Lipid Droplet Isolation from Arabidopsis thaliana Leaves

Abstract

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.

Keywords: Caleosin; Leaf senescence; Lipid droplet isolation; Plant-pathogen interaction; Triacylglycerides.

Figure 1.. Protocol overview.

Collect leaves, cut into small pieces and immediately place in a precooled mortar. After addition of abrasive and Extraction buffer, grind the sample and then filter through 3 layers of Miracloth. Place the filtrate in a 5 ml centrifuge tube, overlay Floating buffer 1 and centrifuge the resulting gradient. LDs will accumulate on top of the tube. Place this LD fraction (FF1) in a new tube and repeat the process using Floating buffer 2. If needed, concentrate the resulting LD fraction (FF2) by centrifugation in a table centrifuge.

Figure 2.. Lipid droplet (LD) extraction from leaves of senescent plants.

A. Image of sucrose gradient after second ultracentrifugation, with Floating fraction 2 (FF2) on top. B. Final LD preparation from wild type senescent plants. C. Lipid droplets from (B), stained with BODIPY^493/503. D. LDs from 35S::CLO3:GFP plants. Nile Red-stained lipids are shown in the core (red), whereas CLO3:GFP locates at the LDs surface (green). E. Protocol tracking of LDs isolated in (D) by western blot with an anti-GFP antibody; BF: bottom fractions, FF: floating fractions. Scale bar = 2 μm.