Plants store oils in the form of triacylglycerols (TAGs) in oil droplets in seeds and fruits, and the potential of these as food, feed, biodiesel and industrial chemicals has been explored (Carlsson et al., 2011; Lu et al., 2011). In addition to accumulation in these dedicated storage organs, TAGs also accumulate in non-seed tissues such as flower petals of Dianthus caryophyllus (Hudak and Thompson, 1997), mature pollen grains of flower plants (Murphy and Vance, 1999), stems and leaves of some plant species (Durrett et al., 2008), and in tubers of Cyperus esculentus (Turesson et al., 2010), albeit in lower abundance. Plant non-seed biomass represents the most abundant feedstock source for renewable bioenergy on earth (Schubert, 2006), and therefore a detailed understanding of TAG metabolism in vegetative tissues has important implications for the development of new energy crops for biofuel production (Durrett et al., 2008; Ohlrogge et al., 2009; Chapman et al., 2013; Troncoso-Ponce et al., 2013). In addition, studies in yeast and mammalian systems suggested that TAG accumulation fulfills specific physiological functions such as protection against cellular lipotoxicity induced by overload of free fatty acids (FFAs) and other lipid intermediates (Ducharme and Bickel, 2008; Yen et al., 2008; Kohlwein, 2010). The pathway and enzymes involved in TAG synthesis and the physiological importance of TAG homeostasis in non-seed tissues of plants remain largely unexplored.
TAG biosynthesis in plants occurs in the endoplasmic reticulum (ER) via the Kennedy pathway, involving three sequential acylations of glycerol-3–phosphate with acyl chains exclusively originating from the plastid (Ohlrogge and Browse, 1995; Chapman and Ohlrogge, 2012; Bates et al., 2013). The first two acylation reactions are shared between TAG and membrane lipid biosynthesis, and result in the generation of phosphatidic acid (PA). Dephosphorylation of PA by PA phosphatase generates diacylglycerol (DAG), which is used for the final acylation reaction to produce TAG, catalyzed by diacylglycerol:acyl CoA acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) (Chapman and Ohlrogge, 2012; Bates et al., 2013). At least three distinct classes of DGATs, namely DGAT1 (Routaboul et al., 1999; Zou et al., 1999), DGAT2 (Shockey et al., 2006) and a soluble DGAT (Saha et al., 2006; Hernández et al., 2012) have been reported in plants, and biochemical and genetic evidence has established DGAT1 as a major player mediating TAG biosynthesis in developing seeds (Routaboul et al., 1999; Zou et al., 1999) and senescent leaves (Kaup et al., 2002; Slocombe et al., 2009) of Arabidopsis. Two close homologs of the yeast PDAT gene were identified in the Arabidopsis genome (Dahlqvist et al., 2000), but only PDAT1 activity has been confirmed by over-expression of PDAT1 in Arabidopsis (Ståhl et al., 2004), and PDAT1 has been shown to play overlapping roles with DGAT1 in TAG assembly in pollen grains and developing seeds (Zhang et al., 2009). Both DGAT1 and PDAT1 genes are expressed in leaves, roots and stems, in addition to developing seeds and flowers (Zou et al., 1999; Ståhl et al., 2004), but the functional role of PDAT in vegetative tissues remains to be elucidated. In addition to serving as an immediate precursor for TAG synthesis, DAG is also a substrate for synthesis of the major phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the ER, catalyzed by aminoalcoholphosphotransferases (Dewey et al., 1994; Ohlrogge and Browse, 1995; Goode and Dewey, 1999).
PC plays a central role in the biosynthesis of TAG and membrane lipids (Bates et al., 2013). It is the major source of DAG for TAG synthesis in developing oilseeds (Bates et al., 2009; Bates and Browse, 2012). Importantly, this phospholipid undergoes constant acyl editing involving a deacylation and reacylation cycle, whereby the nascent fatty acids (FAs) exported from the plastid are desaturated prior to being used for de novo glycerolipid biosynthesis through stepwise glycerol-3–phosphate acylation (Bates et al., 2007, 2009; Tjellström et al., 2012).
The central role of PC in glycerolipid biosynthesis is also indicated by the fact that PC-derived lipids serve as precursors for the synthesis of galactolipids via the eukaryotic pathway involving the ER and the plastid (Roughan and Slack, 1982; Ohlrogge and Browse, 1995), although the extent to which the eukaryotic pathway contributes to thylakoid lipid assembly varies depending on plant species and the various tissues within the plant. For example, in pea plants (Pisum sativum) (Mongrand et al., 1998) and green seeds of Arabidopsis (Xu et al., 2005), the eukaryotic pathway is responsible for the assembly of as much as 90% of thylakoid lipids, whereas in the leaves of Arabidopsis and spinach (Browse et al., 1986), approximately half of the photosynthetic membrane lipids are derived from this pathway. The remainder are assembled by a parallel pathway commonly referred to as the prokaryotic pathway, in which FAs are used directly for stepwise glycerol-3–phosphate acylation to generate PA in the plastid. The resultant PA and its dephospharylated product DAG serve almost exclusively as precursors for the synthesis of thylakoid membrane lipids involving the enzyme machinery present in envelope membranes (Dörmann et al., 1999; Awai et al., 2001; Benning and Ohta, 2005).
The Arabidopsis tgd1–1 mutant, so named because it accumulates unusual oligogalactolipids, particularly trigalactosyldiacylglycerol (TGDG), in leaves, is defective in the transport of ER-derived lipid precursors into plastids due to a point mutation in a gene encoding a permease-like component of an ABC transporter complex (Xu et al., 2003). This results in a drastic decrease in the amounts of thylakoid lipids produced by the eukaryotic pathway, with a compensatory increase in galactolipids produced by the prokaryotic pathway. Intriguingly, the tgd1–1 mutation also leads to accumulation of TAG in oil droplet-like structures in the cytosol in leaves (Xu et al., 2005), and, despite the drastic alterations in FA fluxes in tgd1 mutants, their growth and development are only slightly compromised (Xu et al., 2005). Thus, the tgd1 mutants offer a valuable tool to dissect TAG metabolism and homeostasis in non-seed tissues of plants, particularly at the molecular genetic level. Here we demonstrate that PDAT1 plays a critical role in mediating TAG synthesis in fast-growing tissues. Inactivation of PDAT1 in the tgd1–1 mutant background causes gametophytic defects and premature cell death in developing leaves, probably due to accumulation of cytotoxic FFAs. The possible functional role of PDAT1 in maintaining lipid homeostasis in plant non-seed tissues is discussed.