Galactolipid synthesis in chloroplast envelope membranes. (A) In Arabidopsis, eukaryotic, ER-derived lipid precursors are transported to the chloroplast. Prokaryotic chloroplast-derived and imported eukaryotic diacylglycerols are used for MGDG synthesis by MGD1 in the iEM. In the oEM, MGDG is converted into DGDG by DGD1 (NDGD1CDGD1). NDGD1 binding to PA mediates the association of the iEM and oEM, thereby facilitating the transfer of MGDG from the iEM to the oEM for further galactosylation and of DGDG from the oEM to the iEM. (B) DGDG or GlcGalDG accumulates in dgd1-1 plants complemented with diglycosyllipid synthases (CDGD1, DGD2, or GlcT). Transformation with NM1 fusion constructs (targeting the iEM) relocates diglycosyllipid synthesis to the iEM, resulting in complementation. Transformation with ND2 fusion constructs (targeting the oEM) results in the synthesis of low amounts of DGDG or GlcGalDG without complementation because of the lack of MGDG, DGDG, or GlcGalDG transfer between envelopes. Transformation with DGD2 or GlcT in fusion with NDGD1 results in complementation mediated by PA-dependent aggregations between the iEM and oEM, enabling glycolipid transfer for efficient diglycosyllipid synthesis. Black hexagons indicate galactose; gray hexagons indicate glucose or galactose.