Supplemental Figure 1: Comparison of mRNA expression estimates between different biological replicates of Chlamydomonas strain CC4532 Cells of Chlamydomonas strain CC4532 were collected at the indicated time point after transfer to N-free medium, mRNA was purified and cDNA was generated for RNA-Seq analysis. Expression estimates of the Au10.2 gene models are reported in units of RPKM (Mortazavi et al., 2008). Expression estimates for each of the 12338 expressed genes were compared between two replicates and plotted on logarithmic scales on y and x axis, respectively. The Spearmans rank correlation coefficient (ρ) was calculated and is presented in the figure. Comparison of nitrogen- replete samples (0 h) between 1 h and 8 h time course (A), nitrogen-replete samples (0 h) between 8 h and 48 h time course (B), 30 min nitrogen-deprived samples between 1 h and 8 h time course (C) and 8 h nitrogen-deprived samples between 8 h and 48 h time course (D). Supplemental Figure 1 A 0 h mRNA abundance (S, RPKM) 0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000 mRNA abundance (M, RPKM) ρ = 0.92 ρ = 0.95 mRNA abundance (M, RPKM) 0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000 mRNA abundance (L, RPKM) 0 h B mRNA abundance (S, RPKM) 0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000 mRNA abundance (M, RPKM) ρ = 0.88 0.5 h C mRNA abundance (M, RPKM) 0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000 mRNA abundance (L, RPKM) ρ = 0.92 8 h D Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Transcript
Supplemental Figure 1: Comparison of mRNA expression estimates between different biological replicates of Chlamydomonas strain CC4532Cells of Chlamydomonas strain CC4532 were collected at the indicated time point after transfer to N-free medium, mRNA was purified and cDNA was generated for RNA-Seq analysis. Expression estimates of the Au10.2 gene models are reported in units of RPKM (Mortazavi et al., 2008). Expression estimates for each of the 12338 expressed genes were compared between two replicates and plotted on logarithmic scales on y and x axis, respectively. The Spearmans rank correlation coefficient (ρ) was calculated and is presented in the figure. Comparison of nitrogen-replete samples (0 h) between 1 h and 8 h time course (A), nitrogen-replete samples (0 h) between 8 h and 48 h time course (B), 30 min nitrogen-deprived samples between 1 h and 8 h time course (C) and 8 h nitrogen-deprived samples between 8 h and 48 h time course (D).
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Figure 2: Clustering of 4288 differentially expressed genes identified in the CC4532 RNA-Seq experiments A model-based clustering approach was used to group the 4288 differential expressed genes from the RNA-Seq data in Chlamydomonas strain CC4532 (MBClusterSeq) into 100 clusters. The figure shows the expression patterns for all significantly differentially expressed genes in each cluster for the following 16 time points: +N, 0, 2, 4, 8, 12, 18, 24, 30, 45, 60 min (from the 1 h experiment), 4 h (from the 8 h experiment) and 8, 12, 24, 48 h (from the 48 h experiment). The y-axis represents the log2 fold changes relative to the mean expression across all time points for each gene. Red circles indicate average fold changes within each particular cluster.
1 12 26 61 94 77 25 72 2 9
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37 20 71 39 42 3 5 30 65 32
64 19 57 76 81 21 33 15 93 100
54 43 49 85 68 27 84 96 29 63
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4 66 62 34 60 56 7 14 82 78
90 31 80 36 88 58 91 95 44 52
98 73 59 67 16 47 24 46 8 17
23 11 69 74 35 41 53 89 75 92
Supplemental Figure 2
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Figure 3: Summary of the experimental data gathered from the proteomics time-course experimentA total of 3,724,641 spectra were detected in 218 120 min-long chromatographic separations and subsequent tandem mass spectrometry runs. Using IOMIQS metasearch, that facilitates four different search algorithms (Mascot (Perkins et al., 1999), Sequest (Eng et al., 1994), OMSSA (Geer et al., 2004) and X!Tandem (Craig and Beavis, 2004)), we were able to match 1,636,469 spectra to class 1a peptide sequences, as well as 3,110 SPMs to class 1b, 1,668 SPMs to class 2a, 20,578 SPMs to class 2b, 152,475 spectra to class 3a peptides and 1,667,482 SPMs to class 3b, according to the classification principles of (Qeli and Ahrens, 2010). After filtering at the peptide level by a false positive threshold < 5 %, the protein identifications were further filtered by removing protein or protein groups with fewer than 7 identification out of 9 time points. The filtration resulted in 1,153 mainly class 1a proteins. For peptide quantification 15N metabolic labeling with a uniform 15N-labeled standard (Mühlhaus et al., 2011) and the algorithm ASAPRatio (Li et al., 2003) were used. Analysis of variance (ANOVA) over time revealed 635 protein groups differentially regulated.
Searchdatabase: Aug 10.2 + Chlpst+ mito
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IQMIQS metaseach used engines
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Peptide statistics
1.636 469 no° 1A3 110 no° 1B1 668 no° 2A
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Protein inference
Occam’s razor principle applied classwise to peptide
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Functional annotaion
MapMan for Chlamydomonas v1.0
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Protein statistics
Supplemental Figure 3
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Figure 4: Clustering of 635 differentially accumulating proteins upon transfer to N-free mediaThe centered protein changes were clustered using k-means algorithm with euclidean distance metric. The number of cluster was determined using gap statistic taking into account the original dataset's shape (Tibshirani et al., 2001). Gap statistic suggested two major trends within the protein data which can be further split in 6 clusters for a more detailed visualization of the temporal expression. The clusters are sorted from strong increased (A) to strong decreased abundance (F). Plotted is the 14N/15N ratio on a log2 transformed scale at the indicated time point after transfer to nitrogen-free media of each protein within the cluster (light grey) as well as the median ratio (dark grey). Error bars indicate standard deviation between all proteins within the cluster.
Supplemental Figure 4
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Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Figure 5: mRNA and protein abundances for components of the photosynthetic complexesmRNA and protein abundances are summarized for light harvesting complex II (A), photosystem II (B), cytochrome b6f complex (C), light harvesting complex I (D), photosystem I (E) and plastidic ATP synthase (F).Left: Relative mRNA abundance (% max) in Chlamydomonas strain CC4349, CC4348 (sta6) and CC4532 (48 h time course). See legend for individual gene names (left, italic). Cells were collected at the indicated time point after transfer to N-free medium, mRNA was purified and cDNA was generated for RNA-Seq analysis. Expression estimates of Au10.2 models are reported in units of RPKM (Mortazavi et al., 2008). The maximal RPKM was set to 1 to compare relative expression between individual genes.
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Figure 5 - continuedRight: Abundance of proteins in each sample is compared to a 15N labeled standard (see legend on the right for individual protein names). The 14N/15N ratio was obtained using quantitative LC-MS/MS analysis and is expressed here on a log2 transformed scale. Cells were collected at the indicated time point after transfer to nitrogen-free media. Middle: Overlay of average relative abundance of mRNAs (grey) and proteins (pink) in Chlamydomonas strain CC4532, average protein abundances are drawn similar to Figure 2 in the main body to allow for comparison. Error bars indicate standard deviation between individual proteins/RNAs and lower case protein names on the right indicate origin from the plastid genome.
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Figure 6: mRNA and protein abundance of components of the respiratory complexesPicture composed as described in Supplemental Figure 5 (see legends for individual gene/protein names). Average protein abundances for mitochondrial complexes are drawn in cyan similar to Figure 2 in the main body to allow for comparison. mRNA and protein abundances of NADH ubiquinone oxidoreductase (A, Complex I), succinate dehydrogenase (B, Complex II), ubiquinol cytochrome c oxidoreductase (C, Complex III), cytochrome c oxidase (D, Complex IV) and mitochondrial ATP synthase (E, Complex V).
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Figure 7: mRNA and protein abundance of enzymes involved in Calvin-Benson cyclePicture composed as described in Supplemental Figure 5 (see legends for individual gene/protein names). mRNA and protein abundance of all Calvin-Benson cycle enzymes (A) and overlays of each individual enzyme (B-L), as labelled. For each enzyme only the highest expressed potentially encoding gene and corresponding protein is shown.
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
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Supplemental Figure 7: Calvin-Benson cycle - continued
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Figure 8: mRNA abundances of enzymes involved in tetrapyrrole biosynthesis and degradationRelative mRNA abundance (% max) of enzymes from tetrapyrrole metabolism in Chlamydomonas strain CC4349, CC4348 (sta6) and CC4532 (all experiments), see legends for individual gene names. mRNA was analyzed similar to what was described in Supplemental Figure 5. mRNA abundance of tetrapyrrole biosynthesis enzymes (A) and candidate RNAs potentially involved in chlorophyll degradation (B, 48 h experiment (L)). Absolute mRNA abundance (RPKM) from 0 to 2 h N deprivation in Chlamydomonas strain CC4532, CC4349 and CC4348 (sta6) is given for chlorophyll b reductases (C), chlorophyllases and pheophytinase (D), pheophorbide a oxygenases (E) and CGL61 (F).
Supplemental Figure 8: Tetrapyrrole metabolism
A RNA abundance of enzymes involved in tetrapyrrole biosynthesis
C RNA abundance of potential chlorophyll b reductases
D RNA abundance of potential chlorophyllases and pheophytinase
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Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Figure 8: Tetrapyrrole metabolism - continued
E RNA abundance of candidate pheophorbide a oxygenases
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Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Figure 9: Alignment of putative chlorophyll b reductases from Oryza sativa, Arabidopsis thaliana and Chlamydomonas reinhardtiiAmino acid sequences of NYC1 from Oryza sativa (Os-NYC1, GenBankID: BAF49740.1), its putative orthologs in Arabidopsis thaliana (At-NYC1, TAIR: AT4G13250) and Chlamydomonas reinhardtii (Cr-NYC1, Phytozome: Cre12.g517700) as well as the closely related chlorophyll b reductase NOL from rice (Os-NOL, GenBankID: BAF49741.1) and its putative orthologs At-NOL (AT5G04900) and Cr-NOL (Cre14.g608800) were aligned using the Muscle multiple alignment algorithm (Edgar, 2004) via the web service in Jalview software (version 2.8) (Waterhouse et al., 2009), which was also used for major editing. The sequences are ordered according to their MuscleWS similarity. Residues conserved in either 5 or 6 of the proteins are highlighted with a dark blue background, residues conserved in 3 or 4 of the proteins with a light blue background. Regions with high similarity to short-chain dehydrogenases identified using CDsearch (Marchler-Bauer and Bryant, 2004) are indicated with a black box, important sequence motifs conserved in short chain dehydrogenases are denoted in white bold letters (n = any amino acid, h = hydrophobic amino acid, p = polar or charged amino acid). The two positions indicative for the cofactor that is bound are denoted in black bold letters above the, NADPH is most likely the cofactor when both positions are either lysine or arginine (K/R) (Tanaka et al., 1996). Adobe Illustrator was used for the final editing.
Supplemental Figure 9: Alignment of chlorophyll b reductasesOsNOLATNOLCrNOLCrNYC1AtNYC1OsNYC1
111111
253244367782
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M A A T A A Y L P L R AQ AQ VG L A P L R P SG- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M A TWSG FN V S S S P L L R L R S S S V SN V T K L P F L S- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MM L SQR SG V A A K AH A S T AR A I N PN A V A S L A A S AR GG P A S T S S FGM P V T A V - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R K PD TD N C G P S T SG S S KR - - - - - - - - - G F I N E L L R G PQ P - - - - -- - M T T L T K I Q V Y PQ V L EH R L F FR D P I R VG SR L T C R - - - - - - - - - - ER SN R V Y VH R C E K K V ER KR K V E K F KGN G SWD S L K SG F L G F S K L GM A A A A V VH L S VH GR L R R S P E L H AR P YH R P S L L R C R A F KQ E AD N GG E E A S S S P P P P T T A E AR R R R K - - - - - - - G P L Y K L K A A I QG L AG SR
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S A A AG - - - - - - - - - - - - - - - - - - - - AR L PGR - - - - - - - - - - - - T AR R R L A AR GG - - - - - - - - P E A AG I R A E A V PGG - GG V AR R A AM V P PP I C R R - - - - - - - - - - - - - - - - - - - - R L L A ER FG L - - - - - A T V V V TR QN L T V T P S - - - - - - - - - - - - - - - - S A A V E A - R I SG KR E PM T P PA AG L A - - - - - - - - - - - - - - - - - - - - R A S P AR R G L AQGG S A A AR R AR R S V T V TR A A TQQ T E K P T A A AN G T P A T A T N G - N GGG K A V A PQ A P- - - - - - - - - D L F K AH P K VR Q I N H I L R GR H D R L R G L ER P L G A V V L P A Y L WG S I A A V I G L Q S A L P V L SG A A VGG L F A VMH R VC N R K L WA A PF L S KD - - - - E YN Q K V EN L EM V F S S V A VQ I AR Y I V TM T S T G A I L L I G FQ L SGGD S SMN S L VWY SWL GG I I I G TM T G A - N M V L ED H YR AG PS A A A E A YGG E YQR A V E K A E E I F F S V A TQ VGR Y V I TMM S SG V V L G VG FQ L SGGD SQMN T L I WY SWL GG V I I G TM I G A - N S V L E EH C K AG P
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YN - - - V L I TG S T KG I G Y A L A K E F L K AGD N V V I C SR S A ER V E S A V TD L K K E FG E - - - - - - - - - - - - - - - QH VWG I VC D VR EG KD V K A L VDYN - - - I L I TG S T KG I G Y A L AR E F L K AGD N V V I C SR S A ER V E T A VQ S L K E E FG - - - - - - - - - - - - - - - - EH VWG T KC D V T EG KD VR E L V AYN - - - V V I TG S T KG I GR A L A ED F L R AGD R V V VC SR TGD R V S E T V A E L A AQ YG A - - - - - - - - - - - - - - - D R V KG L A VD V S A PGQ AR Q L ADR Q P L T V V V TGG SR G L G K A L AR E F L A AGD R V L L T SR TQ A A AD A A VR E L R E E V A A L N GC C - - - - - - - - - - PQ V VG V A AD V SD A VG V A A V E AR N - - - V V I TG S TR G L G K A L AR E F L L SGD R V I V T SR S S E S VD M T V K E L EQN L K E I M SN A S E S AR K K L SD A K V VG I AC D VC K P ED V E K L SNR N - - - V V I TG S TR G L G K A L AR E F L L SGD R V V I A SR S P E S V L Q T I N E L E EN I Q EG L S V A K K KQR E I L L H A K V VG T SC D VC K P ED V K K L VN
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F AR D KM K Y I D I W I N N AG SN A Y S Y K P L V E T SD E A L M E V I T TN T L G L M I C C R E A I N MMR N Q PR GGH I FN I D G AG SD GR P T PR F A A YG A T KRY SQ KN L K Y I D I W I N N AG SN A Y S F K P L A E A SD ED L I E V V K TN T L G L M L C C R E AMN MM L T Q SR GGH I FN I D G AG SD GR P T PR F A A YG A T KRF A AQ E L GR VD I W I N N AG T N A YR YG PM A E S TD E E L SQ I VG TN V L G VM L C C K E A I R VMR SQ S TH GH I FN MD G AG AD GN A T PR F A A YG A T KRA A L S S FGR VD AWVN N AG Y SG - S FQ P L V EQ TD AQ I EQ V VR TN L L G T L L C TR Q A V S L MQH Q PGGGH I FN MD G AG AD G F A T PN Y A A YG A T K AF A V K E L G S I N I W I N N AG T N K - G FR P L L E F T E ED I TQ I V S TN L I G S I L C TR G AMD VM SR QH SGGH I FN MD G AG SGG S S T P L T A V YG S T KCF A KD E L G S I D I W I N N AG T N K - G FR P L VN F SD ED I SQ I V S TN L VG S L L C TR E AMN VMQH QQ KGGH V FN MD G AG SGG S S T P L T A V YG S T KC
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S V VH L T K S L Q A E L QMN E VN N VM VH N L S PGM V T T D L L M SG A T T KQ A K F F I N I L A E P AN V V AD Y L V PN I R A I P T - - - - - - N Q SM K P T Y I R FS V VH L T K S L Q A E L QMQD V KN V V VH N L S PGM V T T D L L M SG A T T KQ A K F F I N V L A E P A E V V A E Y L V PN I R A I P A - - - - - - SG SM K P T Y I R FS L AQ L G K S L S A E L GM L G I KN V A VH N L S PGM V T T E L L M AG AN T P T A K F F I N C L A E P A AD V A S F L V PR I R A V P A T A A S P L TG A L Q P V Y I R FG I TQ L TG T L QR E L AD T P I K - - - L H T V S PGM I L TD L L L EG A T T AN KQ A F - N I L C EH P E T V A A F L V PR I K S A V A - - - - - - - R D V SG T Y TR FG L R Q FH G S I V K E SQ K TN VG - - - L H T A S PGM V L T E L L L SG S S I KN KQM F - N I I C E L P E T V AR T L V PR MR V V KG - - - - - - - - - - SG K A VN YG L R Q FQ A S L L K E SR R S K VG - - - VH T A S PGM V L TD L L L SG S S L R N KQM F - N L I C E L P E T V AR T L V PR MR V V KG - - - - - - - - - - SG K A I N Y
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L TG L K A Y SR I F SR I A FG AR R N K Y V A ED - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -L TG I K A Y T K I F SR V A L G AR KN R Y V T E E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -L TQG K A L QR V A AR L L TG SR K SR Y V E E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -L T PG S A L YR L A T A P A - - - R L GR F F D K EGR A V Y P P ER ER L MGR H A K S T AR - - - AQ A A AR R Q SG S L A L A YN L S V L AG V V V L - L A E AQ V L H HL T P PR I L L A I V T SWL - - - R R GR WF D D QGR A L Y A A E AD R L R N - WA EN R T R L S L TD AM EM Y T EN TWV S V F S L S V VC A F I I L Q S T T P S S F PGL T P PR I L L A L V T AWV - - - R R GR WFD E EGR A V Y A A E AD R I R N - WA E SR AR F S F TD AM EM Y T EN TWV S V F S L S V VC A F I I L - S S SGG P L PG
G G GT n n n h
Y n AS STK
D h n p
G n hD h h hNNAG N h n Gh
KR
KR
TP n n nGnhNS nh
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Figure 10: mRNA abundance of enzymes involved in heme biosynthesis and degradationRelative mRNA abundance (% max) of enzymes from heme metabolism in Chlamydomonas strain CC4349, CC4348 (sta6) and CC4532 (48 h experiment), see legends for individual gene names. mRNA was analyzed similar to what was described in Supplemental Figure 5. mRNA abundance of enzymes involved in heme and siroheme biosynthesis (A) and enzymes involved in heme degradation (B).
Supplemental Figure 10: Heme biosynthesis and breakdown
A RNA abundance of enzymes involved in heme and siroheme biosynthesis
0 6 12 18 24 30 36 42 48time in –N (h)
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CC4349 CC4348 (sta6)UPM1HEM15 (FeC)
SIRB
B RNA abundance of enzymes involved in heme breakdown
0 6 12 18 24 30 36 42 48time in –N (h)
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0 6 12 18 24 30 36 42 48time in –N (h)
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CC4349 CC4348 (sta6)HMOX2HMOX1
PCYA
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Figure 11: Abundance of ribosomal proteins and corresponding mRNAsPicture composed as described in Supplemental Figure 5 (see legends for individual gene/protein names). Average protein abundances of cytosolic/plastidic complexes are drawn similar to Figure 6 in the main body to allow for comparison. mRNA and protein abundances of chloroplast ribosomal proteins from the large 50S subunit (A) and small 30S subunit (B) as well as cytosolic ribosomal proteins from the large 60S subunit (C) and small 40S subunit (D). For mitochondrial ribosomal proteins mRNA abundance of the large 50S subunit (E) and small 30S subunit (F) is summarized.
Supplemental Figure 11: Ribosomes - chloroplast
B chloroplast ribosomes: small subunit (30S)
PRPS17PRPS13PRPS6PRPS1 PSRP3PSRP6
PRPS16PRPS1PRPS17 PRPS20
PRPS5PRPS21PRPS6
PRPS10PSRP1
PRPS13PSRP3
PRPS15PSRP4
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CC4532CC4349 CC4348 (sta6) protein (CC4532)overlay (CC4532)
A chloroplast ribosomes: large subunit (50S)
PRPL28 PRPL6 PRPL7/L12PRPL34PRPL35
PRPL15PRPL17PRPL18PRPL19
PRPL21PRPL24PRPL27PRPL28 PRPL33
PRPL29PRPL31PRPL32
PRPL1PRPL4PRPL6PRPL7/L12
PRPL10PRPL11PRPL13
PRPL9
0 6 12 18 24 30 36 42 48time in –N (h)
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0 6 12 18 24 30 36 42 48time in –N (h)
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CC4532CC4349 CC4348 (sta6) protein (CC4532)overlay (CC4532)
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Figure 11: Ribosomes - cytosol
C cytosolic ribosomes: large subunit (60S)
RPL26RPL27RPL30RPL34RPL35RPL36
RPL38
RPL14RPL18RPL19RPL22RPL23RPL24
RPP0RPP1RPL3RPL4RPL6RPL7
RPL11RPL12RPL13RPL13a
RPL10aRPL9RPL27
RPL27ARPL28RPL29RPL30RPL31
RPL10RPL10ARPL11RPL12RPL13RPL13A
RPL14RPL15RPL17RPL18RPL18ARPL19
RPL22RPL23RPL23ARPL24RPL26
RPL21RPP0RPP1RPP2RPL3RPL4RPL5
RPL6RPL7RPL7ARPL7AE
RPL9RPL8
RPL32RPL34RPL35RPL35ARPL36RPL36A
RPL37RPL37ARPL38RPL40
0 6 12 18 24 30 36 42 48time in –N (h)
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CC4532CC4349 CC4348 (sta6) protein (CC4532)overlay (CC4532)
D cytosolic ribosomes: small subunit (40S)
RPS15RPS16RPS18RPS19RPS24RPS25
RPS3RPS3aRPS4RPS5RPS6RPS7
RPS10RPS11RPS13RPS14
RPS8RPS9
RPS26RPS27eRPS28RPSa
RPS27E1RPS27E2RPS28RPS29RPS30
RPS25RPS26RPS27A
RPS23RPS24
RPS11RPS13RPS14RPS15RPS16 RPS21
RPS17RPS18RPS19RPS20
RPS2RPS3RPS3ARPS4RPS5 RPS10
RPS6RPS7RPS8RPS9
RPSA
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CC4532CC4349 CC4348 (sta6) protein (CC4532)overlay (CC4532)
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Aplastidcytosol
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eSupplemental Figure 12: N Sparing
E
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perc
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up regulatedtotal proteomedown regulated
F
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50S
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(cp)
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60S
(cyt
)
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(cyt
)
10-10 0log2 FC
B
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)
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***
**
Ala
Asp Gln
Phe Gly
His Ile Lys
Leu
Met
Pro
Arg
Ser Thr
Val
Tyr
Cys
AsnGlu
Trp
G
prec
ent o
f tot
al
0
8
12
16
20
4
updown
Supplemental Figure 12: Nitrogen sparing mechanisms in ChlamydomonasA: Overlay of cytosolic and chloroplast ribosomal average protein abundance: Relative protein abundance compared to a 15N labeled standard (14N/15N ratio) for each measured protein was averaged for cytosolic ribosomal proteins (blue) and chloroplast ribosomal proteins (green). Error bars indicate standard deviation between individual proteins.B: Total nitrogen content: Nitrogen content (pg/cell) at the indicated time points after transfer to N-free medium measured on a TOC/TN analyzer. Error bars indicate standard deviation of three independent biological replicates.
C: Total non-purgeable organic carbon content: Carbon content (pg/cell) at the indicated time points measured on a TOC/TN analyzer. Error bars indicate standard deviation of three independent biological replicates.D: Nitrogen content of ribosomal proteins: Range (10% quantile (Q10) to 90% quantile (Q90)) of average number of nitrogen atoms in amino acid side chains in the proteome and in ribosomal proteins from different compartments. Color indicates (average) log2 fold change of mRNA abundance (green increase, red decrease) within 48 h of nitrogen deprivation in Chlamydomonas strain CC4532. Nitrogen content in side chains is based on Augustus 10.2 gene models.E: Comparison of sulfur content between up and down-regulated proteins: Quantile distributions of sulfur atoms in amino acid side chains of up (green) or down-regulated (red) proteins. The whole proteome based on Augustus 10.2 gene models was used for reference (grey).F: Comparison of carbon content between up and down-regulated proteins: Picture composed as described in E for distributions of carbon atoms in amino acid side chains.G: Amino acid composition in increasing and decreasing proteins within the proteome: The distributions of the individual amino acids within the proteomics data set is shown, divided into accumulating (up, green) or decreasing (down) proteins upon N starvation. In each data series the total count is normalized to 1. Asterisks indicate statistical significance (t-test, p-value < 0.05) in the differences between the set of accumulating proteins or decreasing proteins (at 48 h time point).
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Figure 13: Abundance of autophagy-related genesA: mRNA abundance of genes involved in autophagy: Relative mRNA abundance (% max) for proteins involved in autophagy in Chlamydomonas strain CC4349, CC4348 (sta6) and CC4532 (48 h time course). mRNA was analyzed similar to what was already described in Supplemental Figure 5. See legend for individual gene names. The gene list derived from (Díaz-Troya et al., 2008).B: Average mRNA abundance of genes involved in autophagy: mRNA from genes in A was averaged, error bars indicate standard deviation between individual genes.
B average RNA abundance of proteins involved in autophagy
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CC4349 CC4348 (sta6) Average mRNA
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Figure 14: mRNA abundance of transporters involved in ammonium importRelative mRNA abundance (% max) of NH4
+ transporters from the AMT family in Chlamydomonas strain CC4349, CC4348 (sta6) and CC4532 (48 h experiment), see legends for individual gene names. mRNA was analyzed similar to what was described in Supplemental Figure 5. mRNA abundance of AMT transporters present in N-replete (A) and absent or very low expressed in N-replete (B).
Supplemental Figure 14
A AMT familiy transporters expressed in N-replete
0 6 12 18 24 30 36 42 48time in –N (h)
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CC4349 CC4348 (sta6)
B AMT familiy transporters low or not expressed in N-replete
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CC4349 CC4348 (sta6)
AMT3AMT6AMT7
AMT1AMT4AMT5AMT8
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
D
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GLN2 (GS2)
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Supplemental Figure 15
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C protein abundance of GS
GS1 GS2
B chloroplast GS
GLN2 GLN3
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A Expression of GLN genes encoding for chloroplast GS
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E cytosolic GS
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F Expression of GLN genes encoding for cytosolic GS
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CC4349 CC4348 (sta6) GLN1GLN4
Supplemental Figure 15: Abundance of glutamine synthases (GS) and GLN RNAsA and B: Relative abundance of GLN RNAs encoding plastidic GS enzymes: Relative abundance (% max) of GLN2 and GLN3 upon transfer to N-free media (A, 48 h) in Chlamydomonas strain CC4349, CC4348 (sta6) and CC4532 (all experiments) and within the first 8 h of N deprivation in Chlamydomonas strain CC4532 (B, 1 h and 8 h experiment). mRNA was analyzed similar to what was already described in Supplemental Figure 5. C: Abundance of cytosolic and chloroplast GS: Relative protein abundances compared to a 15N labeled standard (14N/15N ratio) for GS as described in Supplemental Figure 5. Error bars indicate standard deviation between individual experiements.D: Overlay of protein and mRNA abundance of GS2 (GLN2): The relative abundance of GLN2 mRNA (% max) within the 48 h N deprivation experiment (L) in Chlamydomonas strain CC4532 is displayed in grey. The relative abundance (14N/15N, log2 transformed) for the GS2 protein is given in red.E and F: Relative abundance of GLN RNAs encoding cytosolic GS enzymes: Relative abundance (% max) of GLN1 and GLN4 upon transfer to N-free media (E, 48 h) in Chlamydomonas strain CC4349, CC4348 (sta6) and CC4532 (all experiments) and within the first 8 h of N deprivation in Chlamydomonas strain CC4532 (F, 1 h and 8 h experiment). mRNA was analyzed similar to what was already described in Supplemental Figure 5.
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Figure 16: Abundance of glutamate oxoglutarate amidotransferases (GOGAT) and corresponding mRNAsPicture composed as described in Supplemental Figure 5 (see legends for individual gene/protein names).
Supplemental Figure 16: RNA and protein abundance of GOGAT enzymes
0 6 12 18 24 30 36 42 48time in –N (h)
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Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Figure 17: Abundance of RNAs encoding nitrate or nitrite metabolism functionsmRNA abundance (RPKM) of proteins involved in NO3/NO2 assimilation in Chlamydomonas strain CC4349, CC4348 (sta6) and CC4532 (all experiments), see legends for individual gene names. mRNA was analyzed similar to what was described in Supplemental Figure 5. mRNA abundance of nitrate (NR) and nitrite reductase (NiR) (A), nitrate transporters of the NRT1 family (B), nitrate/nitrite transporters of the NRT2 family (C) and nitrite transporters from the NAR1 family (D) upon transfer to N-free media.
Supplemental Figure 17
A Nitrate and nitrtite reductase
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100
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CC4349 CC4348 (sta6)
B Nitrate transporter of the NRT1 family
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C Nitrate/nitrite transporter of the NRT2 family
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NIA1/NIT1NII1
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NRT2.1NRT2.2NRT2.3NRT2.4NRT2.5NRT2.6NAR2
NAR1.1NAR1.2NAR1.3NAR1.4NAR1.5NAR1.6
D Nitrate/nitrite transporter of the NAR1 family
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Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
DSupplemental Figure 18: Abundance of RNAs encoding (potential) regulators of N assimilationmRNA abundance (RPKM) of proteins (potentially) regulating N assimilation in Chlamydomonas strain CC4349, CC4348 (sta6) and CC4532 (48 h experiment), see legends for individual gene names. mRNA was analyzed similar to what was described in Supplemental Figure 5. mRNA abundance of NIT2 and NRR1 (A), the GLB1 gene encoding the PII protein (B) and three co-regulated transcription factors from Cluster 25 (C) upon transfer to N-free media. N content within side chains is provided for the characterized and potential transcriptional regulators (D). Range (from the 10 % to 90 % quantile) of average number of nitrogen atoms in amino acid side chains in all proteins of the proteome and in all transcription factors as proposed from either Perez-Rodriguez et al. 2010 or Zhang et al. 2011. The color indicates (average) log2 fold changes of mRNA abundance (green - increase, red - decrease) between N-replete conditions and 60 min after the transfer to N-free medium in Chlamydomonas strain CC4532. The nitrogen content in side chains is based on Augustus 10.2 gene models.
Supplemental Data. Schmollinger et al. (2014). Plant Cell 10.1105/tpc.113.122523
Supplemental Table 1: Overlap of differentially accumulating mRNAs between all and individual strains
Comparison of all three strains Chlamydomonas strain
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