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

Abstract

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 [¹⁴ C]-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.

Keywords: chloroplast; enzyme biochemistry; galactolipid; lipid transport; membrane transport; phosphatidic acid; rhomboid protease.

Figure 1. Structure and phenotypes of rbl10 mutant alleles.

(a) Gene model of RBL10 and the two splice forms (At1g25290.1 and At1g25290.2), where in At1g25290.2 exon 6 has 21 fewer nucleotides at the exon’s 5’ end. Positions of the T-DNA insertions in the mutant lines used in this study are shown on the gene model. (b) Quantitative PCR showing reduction in RBL10 gene transcript abundance amplified from cDNA prepared from the rbl10-1 and rbl10-2 knock-out mutants (closed bar) or wild type (WT) (open bar) n=4. (c) Acyl composition of MGDG (carbon # : double bond #) measured in the three rbl10 alleles and WT, showing decreased 16:3 and increased 18:3 relative acyl chain abundance in the mutants n=3. (d) WT acyl chain composition of MGDG restored by expression of the RBL10 coding sequence driven by a 35S promoter in the rbl10-1 background n=3. Error bars report SE but are in most cases too small to show.

Figure 2. Localization of RBL10 to the Inner Envelope Membrane of Chloroplast.

(a) The RBL10 protein was labeled with [³H]-Leucine, imported into chloroplast and subsequently fractionated into various subcellular compartments. The presence of the protein exclusively in the envelope “E”, but not in the stroma “S” or thylakoid “T” fractions confirms its localization. (b) Import and protease protection assays further confirmed that RBL10 was specifically localized to inner envelope membrane. Resistance of imported RBL10 to trypsin (+) digestion is indicated by the presence of a band in the pellet fraction “P”, thus confirming that RBL10 is deeply imbedded into the inner envelope membrane. (c) Homology model of RBL10 made with SwissModel using the GlpG crystal structure as a template (PDB 4njn.1.A). Membrane boundaries are shown for illustration purposes only, predicted catalytic serine and histidine residues are shown in yellow and green respectively.

Figure 3. Radiolabeled pulse chase of detached leaves using ¹⁴C-acetic acid.

A decrease in incorporation of radioactivity into MGDG and increased incorporation into PG and PA of the rbl10-1 mutant (bottom panels) compared to the wild type (WT, top panels) is shown n=3. Error bars report SE but are often smaller than the symbol. Abbreviations: DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PG., phosphatidylglycerol; PI/PE, phosphatidylinositol/phosphatidylethanolamine; SQDG, sulfoquinovosyldiacylglycerol.

Figure 4. Labeling of isolated chloroplasts of rbl10-1 mutant and WT with ¹⁴C-acetate.

(a) Labeling time course with isolated chloroplasts of rbl10-1 (right panel) showing an increased [¹⁴C]-acetate accumulation into PA with an apparent conversion of PA into PG compared to wild type (WT, a, left) which appears to convert PA to MGDG n=3. Error represent SE and are often smaller than the labels. (b) TLC image of the extracted lipids of the chloroplasts labeled with [¹⁴C]-acetate in (a), one representative of three. The accumulation of radiolabel into MGDG of WT chloroplasts is approximately twice the accumulation of radiolabel into MGDG for rbl10-1 chloroplasts. The chloroplasts of both mutant and WT appear to be capable of converting PA to PG. Abbreviations of lipids as in the Figure 3 legend.

Figure 5. Probing PA metabolism in rbl10-1.

(a) Overview of plastid PA metabolism. PA can be converted to PG (ai) or is dephosphorylated to supply diacylglycerol (DAG) pool (aii) for galactolipid biosynthesis (aii, iv). It is important to note that DGDG biosynthesis favors precursors from the ER pathway not shown here (aiv). (b) Activity of the galactolipid enzymes MGD1 and DGD1 in isolated chloroplast using labeled UDP-[¹⁴C]-Gal as precursor. They synthesize MGDG and DGDG respectively and are both functional at wild-type (WT) levels in rbl10-1 isolated chloroplasts n=3. (c) Phosphatidic acid phosphatase activity of envelope membranes supplied with [¹⁴C]-PA n=3. While rbl10-1 chloroplasts cannot readily convert de novo synthesized PA fully to MGDG, mixed envelopes of this mutant did not show decreased [¹⁴C]-PA to [¹⁴C]-DAG dephosphorylation. Error bars report SE. Error bars are often smaller than the symbols.

Figure 6. Proposed hypothesis of PA metabolism in the chloroplast envelope membranes.

(a) Fate of PA assembled at the ER and imported back to the plastid. (b) Fate of PA assembled and metabolized in the plastid. Both panels depict ER and plastid PA serving as a substrate for the same PA phosphatase in the intermembrane space leaflet of the inner membrane of the chloroplast. The import of ER sourced PA is hypothesized to be facilitated by the TGD complex in the plastid outer and inner envelope membranes (a). The plastid PA is assembled on the stromal side of the inner envelope membrane. A transport mechanism is proposed, which translocates PA and inserts it into the leaflet of the inner membrane facing the intermembrane space (b). The rbl10 mutant phenotype can be explained assuming that RBL10 is critical for the function of the inner envelope PA transporter.