1. Lipid extractions [Quench with hot isopropanol and hexane] Heat stress at 42°C [0, 30, 60 min; at an Infors] Collect cells [Centrifuge at 1000 g for 2 min at 4°C] Lipid class quantification Fatty acid composition Lipid molecular species analyses Pre-culture at 25°C [photoautotrophic ; 100 µmoles photons m -2 s -1 at an Infors] being analyzed for Supplemental Figure S1. Experimental setup. Cell cultivation is described in the Materials and Methods section; cells before heat stress was used as the control. Other analyses: - Cell counting - Chlorophyll - Starch - RNA [RNAseq, qRT-PCR]

2. Supplemental Figure S2. Major alterations in membrane lipids were compared using UPLC-MS/MS on total lipid extracts before and after 1 hour heat stress. Values are means ± 95%CI of three biological replicates. * indicates significant difference from the control cells as determined by the Student’s t-test p<0,05. Abbreviations: TAG, Triacylglycerol; MGDG, Monogalactosyldiacylglycerol; DGDG, Digalactosyldiacylglycerol; PtdGro, Phosphatidylglycerol; PtdEtn, Phosphatidylethanolamine; DGTS, Diacylglycerol N’ N’ N’ – trimethylhomoserine; SQDG, Sulfoquinovosyldiacylglycerol; PtdIns, phosphatidylinositol; CI: confidence intervals. * * * *

3. TIC intensity Time TIC intensity A B C D Mass/charge DGTS16:0/18:3 2-Lyso-DGTS16:0 Control Heat stressed Supplemental Figure S3. Identification of lyso-DGTS in control and heat-stressed cells. The relative abundance of DGTS16:0/18:3 and 2-lyso-DGTS16:0 is shown in (A), and (C); and their mass-spectrum patterns are shown in (B) and (D). Abbreviations: TIC: total ion current.

4. Supplemental Figure S4. Over-accumulation of 2-lyso-PtdEtn in control and heat-stressed cells. Value are means of three biological replicates with 95%CI as error bars shown. CI: confidence intervals. * indicates significant difference from the control cells as determined by the Student’s t-test p<0,05. * *

5. 0 min 30 min 60 min Loading origin C17:0 TAG standard Control Heat stressed Supplemental Figure S5. TAG accumulation in heat stressed cells of Chlamydomonas as revealed by TLC. Three replicates at each time point was taken for analyses. Abbreviations: TLC, thin layer chromatography. UPLC-MS/MS identified this band as composed mainly (>80%) of: TAG52:10(18:3/16:4/18:3) TAG52:11(18:3/16:4/18:4) Lipids recovered

6. Detector response (arbitrary unit) Lipid molecular species analysis MGDG DAG TAG Supplemental Figure S6. Lipidomic analysis of the crfad7 mutant provided evidence that DAGs and TAGs accumulated under heat stress are derived from MGDG. Note: The crfad7 mutant is a previously characterized mutant defected in the plastidial  -3 fatty acid desaturase (Nguyen et al 2013). Values are means of three technical replicates.

7. Supplemental Figure S7. qRT-PCR analyses of transcriptional response of selected genes to heat stress. Data are means of three biological replicates, and error bars represent 95%CI. The expression of RACK1 gene is used as a normalization control. Abbreviations: PDAT, phospholipid:diacylglycerol acyltransferase; DGAT1 and DGTT1-4: five diacylglycerol acyltransferase; MLDP, major lipid droplet protein. Ci, confidence interval.

8. Supplemental Figure S8. Comparison of TAG molecular species distribution in photoautrophically (MM)- or mixotrophically (TAP)- grown cells when subjected to nitrogen starvation. Values are means of three technical replicates. Detector response (arbitrary unit) TAG (total number of carbons: total number of unsaturations) Photoautotrophic condition (MM-N) Mixotrophic condition (TAP-N)