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1 3 2.6 TEF30 VIPP1 Fold change Time (min) Supplemental Figure S1. TEF30 protein accumulates in Chlamydomonas cells exposed to high light. Chlamydomonas cc1690 wild-type cells were grown photoautotrophically in a photobioreactor at 41 µmol photons m -2 s -1 and 5% CO 2 for 3 d. Then the light intensity was increased to 145 µmol photons m -2 s -1 for 8 h. Relative changes of protein abundance were measured via shotgun proteomics including a 15 N-labeled universal standard (Mettler et al., 2014). Data for TEF30 and VIPP1 as control from membrane-enriched fractions are shown.

2 A Medicago truncatula Glycine max Arabidopsis thaliana Zea mays Oryza sativa Selaginella moellendorffii Physcomitrella patens Ostreococcus lucimarinus Micromonas pusilla Volvox carteri Chlamydomonas reinhardtii MGSSHHHHHH G Putative TAT signal peptide S Cr : MLAVKPINVVAPSGARPAPVAMLRPLASSRAERTVVASASSVAPKAVSRRQVLCQAQTGTKPAQAATSEA : 70 At : --MSLAPSSYPSLYSSPSLPRTQQTKQNPSLITQSSFISAKSLFLSSNSASLCNTHVAKRRNLALKASETESSAKAEAGGDGEEEEKY : 86 Pp : MASAMVTGSCTAAQVNVVARVVDSPVSSPTVAAVGVQLRHSSYAGAGMQSLKGGSTACETHQRGGRNVAMVVRAMAETSPPSGQPAVK : 88 Cr : EYIELDLPKPLGFKFARGNDGGAYIIEVNP-KAGNIDARVQPGDKIVEISASFGSEVWKAENFGQIMYAIRTRSGTVYMKLKKNYGDL :157 At : ETYEIEVEQPYGLKFRKGRDGGTYIDAILPGGSADKTGKFTVGDRVIATSAVFGTEIWPAAEYGRTMYTIRQRIGPLLMQMEKRNGKA :174 Pp : ETYEVELEKPWGLRFYKGADGGTYIDAVAPGGSADKSGMFTPGDKVIETSAMFGNEMWPAAEYGRTMYTIRQRVGTLLMRMEKRYGVR :176 Cr : SALEEEGLDAAEKQWKKERAGGNYGAGTKEIQARNYVQRKENERKRREMFDDALAKFKENDIQGALVEFENIIAMEPRNFVGDNFSRN :245 At : ---EDTGELTEKEIIRAERNAGYISSRLREIQMQNYLKKKEQKAQREKDLREGLQFSKNGKYEEALERFESVLGSKP T :249 Pp : ---EDRGATAEQ--LSAERNAGSIGDGIREIQVQNYLRKQEQKKQREQELDAGLKLYKQGKYEEALGHFESVLGLKP E :249 TPR domain PDZ domain TPR domain Cr : TPIYKVTQYNIACCYSMLDQVEEAIKSLDAAMLSGFDNYDQIRRDKNLSKARANPKFQAVLDKYDEPVVNWNAVKATFGAFGNMFNKEK:334 At : PEEASVASYNVACCYSKLNQVQAGLSALEEALKSGYEDFKRIRSDPDLETLRKSKDFDPLLKQFDESFINESAINAIKSLFGFN--KK-:335 Pp : AREEAVASYNVACCYSKLNQIESGLQALEEAMEAGFDDYKTVREDPDLALLRQSPGFTPLINKYDEPFINENAMNAIKNVFGLFGGKK-:337 Supplemental Figure S2. Phylogenetic tree and alignment of TEF30 homologs. (A) Phylogram based on an amino acid sequence alignment of TEF30 homologs lacking putative transit peptides. The analysis was performed with the MEGA 5.1 program. () Amino acid sequences of TEF30 homologs from C. reinhardtii (Cr), A. thaliana (At), and P. patens (Pp) were aligned using ClustalW. Amino acids highlighted in black are conserved in all 3 sequences. Residues underlined indicate transit peptides according to ChoroP predictions, lines above sequences depict a putative TAT signal peptide (C. reinhardtii sequence), PDZ and TPR domains (all sequences). The N-terminal translational fusion of a hexahistidine TAG with C. reinhardtii TEF30 lacking potential transit sequences is indicated.

3 SpeI A ARG7 HSP70A RCS2 amirna prec RPL12-3 UTR C_ UTR cw transformants TEF30-amiRNA mirna* 100% 50% 100% TEF30 signal intensity AAGTTCGCTCGCGGTGGCGAA TTCGTTACCGCGAGCGAACTT TEF30 CF1β...K P A Q A A T S E A E Y I E -...CUGGGCUUCAAGUUCGCUCGCGGUAACGACGGUGGUGCCUAC...- -UUCAAGCGAGCGCCAUUGCUU- mirna cw transformants TEF30-amiRNA TEF30 signal intensity TEF30 CF1β Supplemental Figure S3. Screening of TEF30-amiRNA transformants. (A) Artifical microrna (amirna) construct used for the constitutive downregulation of TEF30. The construct is based on the pchlamirna2 plastid described by Molnar et al. (2009), which employs the strong HSP70A-RCS2 tandem promoter to drive amirna expression, a double terminator to assure transcriptional termination from both 5 and 3 directions (RPL12-3 UTR/C_ UTR), and the ARG7 gene as selectable marker. The mirna generated targets the TEF30 coding region. () Immunoblot analysis of TEF30-amiRNA transformants in the cw (upper panel) and cw (lower panel) strain backgrounds. trols were transformed with a construct containing the ARG7 gene alone (). Proteins corresponding to 0.5 µg chlorophyll were separated on a 12% SDS-polyacrylamide gel and TEF30 levels were analyzed by immunoblotting with CF1β as loading control.

4 A TEF30- amirna C TEF30- amirna TAP-NH 4, LL TMP, LL TMP, 2% CO 2, LL TAP-NH 4, 16 h dark TAP-NH 4, HL TMP, HL TMP, 2% CO 2, HL TAP-NH 4, 16 h 10 o C Supplemental Figure S4. TEF30-underexpressing transformants exhibit no obvious growth phenotype under a variety of growth conditions. (A) Light microscopy images of control and a TEF30-underexpressing transformant in the cw strain background grown in TAP-NH 4 medium at ~30 μmol photons m 2 s 1. lack bars = 2 µm. () Growth curve of TEF30-underexpressing transformants (#1.23, #3.13, #4.41) and control () grown as described in (A). Error bars represent SD, n = 3. (C) Visual comparison of cultures with a TEF30-underexpressing transformant and a control transformant () cultivated in parallel under several different growth conditions. TMP = minimal medium. LL= 30 μmol photons m 2 s 1, HL= 800 μmol photons m 2 s 1.

5 Relative fluorescence Relative fluorescence Wavelength [nm] Wavelength [nm] Supplemental Figure S5. PSII fluorescence is more affected by high light in TEF30-amiRNA cells than in control cells. 77K fluorescence spectra normalized at the PSI emission peak (~712 nm) of C. reinhardtii cw control () and TEF30-amiRNA cells before (LL 0 min), during (PI 60 min), and after (Rec 90 and 180 min) the photoinhibition treatment. Photoinhibition was done at 1800 μmol photons m 2 s 1 for 60 min.

6 Fold change /TEF30-amiRNA A TAP-NH 4 TAP-NO 3 Supplemental Figure S6. Reduced induction of LHCSR3 expression in TEF30-amiRNA cells is realized at the transcriptional level and NPQ is not affected under photoautotrophic conditions. (A) Accumulation of LHCSR3.1 transcript levels in cw control and TEF30- amirna cells grown in TAP-NH 4 or TAP-NO 3 and exposed to 600 μmol photons m 2 s 1 for 1 h. LHCSR3.1 transcript levels were quantified by RT-qPCR using CLP2 transcripts as calibrator. Error bars represent SD, n=3. () Measurement of non-photochemical quenching (NPQ) of photoautotrophically grown cw TEF30-amiRNA and control cells in comparison with qe mutant npq4 and state transition mutant stt7. Error bars represent SD, n=2.