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

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.

Lipid trafficking in plant cells.

Plant cells contain unique organelles such as chloroplasts with an extensive photosynthetic membrane. In addition, specialized epidermal cells produce an extracellular cuticle composed primarily of lipids, and storage cells accumulate large amounts of storage lipids. As lipid assembly is associated only with discrete membranes or organelles, there is a need for extensive lipid trafficking within plant cells, more so in specialized cells and sometimes also in response to changing environmental conditions such as phosphate deprivation. Because of the complexity of plant lipid metabolism and the inherent recalcitrance of membrane lipid transporters, the mechanisms of lipid transport within plant cells are not yet fully understood. Recently, several new proteins have been implicated in different aspects of plant lipid trafficking. While these proteins provide only first insights into limited aspects of lipid transport phenomena in plant cells, they represent exciting opportunities for further studies.

REF4 and RFR1, subunits of the transcriptional coregulatory complex mediator, are required for phenylpropanoid homeostasis in Arabidopsis.

The plant phenylpropanoid pathway produces an array of metabolites that impact human health and the utility of feed and fiber crops. We previously characterized several Arabidopsis thaliana mutants with dominant mutations in REDUCED EPIDERMAL FLUORESCENCE 4 (REF4) that cause dwarfing and decreased accumulation of phenylpropanoids. In contrast, ref4 null plants are of normal stature and have no apparent defect in phenylpropanoid biosynthesis. Here we show that disruption of both REF4 and its paralog, REF4-RELATED 1 (RFR1), results in enhanced expression of multiple phenylpropanoid biosynthetic genes, as well as increased accumulation of numerous downstream products. We also show that the dominant ref4-3 mutant protein interferes with the ability of the PAP1/MYB75 transcription factor to induce the expression of PAL1 and drive anthocyanin accumulation. Consistent with our experimental results, both REF4 and RFR1 have been shown to physically associate with the conserved transcriptional coregulatory complex, Mediator, which transduces information from cis-acting DNA elements to RNA polymerase II at the core promoter. Taken together, our data provide critical genetic support for a functional role of REF4 and RFR1 in the Mediator complex, and for Mediator in the maintenance of phenylpropanoid homeostasis. Finally, we show that wild-type RFR1 substantially mitigates the phenotype of the dominant ref4-3 mutant, suggesting that REF4 and RFR1 may compete with one another for common binding partners or for occupancy in Mediator. Determining the functions of diverse Mediator subunits is essential to understand eukaryotic gene regulation, and to facilitate rational manipulation of plant metabolic pathways to better suit human needs.