Activation of the Chloroplast Monogalactosyldiacylglycerol Synthase MGD1 by Phosphatidic Acid and Phosphatidylglycerol

Abstract

One of the major characteristics of chloroplast membranes is their enrichment in galactoglycerolipids, monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG), whereas phospholipids are poorly represented, mainly as phosphatidylglycerol (PG). All these lipids are synthesized in the chloroplast envelope, but galactolipid synthesis is also partially dependent on phospholipid synthesis localized in non-plastidial membranes. MGDG synthesis was previously shown essential for chloroplast development. In this report, we analyze the regulation of MGDG synthesis by phosphatidic acid (PA), which is a general precursor in the synthesis of all glycerolipids and is also a signaling molecule in plants. We demonstrate that under physiological conditions, MGDG synthesis is not active when the MGDG synthase enzyme is supplied with its substrates only, i.e. diacylglycerol and UDP-gal. In contrast, PA activates the enzyme when supplied. This is shown in leaf homogenates, in the chloroplast envelope, as well as on the recombinant MGDG synthase, MGD1. PG can also activate the enzyme, but comparison of PA and PG effects on MGD1 activity indicates that PA and PG proceed through different mechanisms, which are further differentiated by enzymatic analysis of point-mutated recombinant MGD1s. Activation of MGD1 by PA and PG is proposed as an important mechanism coupling phospholipid and galactolipid syntheses in plants.Chloroplast membrane lipids are mostly composed of nonphosphorous galactoglycerolipids i.e. monogalactosyldiacylglycerol (MGDG) 2 and digalactosyldiacylglycerol (DGDG) that represent more than 50 and 30% of thylakoid lipids, respectively. Phosphatidylglycerol is the main phospholipid in plastids, representing about 10% of thylakoid lipids. Each of those glycerolipid classes is represented by a range of molecular species differing in the acyl composition at sn-1 and sn-2 positions of the glycerol backbone. Based on the model of cyanobacterial lipids, the prokaryotic type of glycerolipids contains a 16-carbon fatty acid at the sn-2 position of glycerol. The eukaryotic type contains an 18-carbon fatty acid at the sn-2 position. Some plants, such as Arabidopsis or spinach, have both prokaryoticand eukaryotic-type MGDG, whereas other plants, such as pea or cucumber, have only eukaryotic-type MGDG. DGDG is mostly of eukaryotic type in all plants. Chloroplast PG contains exclusively a prokaryotic-type DAG moiety in contrast to nonplastidial PG. These different chloroplast lipids are assembled in the chloroplast envelope (1). Most enzymes have now been identified, but their functioning and regulation remain largely unknown.MGDG is synthesized by MGDG synthase (MGD), which transfers galactose from UDP-gal to DAG. In Arabidopsis, there are three MGDG synthases, and among them, MGD1, is necessary for synthesis of galactolipids and for development of photosynthetic membranes (2, 3). MGD1 utilizes DAG derived from two main sources, either from a DAG de novo synthesized in plastid envelope by double acylation of glycer...

Role of phosphatidic acid in plant galactolipid synthesis

“…Some DAG kinases produce PA possibly even in plastids since some sequences harbour a putative chloroplast transit peptide. PA can modify the activity of some target proteins including several enzymes involved in the lipid synthesis pathways such as the MGDG synthase, MGD1, in the chloroplast envelope and the soluble phosphoethanolamine N-methyltransferase, PEAMT (indicated by * in the scheme), involved in PC synthesis [2,80]. Demonstrations that specific sources of PA are active at another cell location indicate that PA can be transported between membranes (see [2,26]).…”

“…Regulation of MGDG synthase by phosphatidic acid MGDG synthase, MGD1, is essential for the formation of photosynthetic membranes [76]. Regulation of MGD1 by PA and PG was recently analyzed on native enzyme in leaf extracts and on the recombinant AtD1-137 MGD1 enzyme [2]. No activity was measured when the enzyme was totally depleted of lipids except for the supply of its own substrate DAG.…”

“…increased activation by PA with high salt concentration, and electrostatic interaction of PG, i.e. decreased activation by PG with high salt concentration, and lastly, point mutation of the enzyme allowed determination of 2 amino acids specifically important for activation by PG [2]. It is now important to understand the synergic effect of PA and PG on the MGDG synthase activity in vivo.…”

“…In leaves, PLDz2 is a source of PA that can activate MGDG synthase since MGDG synthase activity is weaker in the pldz2 knock out mutant unless an exogenous source of PA is added [2]. With regards to the role of PLDz2 under the initial stage of Pi deprivation and because PLDz2 is located on the membrane of vacuole where Pi is stored (Fig.…”

“…This step is catalyzed by MGDG synthases (which in Arabidopsis are AtMGD1, AMGD2, AtMGD3) located in the plastid envelope [1]. Diacylglycerol supplied to MGDG synthases results from hydrolysis of PA and recent investigations indicate that in the chloroplast envelope MGD1 is activated by PA [2]. In the chloroplast envelope, PA is also the precursor for the synthesis of phosphatidylglycerol, PG, a phospholipid, which is another essential component of the chloroplast membranes [3].…”