Chloroplast membranes house the photosynthetic machinery and have a distinct lipid composition, with characteristically abundant galactolipids mono- and digalactosyldiacylglycerol (MGDG and DGDG). Both galactolipids are synthesized through both plastid and ER pathways in Arabidopsis, resulting in distinguishable molecular species. Phosphatidic acid (PA) is the first key intermediate formed by the plastid galactolipid biosynthetic pathway. It is further dephosphorylated to diacylglycerol (DAG), which is a substrate for MGDG Synthase (MGD1). MGD1 further adds a galactose to DAG from UDP-Gal. The enzymatic reactions yielding these galactolipids are known, but regulatory factors controlling this process are largely unknown. We identified a predicted rhomboid-like protease 10 (RBL10), located in plastids of Arabidopsis thaliana, that affects galactolipid biosynthesis likely through intramembrane proteolysis. Plants with T-DNA disruptions in RBL10 have greatly decreased 16:3 (acyl carbons : double bonds) and increased 18:3 acyl chain abundance in MGDG of leaves. Additionally, rbl10-1 mutant chloroplasts show reduced [14C]-acetate incorporation into MGDG during pulsechase labeling, indicating a reduced flux through the plastid galactolipid biosynthesis pathway. While plastid MGDG biosynthesis is blocked in rbl10-1 mutants, they are capable of synthesizing PA, as well as producing normal amounts of total MGDG by compensating with ER-derived lipid precursors. These findings link this predicted protease to the utilization or transport of PA for plastid galactolipid biosynthesis potentially revealing a regulatory mechanism for galactolipid biosynthesis in chloroplasts. In addition to serving as a key metabolite, PA also has signaling roles in the cell, making its trafficking important to understanding plant cell metabolism. The substrate(s) of RBL10 are not yet known, but Blue-Native PAGE and FPLC analysis showed that RBL10 is part of a large molecular weight complex (> 660kDa). The protein interactors of RBL10 are currently being probed by using co-immunoprecipitation and split-ubiquitin yeast two-hybrid approaches. Additionally, RBL10 seems to be autoproteolytic toward its own carboxyterminal domain (CTD). The role of complex residency and autolytic activity of RBL10 is not currently clear, but these findings could help uncover the identity of a new transporter of PA in the chloroplast as well as a regulatory mechanism of its activity.
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