Probing Arabidopsis Chloroplast Diacylglycerol Pools by Selectively Targeting Bacterial Diacylglycerol Kinase to Suborganellar Membranes

Diacylglycerol (DAG) is an intermediate in metabolism of both triacylglycerols and membrane lipids. Probing the steady-state pools of DAG and understanding how they contribute to the synthesis of different lipids is important when designing plants with altered lipid metabolism. However, traditional methods of assaying DAG pools are difficult, because its abundance is low and because fractionation of subcellular membranes affects DAG pools. To manipulate and probe DAG pools in an in vivo context, we generated multiple stable transgenic lines of Arabidopsis (Arabidopsis thaliana) that target an Escherichia coli DAG kinase (DAGK) to each leaflet of each chloroplast envelope membrane. E. coli DAGK is small, self inserts into membranes, and has catalytic activity on only one side of each membrane. By comparing whole-tissue lipid profiles between our lines, we show that each line has an individual pattern of DAG, phosphatidic acid, phosphatidylcholine, and triacylglycerol steady-state levels, which supports an individual function of DAG in each membrane leaflet. Furthermore, conversion of DAG in the leaflets facing the chloroplast intermembrane space by DAGK impairs plant growth. As a result of DAGK presence in the outer leaflet of the outer envelope membrane, phosphatidic acid accumulation is not observed, likely because it is either converted into other lipids or removed to other membranes. Finally, we use the outer envelope-targeted DAGK line as a tool to probe the accessibility of DAG generated in response to osmotic stress.Diacylglycerol (DAG) is a central metabolite in plant lipid metabolism. Its glycerol backbone is modified with two acyl chains. If a third acyl chain is added, triacylglycerol (TAG) is formed, whereas if a head group is added, it is converted into polar lipids such as a galactolipid. In green tissues, the majority of DAG is used as an intermediate in galactolipid synthesis, because the extensive thylakoid membrane system consists of approximately 85% galactolipids (Block et al., 1983). Although under normal conditions the galactolipids are exclusively chloroplastic, in Arabidopsis (Arabidopsis thaliana), the DAG used to make galactolipids is derived from assembly pathways in both the chloroplast and the endoplasmic reticulum (ER; Benning, 2009). In both pathways, the bulk of the fatty acids are synthesized in the chloroplast stroma (Browse et al., 1986) in the following order of abundance: 18:1, 16:0, and 18:0 (Wallis and Browse, 2002).In the chloroplast pathway, these fatty acids are directly attached to a glycerol-3-P, generating first lyso-phosphatidic acid (l-PtdOH) and then phosphatidic acid (PtdOH) in the inner leaflet of the chloroplast inner envelope (Fig. 1; Frentzen et al., 1983). The acyltransferases involved are specific to the extent that the sn-2 position of the glycerol backbone predominantly receives a 16:0 fatty acid. PtdOH is then used directly for phosphatidylglycerol (PtdGro) synthesis (Babiychuk et al., 2003) or converted to DAG by a PtdOH phosphatase (Joyard and Douce, 1977). The PtdOH phosphatase activity is known to be associated with the inner envelope, though which leaflet is obscured by the fact that DAG can efficiently flip across membranes (Hamilton et al., 1991) and the actual enzyme has not been unambiguously identified and located (Nakamura et al., 2007). However, the leaflet associations of two other enzymes that use DAG in the inner envelope have been established. MGD1, which uses DAG to synthesize the most abundant galactolipid, monogalactosyldiacylglycerol (MGDG), is on the outer leaflet of the inner envelope membrane (Xu et al., 2005), while SQD2, which uses DAG to generate the less abundant sulfolipid, sulfoquinovosyldiacylglycerol (SQDG), is located on the inner leaflet of the inner envelope membrane (Tietje and Heinz, 1998). Also associated with the inner envelope membrane are a number of fatty acid desaturases, including FAD4, FAD5, FAD6, FAD7, and FAD8 (Joyard et al., 2010). Two of these are specific, generating lipids with signature desaturations: FAD4 desaturates only the 16:0 fatty acid of PtdGro, giving plastidic PtdGro a distinct 16:1 Δ3 trans moiety (Browse et al., 1985; Gao et al., 2009), and FAD5 desaturates primarily the 16:0 fatty acid of MGDG, producing 16:1 Δ7 cis (Kunst et al., 1989). The remaining desaturases are less specific, with little preference for head group or acyl tail. They further desaturate 16:1 or 18:1 in the cis conformation to 16:2 or 18:2 (FAD6; Browse et al., 1989) and on to 16:3 or 18:3 (FAD7 and FAD8; Wallis and Browse, 2002). The combined actions of these FADs result in the highly desaturated fatty acid profiles seen for most chloroplast lipids.Open in a separate windowFigure 1.Overview of DAG pools in the chloroplast envelope membranes. Processes that are known to have activity feeding into or withdrawing from DAG pools in the chloroplast envelope membranes are shown. Enzymes are indicated, and their substrates and products are connected with black arrows. However, for space reasons, not all reactants are shown. Membrane leaflets are indicated, and enzymes with known membrane topology are displayed correctly, while those without known topology are displayed in the center of the appropriate membrane. The acyl group preferred by each l-PtdOH acyltransferase is given in parentheses. Proposed processes transporting lipids from the ER to the chloroplast are shown with dashed arrows. Enzymes are as follows: 1, ATS1; 2, ATS2; 3, lipid phosphate phosphatase γ; 4, MGD1; 5, SQD2; 6, cytosolic phospholipases; 7, MGD2 or MGD3; 8, SFR2; 9, acyl-CoA:glycerol-3-P acyltransferase; 10, l-PtdOH acyltransferase; 11, PtdOH phosphatase; 12, cytidine diphosphate-choline:DAG cholinephosphotransferase; 13, TGD4; and 14, TGD1, TGD 2, TGD3 lipid transport complex. OE, Chloroplast outer envelope membrane; IE, chloroplast inner envelope membrane; ACP, acyl carrier protein. See online article for color version of this figure.]In unstressed plants, DAG seems to be used primarily in the inner chloroplast envelope. However, several conditions are known to cause extensive DAG use in the chloroplast outer envelope. During phosphate deprivation, MGD2 and MDG3 synthesize MGDG from DAG on the outer leaflet of the outer envelope membrane (Kobayashi et al., 2009). The DAG backbones are probably supplied from the phosphatidylcholine (PtdCho) pool by phospholipase activity, which was shown to be simultaneously up-regulated (Andersson et al., 2004; Nakamura et al., 2005). DAG is also generated during freezing stress by a galactolipid:galactolipid galactosyltransferase named Sensitive to FReezing2 (SFR2). This enzyme transfers the galactosyl head group of MGDG onto another MGDG, giving rise to digalactosyldiacylglycerol (DGDG) and DAG (Moellering et al., 2010). The DAG is subsequently sequestered into a lipid droplet by formation of TAG by an as yet unidentified enzyme.In the ER pathway, fatty acids synthesized in the chloroplast stroma are exported through a still poorly defined mechanism to the ER and activated to acyl-CoAs. Acyltransferases sequentially catalyze formation of l-PtdOH and PtdOH from glycerol-3-P and acyl-CoAs. Again, the acyltransferase working on the sn-2 position of the glycerol backbone is specific, but unlike the chloroplast isoform, it prefers an 18:1 carbon fatty acid (Frentzen et al., 1983). Newly generated PtdOH can be converted to PtdGro or phosphatidyl inositol (PtdIns) (Collin et al., 1999) or hydrolyzed to DAG (Shimojima et al., 2009). DAG can then be further metabolized to TAG and PtdCho. PtdCho acyl groups (18:1/18:1 and 18:1/16:0) are desaturated sequentially by desaturases FAD2 (Okuley et al., 1994) and FAD3 (Browse et al., 1993). These desaturases prefer PtdCho as substrate. The acyl chains modified on PtdCho are transferred to other ER lipids, including DAG, as a result of continual acyl editing of the PtdCho pool (Bates et al., 2012). Furthermore, PtdOH and many of the other extraplastidic phospholipids can be converted to DAG by action of phospholipases (Shimojima et al., 2009). These have as yet partially defined roles in response to stress or recycling of membrane lipids (Testerink and Munnik, 2005).Glycerolipid precursors generated by de novo synthesis, acyl editing, and possibly stress conditions in the ER are transported to the chloroplast by a mechanism that is likely to involve at least two putative lipid transporters: trigalactosyldiacylglycerol4 (TGD4) in the chloroplast outer envelope membrane and the TGD1, TGD2, and TGD3 complex in the inner envelope membrane (Wang and Benning, 2012). The actual lipid species transported remains unclear, but PtdCho, lyso-phosphatidylcholine, PtdOH, and DAG have been discussed in the literature (Andersson and Dörmann, 2009). The DAG moieties are then fully incorporated into all plastidic lipids except PtdGro, presumably using the same pathways that metabolize plastidic DAG, described above. Because of the preference of chloroplast and ER sn-2 acyltransferases for 16 or 18 carbon fatty acids, respectively, the origin of the DAG moieties can be distinguished by positional analysis of the acyl groups on the glycerol backbone (Roughan and Slack, 1982). In Arabidopsis, the chloroplast and ER lipid synthesis pathways contribute nearly equally to mature chloroplast lipids (Browse et al., 1986; Mongrand et al., 1998). Thus, the DAG pools described so far in the chloroplast inner and outer envelope membranes are each of dual origin.A challenge for the analysis of the different DAG pools is that this compound is not a bilayer-forming lipid and thus does not accumulate stably to high levels. Furthermore, during any lengthy fractionation procedure, its levels can be expected to alter, as DAG-modifying enzymes exist in multiple membranes. Moreover, because DAG is quickly metabolized and may have efficient transport systems (Dong et al., 2012), it is difficult to confirm whether metabolizing enzymes are accessing the same or separate DAG pools.To probe different DAG pools of chloroplast membranes in vivo, we have generated a series of stable transgenic Arabidopsis lines in which we target an Escherichia coli DAG kinase (DAGK) to each leaflet of the chloroplast envelope membranes. The basic utility of this approach was previously shown by targeting a DAGK to the chloroplast in tobacco (Nicotiana tabacum) using a single construct fusing the bacterial protein to the Rubisco small subunit N-terminal peptide (Fritz et al., 2007). Here, we present a full phenotypic analysis of these lines, determining which chloroplast membranes show steady-state alterations of DAG and PtdOH levels predicted by ectopic DAGK activity. We further determine the accessibility of DAG pools generated on the outer leaflet of the chloroplast outer envelope membrane during osmotic stress. Having this system established in Arabidopsis will allow characterization of DAG pools in multiple lipid mutant lines.