A predicted plastid rhomboid protease affects phosphatidic acid metabolism in Arabidopsis thaliana

Abstract

Summary The thylakoid membranes of the chloroplast harbor the photosynthetic machinery that converts light into chemical energy. Chloroplast membranes are unique in their lipid makeup, which is dominated by the galactolipids mono‐ and digalactosyldiacylglycerol (MGDG and DGDG). The most abundant galactolipid, MGDG, is assembled through both plastid and endoplasmic reticulum (ER) pathways in Arabidopsis, resulting in distinguishable molecular lipid species. Phosphatidic acid (PA) is the first glycerolipid formed by the plastid galactolipid biosynthetic pathway. It is converted to substrate diacylglycerol (DAG) for MGDG Synthase (MGD1) which adds to it a galactose from UDP‐Gal. The enzymatic reactions yielding these galactolipids have been well established. However, auxiliary or regulatory factors 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 mutants show reduced [14C]–acetate incorporation into MGDG during pulse−chase 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 MGDG by compensating with ER‐derived lipid precursors. These findings link this predicted protease to the utilization of PA for plastid galactolipid biosynthesis potentially revealing a regulatory mechanism in chloroplasts.

Proteins associated with the Arabidopsis thaliana plastid rhomboid‐like protein RBL10

“…The causal relationship between the observed lipid phenotype in the rbl10 mutants and the RBL10 gene was previously shown by rescuing the mutant line with a wild-type copy of RBL10 (Lavell et al, 2019). This provided a possible opportunity to test whether the proteolytic function of RBL10 is important to its role in lipid homeostasis.…”

“…The interaction of CTI1 with ACCase was found to mediate its docking to the plastid envelope membrane, thereby attenuating fatty acid biosynthesis (Ye et al, 2020). The identified interaction between RBL10 and CTI1 might play a role in retaining CTI1 in the envelope membrane, since the abundance of CTI1 is reduced by up to 50% in the envelope membrane of the rbl10 rbl11 double mutant line (Knopf et al, 2012), taking into account that RBL11 has no known function in lipid metabolism (Lavell et al, 2019).…”

“…However, it seems that not all members fall perfectly into each category, leaving room for divergent members in each subtype. Initial studies investigating RBL10 assumed a six-TM topology with both the amino-and carboxy-termini facing the chloroplast stroma and those six helices homologous to the Escherichia coli GlpG TMs (Lavell et al, 2019). These six TMs only represent about half of the amino acids in the RBL10 sequence (Figure S2).…”

“…A number of publications probed the biological relevance of RBL10 as well as its mechanism, showing that loss-of-function of RBL10 causes altered pollen morphology and floral defects and possibly affects jasmonic acid (JA) signaling (Knopf et al, 2012;Thompson et al, 2012). More recently, RBL10 has been implicated in Arabidopsis lipid metabolism and the utilization of chloroplast-assembled phosphatidic acid (PA) for galactolipid biosynthesis (Lavell et al, 2019). Though the involvement of RBL10 in lipid metabolism narrowed its potential biological function, the exact mechanism through which this rhomboid-like protein affects PA metabolism or transport is not clear.…”

“…This provided a possible opportunity to test whether the proteolytic function of RBL10 is important to its role in lipid homeostasis. Towards this end, predicted active site residue point mutants, RBL10 S240A and RBL10 S240A;H293A , were generated using PCR mutagenesis and the respective coding fragments were inserted into the same pEARLEYGATE101 plant expression vector that was used for the wild-type sequence, which contained a BASTA herbicide marker for screening transformants (Lavell et al, 2019). Contrary to the wild-type construct, the floral dipping transformation protocol for the point mutant constructs of RBL10 using Agrobacterium tumefaciens did not yield any transformants in the rbl10-1 mutant background in two independently conducted experiments and after thousands of seeds screened with BASTA selection.…”