Algal Lipid Bodies: Stress Induction, Purification, and Biochemical Characterization in Wild-Type and StarchlessChlamydomonas Reinhardtii

ABSTRACTWhen the unicellular green soil algaChlamydomonas reinhardtiiis deprived of nitrogen after entering stationary phase in liquid culture, the cells produce abundant cytoplasmic lipid bodies (LBs), as well as abundant starch, via a pathway that accompanies a regulated autophagy program. After 48 h of N starvation in the presence of acetate, the wild-type LB content has increased 15-fold. When starch biosynthesis is blocked in thesta6mutant, the LB content increases 30-fold, demonstrating that genetic manipulation can enhance LB production. The use of cell wall-less strains permitted development of a rapid “popped-cell” microscopic assay to quantitate the LB content per cell and permitted gentle cell breakage and LB isolation. The highly purified LBs contain 90% triacylglycerol (TAG) and 10% free fatty acids (FFA). The fatty acids associated with the TAGs are ∼50% saturated (C16and C18) fatty acids and ∼50% unsaturated fatty acids, half of which are in the form of oleic acid (C18:1). The FFA are ∼50% C16and ∼50% C18. The LB-derived TAG yield from a liter ofsta6cells at 107cells/ml after starvation for 48 h is calculated to approach 400 mg. The LB fraction also contains low levels of charged glycerolipids, with the same profile as whole-cell charged glycerolipids, that presumably form LB membranes; chloroplast-specific neutral glycerolipids (galactolipids) are absent. Very low levels of protein are also present, but all matrix-assisted laser desorption ionization-identified species are apparent contaminants. Nitrogen stress-induced LB production inC. reinhardtiihas the hallmarks of a discrete pathway that should be amenable to additional genetic and culture condition manipulation.