In vivo lipid ‘tag and track’ approach shows acyl editing of plastid lipids and chloroplast import of phosphatidylglycerol precursors in Arabidopsis thaliana

Abstract

In plant lipid metabolism, the synthesis of many intermediates or end products often appears overdetermined with multiple synthesis pathways acting in parallel. Lipid metabolism is also dynamic with interorganelle transport, turnover, and remodeling of lipids. To explore this complexity in vivo, we developed an in vivo lipid 'tag and track' method. Essentially, we probed the lipid metabolism in Arabidopsis thaliana by expressing a coding sequence for a fatty acid desaturase from Physcomitrella patens (Δ6D). This enzyme places a double bond after the 6th carbon from the carboxyl end of an acyl group attached to phosphatidylcholine at its sn-2 glyceryl position providing a subtle, but easily trackable modification of the glycerolipid. Phosphatidylcholine is a central intermediate in plant lipid metabolism as it is modified and converted to precursors for other lipids throughout the plant cell. Taking advantage of the exclusive location of Δ6D in the endoplasmic reticulum (ER) and its known substrate specificity for one of the two acyl groups on phosphatidylcholine, we were able to 'tag and track' the distribution of lipids within multiple compartments and their remodeling in transgenic lines of different genetic backgrounds. Key findings were the presence of ER-derived precursors in plastid phosphatidylglycerol and prevalent acyl editing of thylakoid lipids derived from multiple pathways. We expect that this 'tag and track' method will serve as a tool to address several unresolved aspects of plant lipid metabolism, such as the nature and interaction of different subcellular glycerolipid pools during plant development or in response to adverse conditions.

Metabolically distinct pools of phosphatidylcholine are involved in trafficking of fatty acids out of and into the chloroplast for membrane production

“…This model is also supported by recent characterization of an unusual D6 desaturated fatty acid produced transgenically in Arabidopsis leaves at only the sn-2 position of PC. However, the D6D fatty acid was redistributed approximately equally to the sn-1 and sn-2 positions of MGDG (Hurlock et al, 2018). Removal of the D6D fatty acid from PC by acyl editing ( Figure 11A) and its subsequent incorporation into both positions of de novo DAG by GPAT/LPAT activities of the eukaryotic pathway before MGDG synthesis is consistent with our new model of acyl flux.…”

Plant Unsaturated Fatty Acids: Biosynthesis and Regulation

“…In their model, the neosynthesized FAs are largely channeled by LPCAT into PC for further desaturation, whereas the exchanged ones (cleaved by PLA 2 or LPCAT) instead enter the de novo eukaryotic pathway. Interestingly, a support is offered by an in vivo lipid 'tag and track' approach established via introducing an ER-resident 6 FAD that specifically acts on the sn-2 acyl chain of PC (Hurlock et al, 2018). A notable outcome was that ∼10% of the unusual 6 FAs, e.g., 18:4( 6, 9, 12, 15), in PC switched to sn-1, suggesting that they were released via acyl editing and then reused for de novo synthesis.…”

“…However, unless 16:0-containing PC is excluded from the transport and 16:0-CoA is unavailable for GPCAT, the C18 criterion will be challenged, as 16:0 may be transferred to sn-2, although 16:0-lysoPC is a preferred acyl acceptor (Lager et al, 2015). Moreover, a specific pool or compartmentalization might also be required, otherwise it would be incompatible with the observation that there is little change in the stereospecificity of the 6 FAs in chloroplastic PC (still ∼90% at sn-2) (Hurlock et al, 2018).…”

“…However, there is a possibility that PA is a functional component of TGD, acting as a membrane destabilizer to reduce the energy barrier for transport (LaBrant et al, 2018). Of note, the presence of ER-derived acyl chains in chloroplastic PG was tracked by using the 6 tag (Hurlock et al, 2018). This might result from PA import, because PA can be converted to not only DAG but PG in plastids (Figure 1), or PA generation from the imported DAG.…”

“…Surprisingly, a great discrepancy existed in the distribution of the 6 tag in the chloroplastic glycerolipid species examined -∼90% in PC, ∼30% in PA, and ∼50% in the four thylakoid lipids occupied sn-2, rendering the precursorproduct relationship elusive. This observation, together with the coexistence of eukaryotic 18:4 and prokaryotic 16:3 in the same MGDG molecule, led to the assumption that extensive acyl editing occurred in thylakoid lipids, which might be triggered by the presence of unusual acyl groups (Hurlock et al, 2018). However, if a proportion of the imported PA undergoes sn-2 deacylation and thus the characteristic 16:0 acylation, the phenomena can be accounted for.…”