Activation of the chloroplast monogalactosyldiacylglycerol synthase MGD1 by phosphatidic acid and phosphatidylglycerol.

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.

Phosphate availability affects the tonoplast localization of PLDzeta2, an Arabidopsis thaliana phospholipase D.

Under phosphate deprivation, higher plants change their lipid composition and recycle phosphate from phospholipids. A phospholipase D, PLDzeta2, is involved in this recycling and in other cellular functions related to plant development. We investigated the localization of Arabidopsis PLDzeta2 by cell fractionation and in vivo GFP confocal imaging. AtPLDzeta2 localizes to the tonoplast and the Nter regulatory domain is sufficient for its sorting. Under phosphate deprivation, AtPLDzeta2 remains located in the tonoplast but its distribution is uneven. We observed PLDzeta2-enriched tonoplast domains preferentially positioned close to mitochondria and beside chloroplasts. In absence of PLDzeta2, membrane developments were visualized inside vacuoles.

In vitro reconstitution of monogalactosyldiacylglycerol (MGDG) synthase regulation by thioredoxin.

Monogalactosyldiacylglycerol (MGDG), a major membrane lipid of chloroplasts, is synthesized by MGDG synthase (MGD) localized in chloroplast envelope membranes. We investigated whether MGD activity is regulated in a redox-dependent manner using recombinant cucumber MGD overexpressed in Escherichia coli. We found that MGD activity is reversibly regulated by reduction and oxidation in vitro and that an intramolecular disulfide bond(s) is involved in MGD activation. Because thioredoxin efficiently reduced disulfide bonds to enhance MGD activity in vitro, MGD is potentially an envelope-bound thioredoxin target protein in higher plants.

Digalactosyldiacylglycerol is a major glycolipid in floral organs of Petunia hybrida.

In higher plants, glycolipids such as monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are major components of chloroplast membranes in leaves. A recent study identified an isoform of MGDG synthase that is expressed specifically in floral organs, suggesting a novel function for glycolipids in flowers. To elucidate the localization and developmental changes of glycolipids and their biosynthetic activities in flowers, we carried out a series of analytical studies with Petunia hybrida. The results showed that the biosynthetic activities of galactolipid synthesis, particularly for DGDG, increased during flower development. Among the floral organs, the pistil had the highest galactolipid synthetic activity. Its specific activity for incorporation of UDP-galactose to yield galactolipids was estimated to be more than twice that of leaves, which are the major site of galactolipid synthesis in plant tissues. Analysis of lipid contents of pistils revealed that they contained higher amounts of galactolipids than other floral organs. Moreover, DGDG was more abundant than MGDG in both pistils and petals. These results show that DGDG is a major glycolipid in floral organs and that DGDG biosynthetic activity is highly upregulated in the pistils and petals of Petunia flowers.

Light and cytokinin play a co-operative role in MGDG synthesis in greening cucumber cotyledons.

The current research investigated the regulation of monogalactosyldiacylglycerol (MGDG) biosynthesis, catalyzed by MGDG synthase (MGD) (UDP-galactose:1,2-diacylglycerol 3-beta-D-galactosyltransferase; EC 2.4.1.46), during chloroplast development in cucumbers (Cucumis sativus L. cv. Aonagajibai). In etiolated seedlings, white light induced a transient increase in MGD mRNA, followed by a subsequent increase in enzyme activity. MGDG, digalactosyldiacylglycerol (DGDG), and linolenic acid (18 : 3) of both MGDG and DGDG accumulated in a light-dependent manner. Early light-dependent induction of MGD protein was also identified in isolated chloroplasts. When cotyledons were detached from seedlings, these light-induced changes diminished. However, when a synthetic cytokinin, benzyladenine, was added to the detached cotyledons, a transient increase in MGD mRNA and a linear increase in the enzyme activity were induced even in the dark. Galactolipids subsequently accumulated to some extent and 18 : 3 content also increased. MGDG fully accumulated in detached cotyledons with co-treatment of light and a cytokinin. Red light (>600 nm) and far-red light (>700 nm) both induced an increase in MGD mRNA and enzyme activity but far-red light did not induce an accumulation of MGDG. These results suggest that (1). galactolipid biosynthesis is regulated by the cooperation of light and a cytokinin; (2). the accumulation of MGDG requires cytokinin in addition to light; (3). a red light (600-700 nm) dependent factor is necessary for the maximal galactolipid accumulation in addition to increase in MGD transcript and activity.