Protein Profiles of Lipid Droplets during the Hypersensitive Defense Response of Arabidopsis against Pseudomonas Infection.

Lipid droplets (LDs) have classically been viewed as seed storage particles, yet they are now emerging as dynamic organelles associated with developmental and stress responses. Nevertheless, their involvement in plant immunity has still been little studied. Here, we found LD accumulation in Arabidopsis thaliana leaves that induced a hypersensitive response (HR) after Pseudomonas infection. We established a protocol to reproducibly isolate LDs and to analyze their protein content. The expression of GFP fusion proteins in Nicotiana benthamiana and in transgenic Arabidopsis lines validated the LD localization of glycerol-3-phosphate acyltransferase 4 (GPAT4) and 8 (GPAT8), required for cutin biosynthesis. Similarly, we showed LD localization of α-dioxygenase1 (α-DOX1) and caleosin3 (CLO3), involved in the synthesis of fatty acid derivatives, and that of phytoalexin-deficient 3 (PAD3), which is involved in camalexin synthesis. We found evidence suggesting the existence of different populations of LDs, with varying protein contents and distributions. GPAT4 and GPAT8 were associated with LDs inside stomata and surrounding cells of untreated leaves, yet they were mainly confined to LDs in guard cells after bacterial inoculation. By contrast, α-DOX1 and PAD3 were associated with LDs in the epidermal cells of HR-responding leaves, with PAD3 mostly restricted to cells near dead tissue, while CLO3 had a more ubiquitous distribution. As such, the nature of the proteins identified, together with the phenotypic examination of selected mutants, suggests that LDs participate in lipid changes and in the production and transport of defense components affecting the interaction of plants with invading pathogens.

Lipid Droplet Isolation from Arabidopsis thaliana Leaves.

Lipid droplets (LDs) are neutral lipid aggregates surrounded by a phospholipid monolayer and specific proteins. In plants, they play a key role as energy source after seed germination, but are also formed in vegetative tissues in response to developmental or environmental conditions, where their functions are poorly understood. To elucidate these, it is essential to isolate LDs with good yields, while retaining their protein components. LD isolation protocols are based on their capacity to float after centrifugation in sucrose gradients. Early strategies using stringent conditions and LD-abundant plant tissues produced pure LDs where core proteins were identified. To identify more weakly bound LD proteins, recent protocols have used low stringency buffers, but carryover contaminants and low yields were often a problem. We have developed a sucrose gradient-based protocol to isolate LDs from Arabidopsis leaves, using Tween-20 and fresh tissue to increase yield. In both healthy and bacterially-infected Arabidopsis leaves, this protocol allowed to identify LD proteins that were later confirmed by microscopy analysis.