Full Legends for Supporting Information
Figure S1. Physiological measures of cw15 under nitrogen deprivation. (a) Growth curve.
OD750, optical density at 750 nm. Circle: with nitrogen. Square: without nitrogen. (b) Flow
cytometry to show cell size distributions.
Figure S2. Change of C/N ratios under N starvation across the time course.
Figure S3. Decrease of total RNA under N starvation across the time course. Means (n=2, SE)
are shown.
Figure S4. The response of the PII regulated arginine biosynthesis pathway under N deprivation
in C. reinhardtii. (a) VANTED diagram displaying the arginine biosynthesis pathway that
responds to N deprivation. Transcript levels are displayed above the abbreviated enzyme name,
with protein levels below them, and with levels relative to time 0 (control) going from left to
right being, 1, 2, 4, 6, 12, 24 h, respectively. Blue color in the boxes means an increase in
expression/level and yellow means a corresponding decrease, with fold-change (log2 scale)
indicated by color intensity as shown in the key. Two lanes are displayed for enzymes where two
isoforms were measured. Enzyme names were abbreviated and capitalized, with red and blue
names indicating, respectively, that both transcript and protein ratios were acquired or that only
data for transcripts was obtained. Metabolite names are in black. For metabolites that were
quantified, graphs displaying their changes in relative concentrations (standard errors indicated
as shading above/below the graph line) are shown under the compound name abbreviation.
Abbreviations are as follows: GLB1, pii protein; LCI8, n-acetylglutamate synthase; AGK1, nacetylglutamate kinase; NAGPR, n-acetyl-gamma-glutamyl-phosphate reductase; ARG9,
acetylornithine aminotransferase; AOD1, acetylornithine deacetylase; ODC2, ornithine
decarboxylase; CMPS1, carbamoyl-phosphate synthase; OTC1, ornithine carbamoyltransferase;
AGS1, argininosuccinate synthase; ARG7, argininosuccinate lyase; ADC, arginine
decarboxylase; ADI1, arginine deiminase; AIH2, agmatine deiminase; CPA1, ncarbamoylputrescine amidase; AMX4, copper amine oxidase; SPD1, spermidine synthase. (b)
Arginine pool size measured by HPLC under N starvation from 0, 0.5, 1, 2, 4, 6, 12 and 24h time
points. Means (n=3, SE) were shown. (c-e) 13-C Incorporation into arginine and glutamine after
nitrogen deprivation in TAP media with 13-C acetate expressed as fraction of molecules that
contain 13C. (c) & (d) 13C incorporation into glutamine and arginine during the first 6 hours of
nitrogen deprivation when exponentially growing cells are resuspended in nitrogen deprived
TAP media with 13C-acetate. (e) & (f) 13C incorporation into glutamine and arginine after 6
hours of nitrogen deprivation when a pulse of 13C-acetate was added to nitrogen deprived cells in
TAP media. Means (n=3, SE) were shown. White circles: Control Chlamydomonas cells grown
in TAP media with nitrogen. Black squares: Chlamydomonas cells deprived of nitrogen.
Figure S5. Changes of polyamines in C. reinhardtii cw15 with and/or without N starvation.
(a) putrescine measured by GC-MS, means (n=2, SE) are shown. (b) spermidine, (c) spermine
measured by HPLC, means (n=4, SE) are shown.
Figure S6. The response of amino acid metabolism to N deprivation in C. reinhardtii. (a)
Decrease of total amino acids from hydrolyzed proteins under N starvation across the time
course. Means (n=2, SE) are shown. (b) Decrease of total free amino acids under N starvation
across the time course. Means (n=3, SE) are shown.
Figure S7. The response of protein metabolism to N deprivation in C. reinhardtii. Heat map
showing the relationships between 114 protein metabolism proteins and their corresponding
transcripts across the time course under N starvation. Blue means an increase in expression/level
and yellow means a corresponding decrease, with fold-change indicated by color intensity as
shown in the key.
Table S1. Biological properties of cw15 in the N deprivation time course, based on dried cell
weight.
Table S2. Summary statistics for RNA-seq data.
Table S3. Seven of the 10 most up-regulated proteins are involved in nitrogen metabolism.
Data S1. Total RPKM values, and ratios, ratio averages and standard deviations (STD Dev)
relative to 0 h control for all genes detected in the RNA-seq experiments.
Data S2. List of cw15 proteins (3422) that were identified in three biological replicates with
protein FDR of 0.4%, protein probablity of 99% and at least 2 peptides matched to one protein.
Data S3. A total of 933 proteins that were significantly changed across the N deprivation time
course and quantified by iTRAQ labeling are listed.
Data S4. Relative concentrations of metabolites from central metabolim detected the metabolite
profiling experiments.
Data S5. Overview of RNA-seq and quantitative proteomics data related to central metabolism.
Data S6. Overview of RNA-seq and quantitative proteomics data related to lipid biosynthesis.
Data S7. Overview of RNA-seq and quantitative proteomics data related to N metabolism.
Methods S1. Extraction method, sample preparation and data analysis of proteins.