A Land-Plant-Specific Glycerol-3-Phosphate Acyltransferase Family in Arabidopsis: Substrate Specificity, sn-2 Preference, and Evolution

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.sn-Glycerol-3-phosphate 1-O-acyltransferase (GPAT; Enzyme Commission [EC] 2.3.1.15) is the first enzyme in the pathway for the de novo synthesis of membrane and storage lipids. It catalyzes the transfer of an acyl group from acyl-CoA or acyl-ACP to the sn-1 position of sn-glycerol-3-phosphate (G3P). This reaction has been extensively characterized in bacteria, fungi, animals, and plants (Murata and Tasaka, 1997; Zheng and Zou, 2001; Gimeno and Cao, 2008; Zhang and Rock, 2008; Wendel et al., 2009; Chen et al., 2011a). In the Arabidopsis (Arabidopsis thaliana) genome, there are 10 genes annotated as GPATs. One of these is the soluble, plastid-localized GPAT (At1g32200) that utilizes acyl-ACP substrates and exhibits sn-1 acyl transfer regiospecificity (Nishida et al., 1993). A second enzyme is GPAT9 (At5g60620), which is localized to the endoplasmic reticulum (Gidda et al., 2009) and may be an acyl-CoA-dependent sn-1 GPAT that enables nonplastid glycerolipid synthesis. The remaining eight GPATs cluster together in a family (Zheng et al., 2003; Beisson et al., 2007; Gidda et al., 2009) that is not required for membrane or storage lipid biosynthesis; instead, several members of the family clearly affect the composition and quantity of cutin or suberin (Beisson et al., 2012).Cutin and suberin are extracellular lipid barriers deposited by certain types of plant cells. These insoluble polymers, and associated waxes, function to control water, gas, and ion fluxes and serve as physical barriers to protect plants from pathogen invasion (Kolattukudy, 2001; Schreiber, 2010; Ranathunge et al., 2011). From an evolutionary perspective, the appearance of these lipid barriers was likely a requirement for the adaptation of plants to a terrestrial environment (Rensing et al., 2008). ω-oxidized fatty acids and glycerol are usually major constituents of both polymers (Bernards, 2002; Graça and Santos, 2007; Pollard et al., 2008). The detailed structures of cutin and suberin polymers are still largely unknown (Pollard et al., 2008), but direct esterification of fatty acids to glycerol and to each other has been demonstrated in a large number of partial depolymerization studies of cutin and suberin (Graça and Santos, 2007; Graça and Lamosa, 2010). In Arabidopsis, GPAT4 and GPAT8 are required for the accumulation of C16 and C18 ω-hydroxy fatty acid (ω-OHFA) and α,ω-dicarboxylic acid (DCA) cutin monomers in stems and leaves (Li et al., 2007a). GPAT6 is required for the incorporation of the following C16 monomers: 10,16-dihydroxypalmitate (10,16-diOH C16:0-FA), hexadecane-1,16-dioic acid (C16:0-DCA), and 16-hydroxypalmitate (16-OH C16:0-FA), into flower cutin (Li-Beisson et al., 2009). (For a fatty acid, the abbreviation used is Cm:n-FA, where m is the number of carbon atoms and n is the number of double bonds. The position and number of hydroxyl groups precedes this notation. The same nomenclature is used for DCAs.) GPAT5 controls the accumulation of C22:0- and C24:0-FA, ω-OHFA, and DCA monomers in the suberin of roots and seed coats (Beisson et al., 2007). Recently, we demonstrated that GPAT4 and -6 are bifunctional enzymes that possess sn-2 acyltransferase and phosphatase activities (Yang et al., 2010) and that therefore produce sn-2 monoacylglycerols (MAGs) as the major product. GPAT5 also exhibits strong preference for sn-2 acylation but lacks phosphatase activity; thus, sn-2 lysophosphatidic acids (LPAs) are its major product (Yang et al., 2010).These observations all attest to the fact that several members of the GPAT1 to -8 family are enzymatically very distinct from the GPATs required for membrane and storage lipid biosynthesis. Indeed, they represent a new acylglycerol biosynthesis pathway that provides precursors for cutin and suberin biosynthesis. To better understand the early steps in polyester synthesis and the roles contributed by GPAT4 to -8, and to determine whether the clade of GPAT1 to -3 has distinct or similar activity, we have characterized the regiochemistry and acyl substrate specificity of GPATs representing all three clades. We show that the cutin-associated GPAT8 is a bifunctional sn-2 acyltransferase/phosphatase, while GPAT1, an isozyme with uncertain function but important for tapetum and anther development (Zheng et al., 2003; Li et al., 2012), possesses sn-2 acyltransferase activity but not phosphatase activity. An important issue in defining the pathway of cutin/suberin biosynthesis is whether to place the P450 oxidation reactions before or after the G3P acylation reactions. As discussed (Pollard et al., 2008), previous evidence has not allowed definitive determination of the alternative pathways. However, conducting a GPAT substrate specificity study, particularly with a range of ω-oxidized and unmodified acyl-CoAs can help clarify the situation. Here, we show the acyl-CoA specificities of GPAT4, -5, -6, and -8 are concordant with the compositions of their respective cutins and suberins and the resulting changes in corresponding gpat mutants and overexpression lines. Furthermore, the results provide strong evidence that acyl transfer to glycerol by GPAT occurs after ω-oxidation of acyl chains, thus increasing our limited understanding of the biochemical pathway for cutin and suberin polymer assembly.A phylogenetic analysis of Arabidopsis GPATs with vascular and nonvascular land-plant homologs provides an evolutionary view of the expansion and divergence of the gene family into three distinct clades that are associated with morphological and functional evolution and with the loss of phosphatase activity.