7 expression is induced by ABA, a hormone involved in various stress responses . Research questions to explore include:

• How does GPAT7 respond to abiotic stresses like drought, salinity, or temperature extremes?

• Is GPAT7 involved in pathogen defense through suberin deposition?

• How does GPAT7 expression interact with other stress-responsive pathways?

Methodological approaches could include stress treatments of wild-type and gpat7 mutant plants, transcriptome analysis under various stresses, and phenotypic characterization of stress responses.

How does the substrate specificity of GPAT7 contribute to suberin composition?

The substrate specificity of GPAT7 appears to influence suberin composition, particularly regarding very-long-chain fatty acids. In the gpat5 gpat7 double mutant, C22 monomers are drastically reduced by 80-90% . To further investigate this relationship:

• Perform in vitro enzyme assays with purified recombinant GPAT7 and a comprehensive range of acyl-CoA substrates

• Create GPAT7 variants with altered substrate binding sites and express them in gpat7 backgrounds

• Analyze resulting changes in suberin composition using GC-MS

• Compare natural variation in GPAT7 sequences and corresponding suberin compositions across Arabidopsis accessions

This research would clarify how GPAT7's substrate preferences directly influence the final composition of suberin polymers in planta.

How does GPAT7 differ functionally from other GPAT family members?

GPAT7 differs from other GPAT family members in several key aspects:

This comparative analysis highlights GPAT7's specialized role in stress-induced suberin synthesis with distinct substrate preferences and regulatory patterns .

How can researchers distinguish between the activities of GPAT5 and GPAT7 experimentally?

Given the high sequence similarity and functional overlap between GPAT5 and GPAT7, distinguishing their specific activities requires careful experimental design:

• Tissue-specific expression analysis: GPAT7 is more strongly wound-inducible, while GPAT5 shows stronger expression in root endodermis and seed coat

• Timing of expression: Analyze expression patterns during development vs. stress responses

• Single mutant analysis: Compare phenotypes of gpat5 and gpat7 single mutants to identify specific defects

• Complementation studies: Express GPAT7 under GPAT5 promoter in gpat5 mutant and vice versa to test functional equivalence

• Chimeric protein analysis: Create fusion proteins swapping domains between GPAT5 and GPAT7 to identify regions responsible for specific functions

These approaches would help delineate the unique contributions of each enzyme despite their high sequence similarity.

What are the most promising areas for future GPAT7 research?

Based on current knowledge gaps, promising research directions include:

• Structural biology of GPAT7 to understand substrate binding and catalytic mechanisms

• Interaction partners of GPAT7 in suberin biosynthetic complexes

• Regulatory networks controlling GPAT7 expression under different stresses

• Engineering GPAT7 to modify suberin composition for enhanced stress resistance

• Comparative studies of GPAT7 orthologs across species to understand evolutionary adaptations in suberin synthesis

These research areas would significantly advance our understanding of plant lipid polymer biosynthesis and potentially lead to applications in crop improvement for stress resilience.

How might GPAT7 be utilized in engineering plants with enhanced stress resistance?

GPAT7's role in wound-induced suberin formation suggests potential applications in engineering plants with enhanced stress resistance. Research approaches could include:

• Overexpression of GPAT7 to increase suberin deposition in response to stress

• Modification of GPAT7 promoter elements to alter its stress responsiveness

• Engineering GPAT7 substrate specificity to alter suberin composition

• Expression of GPAT7 in heterologous plant species that lack efficient wound-induced suberization

When designing such experiments, researchers should consider potential trade-offs between enhanced suberization and other physiological processes, as well as comprehensive phenotypic characterization under multiple stress conditions.