DGD2, an Arabidopsis Gene Encoding a UDP-Galactose-dependent Digalactosyldiacylglycerol Synthase Is Expressed during Growth under Phosphate-limiting Conditions

Abstract

The galactolipid digalactosyldiacylglycerol (DGDG), one of the main chloroplast lipids in higher plants, is believed to be synthesized by the galactolipid:galactolipid galactosyltransferase, which transfers a galactose moiety from one molecule of monogalactosyldiacylglycerol (MGDG) to another. Here, we report that Arabidopsis as well as other plant species contain two genes, DGD1 and DGD2, encoding enzymes with DGDG synthase activity. Using MGDG and UDP-galactose as substrates for in vitro assays with DGD2 we could for the first time measure DGDG synthase activity of a heterologously expressed plant cDNA. UDP-galactose, but not MGDG, serves as the galactose donor for DGDG synthesis catalyzed by DGD2, providing clear evidence for the existence of a UDP-galactose-dependent DGDG synthase in higher plants. In in vitro assays, DGD2 was capable of galactosylating DGDG, resulting in the synthesis of an oligogalactolipid tentatively identified as trigalactosyldiacylglycerol. DGD2 mRNA expression in leaves was very low but was strongly induced during growth under phosphate-limiting conditions. This induction correlates with the previously described increase in DGDG during phosphate deprivation. Therefore, in contrast to DGD1, which is responsible for the synthesis of the bulk of DGDG found in chloroplasts, DGD2 apparently is involved in the synthesis of DGDG under specific growth conditions. In contrast to animals, yeast, and many bacteria, plant membranes contain high amounts of the two galactolipids, monogalactosyldiacylglycerol (MGDG) 1 and digalactosyldiacylglycerol (DGDG) (1-3). These galactolipids are highly abundant in thylakoids, intricate membrane systems in chloroplasts containing the complexes of photosystem I and photosystem II. For a long time the elucidation of the biosynthesis of plant galactolipids has proven extremely difficult. Biochemical labeling experiments with chloroplast membranes suggested that MGDG and DGDG are synthesized by sequential galactosylation of diacylglycerol (e.g. Refs. 4 and 5). Only in the recent past, the first genes encoding MGDG synthase and DGDG synthase were isolated (for reviews see Ref. 6 and Footnote 2). In Arabidopsis thaliana, three genes (MGD1, MGD2, MGD3) are known encoding MGDG synthases. The corresponding enzymes transfer a galactose moiety from UDP-galactose to diacylglycerol (8 -11). The synthesis of DGDG in plants is assumed to involve the galactosylation of MGDG by the galactolipid: galactolipid galactosyltransferase (EC 2.4

Phospholipase DZ2 plays an important role in extraplastidic galactolipid biosynthesis and phosphate recycling in Arabidopsis roots

“…These studies suggest the existence of metabolic pathways that are activated under Pi starvation, which perform two major reactions: (i) the hydrolysis of phospholipids to generate DAG, which necessarily involves the enzymatic action of a phospholipase, and (ii) the use of DAG for the synthesis of galactolipids and sulfolipids. Although the participation and induction of genes involved in SQDG (SQD1 and SQD2), DGDG (DGD1 and DGD2), and MGDG (MGD2 and MGD3) biosynthesis in both roots and leaves as a response to Pi starvation have been experimentally demonstrated (12,13), no direct evidence has been reported to our knowledge on the existence of specific phospholipases that hydrolyze phospholipids to provide DAG for the synthesis of nonphosphorus lipids. Two potential pathways for the hydrolysis of phospholipids to release Pi from its storage source and to produce DAG, the substrate for galactolipid synthesis, have been proposed: a direct pathway involving phospholipase C and an indirect pathway involving PLD and PAP activities (14,19,20).…”

“…Under Pilimiting conditions it has been shown that the expression of genes encoding MGDG synthase and DGDG synthase significantly increases and correlates with the accumulation of DGDG (12,13). It has been proposed that part of the DAG needed for the increased biosynthesis of nonphosphorus lipids is obtained by hydrolysis of phospholipids, although this has not been demonstrated experimentally.…”

“…Several studies have demonstrated that Pi availability alters the lipid composition in diverse plant tissues (12,13). In Arabidopsis leaves it has been reported that major changes include a decrease in phospholipids and a concomitant accumulation of the galactolipid DGDG (8).…”

Quantitative Profiling of Arabidopsis Polar Glycerolipids in Response to Phosphorus Starvation. Roles of Phospholipases Dζ1 and Dζ2 in Phosphatidylcholine Hydrolysis and Digalactosyldiacylglycerol Accumulation in Phosphorus-Starved Plants

“…For the synthesis of DGDG, three MGDG synthases (MGD1-3) and two DGDG synthases (DGD1/2) have been identified. During phosphorus starvation, expression of MGD2/3 and DGD1/2 are induced, activating the galactolipid biosynthetic pathway leading to DGDG accumulation (Kelly and Dö rmann, 2002). This pathway is crucial for DGDG accumulation because the dgd1dgd2 double knockout mutants show undetectable accumulation of DGDG even under phosphorus starvation conditions (Kelly et al, 2003).…”

Lipid Biosynthesis and Protein Concentration Respond Uniquely to Phosphate Supply during Leaf Development in Highly Phosphorus-Efficient Hakea prostrata

“…In Arabidopsis, the plastidic pool is synthesized by the sequential activities of the type A MGDG synthase MGD1, in the inner envelope membrane, and the DGDG synthase DGD1, in the outer envelope membrane (Dörmann et al, 1999;Dubots et al, 2010). The extraplastidic pool is synthesized via the inducible type B MGDG synthases MGD2 and MGD3 as well as DGD2, which are all found in the outer envelope membrane (Kelly and Dörmann, 2002;Kelly et al, 2003;Benning and Ohta, 2005;Kobayashi et al, 2009b). For this low-P-dependent lipid remodeling to occur, extraplastidic phospholipids are hydrolyzed by either PHOSPHOLIPASE C (PLC) or PLD to produce the substrate for MGDG synthases, diacylglycerol (DAG).…”