EC Accepts, published online ahead of print on 30 October 2009

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Eukaryotic Cell doi:10.1128/EC.00272-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Algal Lipid Bodies: Stress Induction, Purification, and Biochemical Characterization in Wild-type and Starch-less Chlamydomonas reinhardtii

Zi Teng Wang, Nico Ullrich, Sunjoo Joo, Sabine Waffenschmidt, and Ursula Goodenough^*

Department of Biology, Washington University, St. Louis MO 63130; and Institut für Biochemie, Universität zu Köln, Köln 4750674, Germany

^* To whom correspondence should be addressed. Email: goodenough{at}wustl.edu.

When the unicellular green soil alga Chlamydomonas reinhardtii is 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 hr of N-starvation in the presence of acetate, wild-type LB content has increased 15-fold. When starch biosynthesis is blocked in the sta6 mutant, LB content increases 30-fold, documenting that genetic manipulation can enhance LB production. Use of cell-wall-less strains permitted development of a rapid "popped-cell" microscopic assay to quantitate 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 (C:16 and C:18) and 50% unsaturated, half of which is oleic acid (C:18:1). The FFA are 50% C:16 and 50% C:18. LB-derived TAG yield from a liter of 48-hr-starved sta6 cells at 10⁷ cells/ml 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 MALDI-identified species are apparent contaminants. Nitrogen-stress-induced LB production in C. reinhardtii has the hallmarks of a discrete pathway that should be amenable to additional genetic and culture-condition manipulation.