Algal Lipid Bodies: Stress Induction, Purification, and Biochemical Characterization in Wild-Type and Starchless Chlamydomonas reinhardtii Zi T. Wang*, Nico Ullrich, Sunjoo Joo*, Sabine Waffenschmidt, and Ursula Goodenough* ( Washington University*, Institut fur Biochemie, Universitat zu Koln, Germany) Eukaryotic Cell, 2009, 8:1856–1868 Ursula Goodenough Sabine Waffenschmidt

BIOFUELS (A role for algae?) Potential to have no net increase in [CO 2 ] in atmosphere Renewable and sustainable Ethanol – Produced now from corn starch (sugar cane in Brazil) by fermentation w/yeast or bacteria and distillation Biodiesel – Produced now from vegetable oil (triacylglycerols or triglycerides), less polluting than Petrodiesel Other possible fuels – Butanol – Long-chain hydrocarbons – Hydrogen (H 2 ) (combustion does not produce any greenhouse gas)

Soybean Corn Sunflower Safflower Peanut Cottonseed Rapeseed (canola) Olive Palm Coconut

Chemical conversion step: Oil (triglycerides) to Biodiesel Petro Diesel: C 10 H 20 to C 15 H 28 R= carbons

Chlamydomonas reinhardti: A model system for Biofuel production

Chlamydomonas reinhardtii Model genetic organism for photosynthesis/bioenergetics and cell motility Grows rapidly, autotrophically or heterotrophically Controlled sexual or asexual reproduction All 3 genomes have been sequenced and are transformable: 1.Nuclear (125,000 KB; 15,000 genes) 2.Chloroplast (200 KB; 100 genes) 3.Mitochondrion (16 KB; 12 genes) Can knock-down genes with RNAi

Wang, Z. T. et al Eukaryotic Cell 8(12): FIG. 1. Confocal microscopy surveys of cw15 (A) and cw15 sta6 (B) cell samples starved for N for 24 h. Red, chlorophyll autofluorescence; yellow, Nile Red fluores cence cw15 cw15 sta6 (no starch synthesis) Conclusion: nitrogen starvation increases lipid bodies (LBs), and so does knocking out starch synthesis

Wang, Z. T. et al Eukaryotic Cell 8(12): FIG. 2. (A) Optical sections of cw15 cells (top) and cw15 sta6 cells (bottom) starved for N for 24 h. (B) Three-dimensional reconstructions of through-focal optical sections of cw15 cells (top) and cw15 sta6 cells (bottom) starved for N for 24 h. Red, chlorophyll autofluorescence; yellow, Nile Red fluorescence cw15 sta6 LBs are in the cytoplasm, sticking to the chloroplast surface

Movies Figures A1 & A2. Through-focal optical sections of two cw15sta6 cells N-starved for 48 h. Red, chlorophyll autofluorescence; yellow, Nile-Red fluorescence.

FIG. 3. Size distributions of LBs from: (A) through-focal optical sections of cw15 cells starved for N for 24 h and cw15 sta6 cells starved for N for 24 and 48 h Popped cells after 24 h of N starvation (B), and washed LBs after 18 h of N starvation (C)‏

FIG. 4. Confocal fluorescence microscopy images of cw15 sta6 cells popped in situ Used this Popped-cell assay to do relative quantification of LBs.

Wang, Z. T. et al Eukaryotic Cell 8(12): FIG. 5. Popped cw15 and cw15 sta6 cells stained with Nile Red after 24 and 48 h of N starvation. Nos. are based on summed area of fluorescence pixels/cell

FIG. 6. Pooled distributions of LB contents in popped cw15 and cw15 sta6 cells after 0, 24, and 48 h of N starvation sta6 data skewed to the right; authors suggest LB production may be limited by cell autolysis/autophagy in the 48 h culture. “moribund”

FIG. 7. Washed LB preparation

FIG. 8. (A and B) MS-GC spectra of Fatty acids derived from TAG, in washed LB preparations from cw15 (A) and cw15 sta6 (B) cells Conclusions: 90% of the LB is TAG, 10% C16-C18 species only, no longer-chain FA

FIG. 10. Thin-layer chromatographs (TLC) of NGLs and CGLs from cw15 cells and from initial LB preparations from cw15 sta6 cells LBs do not have much contamination with plastid lipids (NGLs), consistent with an ER origin.

FIG. 11. TAG contents of five independent washed LB preparations from cw15 sta6 and cw15 cells after 18 h of N starvation Estimate yield of ~ 400 mg TAG/liter of culture (10 7 cells/ml) with cw15 sta6

Strengths and weaknesses of this paper: Strengths: 1. Quality of data/results is high 2. Novel finding (Chlamy was thought to not be a good TAG accumulator) 3. Variety of methods used (and developed) 4. It is relevant to Biofuels. Weaknesses: 1. Have to centrifuge cells and replace the medium (not practical at large scale). 2. Did not detect proteins clearly assoc. with LBs. 3. Use of vague term (“Moribund”) cells to explain LB size distribution in sta6 after 48 h.