A land-plant-specific glycerol-3-phosphate acyltransferase family in Arabidopsis: substrate specificity, sn-2 preference, and evolution

Abstract

Arabidopsis (Arabidopsis thaliana) has eight glycerol-3-phosphate acyltransferase (GPAT) genes that are members of a plant-specific family with three distinct clades. Several of these GPATs are required for the synthesis of cutin or suberin. Unlike GPATs with sn-1 regiospecificity involved in membrane or storage lipid synthesis, GPAT4 and -6 are unique bifunctional enzymes with both sn-2 acyltransferase and phosphatase activity resulting in 2-monoacylglycerol products. We present enzymology, pathway organization, and evolutionary analysis of this GPAT family. Within the cutin-associated clade, GPAT8 is demonstrated as a bifunctional sn-2 acyltransferase/phosphatase. GPAT4, -6, and -8 strongly prefer C16:0 and C18:1 ω-oxidized acyl-coenzyme As (CoAs) over unmodified or longer acyl chain substrates. In contrast, suberin-associated GPAT5 can accommodate a broad chain length range of ω-oxidized and unsubstituted acyl-CoAs. These substrate specificities (1) strongly support polyester biosynthetic pathways in which acyl transfer to glycerol occurs after oxidation of the acyl group, (2) implicate GPAT specificities as one major determinant of cutin and suberin composition, and (3) argue against a role of sn-2-GPATs (Enzyme Commission 2.3.1.198) in membrane/storage lipid synthesis. Evidence is presented that GPAT7 is induced by wounding, produces suberin-like monomers when overexpressed, and likely functions in suberin biosynthesis. Within the third clade, we demonstrate that GPAT1 possesses sn-2 acyltransferase but not phosphatase activity and can utilize dicarboxylic acyl-CoA substrates. Thus, sn-2 acyltransferase activity extends to all subbranches of the Arabidopsis GPAT family. Phylogenetic analyses of this family indicate that GPAT4/6/8 arose early in land-plant evolution (bryophytes), whereas the phosphatase-minus GPAT1 to -3 and GPAT5/7 clades diverged later with the appearance of tracheophytes.

Figure 1.

GPAT8 is a sn-2 acyltransferase. GPAT8 enzyme produced with the wheat germ cell-free translation system was assayed with 16-OH C16:0-CoA, 18-OH C18:1-CoA, C16:0-DCA-CoA, or C18:1-DCA-CoA as acyl donor and [¹⁴C]G3P as acyl acceptor. A, Representative radio-TLC image of regiospecificity of ω-oxidized MAGs formed in GPAT8 assays. MAGs from GPAT8 assays were identified by comparison with R_f value of MAGs generated from parallel GPAT6 assays. The large spots at the origin are unreacted [¹⁴C]G3P. Radioactive bands migrating above MAGs represented less than 5% of products and were not identified. B, Quantification of MAG products from GPAT8 assays by autoradiography. The values represent means ± range of two independent enzyme preparations.

Figure 2.

Substrate specificity of GPAT8. Wheat germ translation reaction expressing GPAT8 was used as the enzyme source. GPAT assays were conducted with different acyl-CoA species as acyl donors and [¹⁴C]G3P as the acyl acceptor. Products (nmol) from vector control were subtracted from those of GPAT8 reactions. The values represent means ± range of two independent enzyme preparations.

Figure 3.

Substrate specificity of GPAT6. Wheat germ translation reaction expressing GPAT6 was used as the enzyme source. GPAT assays were conducted with acyl-CoA species shown as acyl donors and [¹⁴C]G3P as the acyl acceptor. Products (nmol) from vector control were subtracted from those of GPAT6 reactions. The values represent means ± range of two independent enzyme preparations.

Figure 4.

Fatty acid selectivity of GPAT6. Wheat germ translation reaction expressing GPAT6 was used as the enzyme source. GPAT assays were conducted with equimolar mixtures of 16-OH C16:0-CoA and 10,16-diOH C16:0-CoA species as acyl donors and [¹⁴C]G3P as the acyl acceptor. After assay, the quenched reaction mixture was directly applied to a TLC plate (K6) and developed with chloroform:methanol:acetic acid:water (52:15:10:3.5). A, Radio-TLC image of product distribution. The large spot at the origin is unreacted [¹⁴C]G3P. Radioactive bands below MAG were present in vector controls and represent impurities in the [¹⁴C]G3P. Radioactive bands migrating above MAG represented less than 10% of products and were not identified. B, Quantification of the GPAT6 MAG product. The values represent means ± range of two independent enzyme preparations.

Figure 5.

Substrate specificity of GPAT5. Microsomes from the yeast gat1Δ strain expressing GPAT5 were used as the enzyme source. GPAT assays were conducted with acyl-CoA species shown as acyl donors and [¹⁴C]G3P as the acyl acceptor. GPAT activities from vector control were subtracted from those of GPAT5 reactions. The values represent means ± sd of three independent enzyme preparations.

Figure 6.

GPAT7 overexpression produces MAGs and free fatty acids in soluble chloroform-extractable surface lipids of the seeds (A) and stems (B). Three independent lines are averaged for the 35S:GPAT7 overexpressors, and error bars represent sd. ALD, Aldehyde; ALK, alkane; FFA, free fatty acid; FW, fresh weight; KET, ketone; PA, primary alcohol; SA, secondary alcohol; WT, wild type.

Figure 7.

GPAT7 expression is induced by wounding, and gpat7 mutants are impaired in wound response. A to C, GPAT7 promoter::GUS responds to wounding. Strong GUS staining was detected only in wounded regions of leaves. D to F, Compared with wild-type leaves, the gpat7-1 mutant fails to exclude toluidine blue after wounding. Rosette leaves (6 weeks old) were wounded with tweezers and kept in standard growth conditions for 48 h. Leaves were then detached from plants and stained with toluidine blue (0.05%) to test tissue-sealing capacities. D, Freshly wounded wild-type leaves are permeable to toluidine blue. E, After 48 h, wild-type leaves are impermeable to toluidine blue, indicating suberin-type wound-sealing response. F, After 48 h, gpat7-1 leaves are still permeable to toluidine blue. Similar observations were also made for other gpat7 alleles (data not shown). Plant tissue images presented in this study were taken with a light microscope (Leica MZ 12.5) coupled with a digital camera.

Figure 8.

Phylogenies of Arabidopsis GPATs from green plants. Each phylogeny illustrates the phylogenetic relationships between the eight members of the Arabidopsis GPAT family and their homologs in angiosperms (rice), early vascular plants (S. moellendorffii), and early land plants (P. patens). In each phylogeny, the Arabidopsis GPATs are identified as GPAT1 to GPAT8, while the genes from the other species are identified by numbers (for annotations, see “Materials and Methods”). The asterisk beside some genes indicates that it has a phosphatase domain, as identified by InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/). The three clades of sn-2-GPATs are highlighted in different colors, representing the GPAT4/6/8 clade (blue), the GPAT5/7 clade (orange), and the GPAT1/2/3 clade (gray). The scale to the left represents the traits that are thought to have evolved in the representative species.

Figure 9.

Substrate specificity and regiospecificity of GPAT1. A, Substrate specificity of GPAT1. Microsomes from the yeast gat1Δ strain expressing GPAT1 were used as the enzyme source. GPAT assays were conducted using the acyl-CoA species shown and [¹⁴C]G3P as substrates. GPAT activities from vector control were subtracted from those of GPAT1 reactions. The values represent means ± sd of three independent enzyme preparations. B, GPAT1 is a sn-2 acyltransferase. The reaction mixture containing C18:0-LPA, C20:0-LPA, or C22:0-FA-LPA was treated with 1 unit of alkaline phosphatase and analyzed by borate-TLC by comparison with C22:0 or C22:0-DCA sn-2 MAG generated from the alkaline phosphatase treatment of GPAT5 assay.