Development of Oat Prothylakoids into Thylakoids during Greening Does Not Change Transmembrane Galactolipid Asymmetry but Preserves the Thylakoid Bilayer.

The lipase from Rhizopus arrhizus and the lipolytic acyl hydrolase from potato tubers have been used to determine the transmembrane distribution of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) in prothylakoids and thylakoids from oat (Avena sativa). Both galactolipids were found to be asymmetrically distributed. The molar outside/inside distribution was 70 +/- 8/30 +/- 8 for MGDG and 10 +/- 4/90 +/- 4 for DGDG in the prothylakoid membrane. Mature thylakoids presented a similar distribution, i.e. 63 +/- 4/37 +/- 4 for MGDG and 12 +/- 3/88 +/- 3 for DGDG. This distribution has been assessed under a variety of different conditions, namely (a) in media favoring thylakoid stacking or unstacking and inducing various membrane surface potentials, (b) in the presence of defatted bovine serum albumin which removed free fatty acids and partially lyso-galactolipids, (c) under various temperature conditions which resulted in different hydrolysis rates and degrees of fluidity of the membrane, and (d) in the presence of different enzyme concentrations which influenced the hydrolysis rate. The above distribution was found to be independent of the type of conditions used. Nonbilayer forming/bilayer forming lipid ratios suggest that both monolayers of the prothylakoid and the inner monolayer of oat thylakoid membranes should display lamellar structures (e.g. ratios <2.5). In contrast the outer monolayer of the thylakoid membrane should display non-lamellar configurations (e.g. ratio >2.5). Thus, it is concluded that the incorporation of chlorophyll-protein complexes into the nascent thylakoid membrane modifies neither the galactolipid nor the phospholipid transmembrane distribution. However, these complexes appear to be crucial to preserve a bilayer configuration to the greening membrane which, otherwise, would adopt nonlamellar structures. The possible origin of galactolipid transversal asymmetry which appears very early during the biogenesis of oat thylakoid membranes is discussed.