Recovery from N Deprivation Is a Transcriptionally and Functionally Distinct State in Chlamydomonas1 OPEN
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Recovery from N Deprivation Is a Transcriptionally and
Functionally Distinct State in Chlamydomonas1[OPEN]
Chia-Hong Tsai,a,b,2 Sahra Uygun,a,c Rebecca Roston,d,3 Shin-Han Shiu,b,c and Christoph Benning a,b,d,4
a
Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824
b
Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824
c
Genetics Program, Michigan State University, East Lansing, Michigan 48824
d
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824
ORCID IDs: 0000-0002-3063-5002 (R.R.); 0000-0001-6470-235X (S.-H.S.); 0000-0001-8585-3667 (C.B.).
Facing adverse conditions such as nitrogen (N) deprivation, microalgae enter cellular quiescence, a reversible cell cycle arrest
with drastic changes in metabolism allowing cells to remain viable. Recovering from N deprivation and quiescence is an active
and orderly process as we are showing here for Chlamydomonas reinhardtii. We conducted comparative transcriptomics on this
alga to discern processes relevant to quiescence in the context of N deprivation and recovery following refeeding. A mutant with
slow recovery from N deprivation, compromised hydrolysis of triacylglycerols7 (cht7), was included to better define the regulatory
processes governing the respective transitions. We identified an ordered set of biological processes with expression patterns that
showed sequential reversal following N resupply and uncovered acclimation responses specific to the recovery phase.
Biochemical assays and microscopy validated selected inferences made based on the transcriptional analyses. These comprise
(1) the restoration of N source preference and cellular bioenergetics during the early stage of recovery; (2) flagellum-based
motility in the mid to late stage of recovery; and (3) recovery phase-specific gene groups cooperating in the rapid replenishment
of chloroplast proteins. In the cht7 mutant, a large number of programmed responses failed to readjust in a timely manner.
Finally, evidence is provided for the involvement of the cAMP-protein kinase A pathway in gating the recovery. We conclude
that the recovery from N deprivation represents not simply a reversal of processes directly following N deprivation, but a
distinct cellular state.
The ability of cells to withdraw temporarily from the maintenance of tissue homeostasis in multicellular or-
cell division cycle is essential for survival during ad- ganisms. This nondividing state is termed quiescence
verse conditions in unicellular organisms, and for the and distinguished from senescence or terminal differ-
entiation by its reversibility (Gray et al., 2004; Valcourt
1
et al., 2012). Signals that promote quiescence can vary
This work was supported in part by the National Science Foun-
across different cell types. For instance, bacteria and
dation (grant no. MCB-1515169) and by MSU AgBioResearch. Addi-
tional support was provided by a grant from the Chemical Sciences,
yeast enter the stationary phase upon carbon exhaus-
Geosciences, and Biosciences Division, Office of Basic Energy Sci- tion or in response to the deprivation from a specific
ences, Office of Science, U.S. Department of Energy (DE-FG02- nutrient, such as nitrogen (N), sulfur, or phosphate
98ER2035) and National Science Foundation grants DEB-1655386 (Thevelein et al., 2000). Quiescence also occurs in the
and IOS-1546617. context of development. In plant root meristems, stem
2
Current address: Amyris, 5885 Hollis Street, Suite 100, Emery- cells surround a small group of organizing cells, re-
ville, CA 94608. ferred to as the quiescent center (Wildwater et al., 2005).
3
Current address: Department of Biochemistry, University of In mammals, quiescence is seldom induced by starva-
Nebraska-Lincoln, Lincoln, NE 68588.
4 tion; fibroblasts, lymphocytes, and stem cells typically
Address correspondence to benning@msu.edu.
The author responsible for distribution of materials integral to the become quiescent unless they are exposed to prolifer-
findings presented in this article in accordance with the policy de- ative signaling molecules (e.g. mitogens and antigens)
scribed in the Instructions for Authors (www.plantphysiol.org) is: or situational cues (e.g. tissue wounding; Valcourt et al.,
Christoph Benning (benning@msu.edu). 2012). Despite these differences, many quiescence re-
C.-H.T. designed the experiments, conducted the bench experi- sponses appear to be universal, including condensed
ments, analyzed the RNA-seq data, and wrote the first draft of the chromosomes, reduced transcription and translation,
manuscript; S.U. analyzed the RNA-seq data and edited the manu- reduced synthesis of rRNA and ribosomal proteins, and
script; R.R. contributed to the experimental design of the study, an-
high catabolism versus low anabolism (Gray et al.,
alyzed the data, and edited the manuscript; S.-H.S. analyzed the
RNA-seq data and edited the manuscript; C.B. conceived and super-
2004; Wu et al., 2004; Coller et al., 2006; Miller et al.,
vised the study, designed experiments, analyzed the data, and edited 2010; Valcourt et al., 2012; Gifford et al., 2013). A re-
and supervised the writing of the manuscript. markable exception in microbial quiescence is the ac-
[OPEN]
Articles can be viewed without a subscription. cumulation of carbon storage compounds. Examples
www.plantphysiol.org/cgi/doi/10.1104/pp.17.01546 are glycogen, trehalose, and triacylglycerol (TAG) in
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yeast (Lillie and Pringle, 1980; Hosaka and Yamashita, hydrolysis of TAG7 (cht7), has been isolated (Tsai et al.,
1984), wax and polyhydroxyalkanoates in bacteria 2014). CHT7 encodes a putative DNA binding protein.
(Daniel et al., 2004; Kadouri et al., 2005; Sirakova et al., In the absence of CHT7, a fraction of transcriptional
2012), or starch and TAG in microalgae (Moellering and changes that are characteristic for N deprivation-
Benning, 2010). Notably, there is an inverse relationship induced quiescence spontaneously occurs under
between growth and the buildup of carbon stores, N-replete conditions, pointing toward a possible role of
which has long hampered the advancement of indus- CHT7 in governing processes relevant during cellular
trial uses of microorganisms as carbon factories. It ap- quiescence or during the transition between the regular
pears to result from redirection of acetyl-CoA from the cell cycle and quiescence and its reverse.
tricarboxylic acid cycle to the synthesis of fatty acids To better understand how photosynthetic cells
(FAs), which are then stored in the form of TAGs (Baek reinitiate growth and proliferation as they exit quies-
et al., 2011). Yeast mutants unable to produce glycogen cence, we applied N deprivation and resupply to in-
or trehalose persist with high tricarboxylic acid fluxes duce the entry and subsequent exit from quiescence,
during stationary phase (Silljé et al., 1999). The pro- respectively. We undertook comparative tran-
pensity of microorganisms to channel acetyl-CoA into scriptomics of the cht7 mutant and its parental line (PL),
reduced carbon storage compounds after entering and conducted metabolite measurements to validate
quiescence seems to be an almost universal phenome- some of the transcriptomics-based findings.
non accompanying impaired growth.
During quiescence, a plethora of metabolic adjust-
ments has to take place. For example, because quiescent RESULTS
cells do not grow they cannot dilute out reactive oxygen Cytological Parameters for Setting up the RNA-
species (ROS) as readily as actively growing and di- Seq Experiments
viding cells. These are toxic to proteins or other mac-
romolecules that cannot be replaced by rapid Previously, we conducted Illumina RNA sequencing
resynthesis during quiescence. Therefore, quiescent (RNA-seq) on the cht7 mutant and the respective PL
cells require specialized ROS-dissipating mechanisms grown under N-replete (midlog phase of a culture in
to maintain redox homeostasis. Autophagy under most standard Tris-acetate phosphate [TAP] medium) and
conditions is very limited, but is drastically elevated N-deprived (48 h in TAP lacking N) conditions under
during quiescence, allowing for degradation and recy- continuous light (70–80 mmol$m22$s21; Tsai et al.,
cling of cellular components (Gray et al., 2004). For 2014). Here, we expand this study by including two
photosynthetic organisms, there is an additional chal- additional conditions: 6 and 12 h of N resupply fol-
lenge when entering quiescence: to reduce the highly lowing 48 h of N deprivation (abbreviated throughout
redox-susceptible photosynthetic machinery in a way as NR6 and NR12, respectively; Supplemental Fig. S1).
that it can be restored rapidly as conditions improve. The NR6 and NR12 RNA samples were harvested,
These include transcriptional modifications, such as prepared, and sequenced alongside with the N-replete
down-regulation of photosynthetic genes, and protein and N-deprived samples published previously, allow-
degradation, for example of light-harvesting com- ing for accurate cross comparisons. The timing of
plexes, but also the degradation of photosynthetic sampling during N resupply (i.e. NR6 and NR12) was
membrane lipids and subsequent storage of acyl based on observations of cytological changes, and
groups in TAG. In addition, diverting photosynthate to typically falls into the period of key transitions during
complex carbohydrates is believed to be a necessary the recovery from N deprivation. It is known that
adjustment to avoid harmful ROS production in mRNAs are rapidly degraded in dying cells (Thomas
microalgae (Li et al., 2012b; Juergens et al., 2016). et al., 2015). To be certain that dying cells potentially
Conversely, it is plausible to expect highly coordinated arising during N deprivation would not confound
processes that safeguard the successful recovery from subsequent analyses, RNA integrity was examined
quiescence in photosynthetic organisms. However, our with a 2100 Bioanalyzer (Agilent Technologies), and
current understanding of these coordinated recovery every sample had a similar RNA integrity number
mechanisms is lacking. .7.0, indicating that these RNA samples likely cap-
Chlamydomonas reinhardtii, a unicellular green alga, tured the viable cells.
offers several advantages for researching life cycle During N resupply, cell number, cell size distribu-
transitions in photosynthetic eukaryotes. First, the two tion, and changes in DNA ploidy were monitored using
states of interest (i.e. quiescence and cell division) can FACS to test for the uniformity of the cells in culture
be discretely defined and controlled by N availability. and particularly during recovery from N deprivation.
Second, the cell cycle arrest caused by N deprivation At 48 h of N deprivation (0 h of N resupply), PL cells
has all the hallmarks of quiescence, including revers- had a mean volume of 76.4 mm3, which increased to
ibility (Bölling and Fiehn, 2005; Miller et al., 2010; 106.3 mm3 after 9 h of N resupply and to a peak value of
Moellering and Benning, 2010; Work et al., 2010; 122.1 mm3 within the first 12 h (Fig. 1A; Supplemental
Nguyen et al., 2011; Li et al., 2012b; Blaby et al., 2013; Fig. S2). On average, cht7 cells did not enlarge to the
Schmollinger et al., 2014). Third, a mutant showing a same extent as PL cells. Past 12 h post N resupply, PL
delay in recovery from N deprivation, compromised cells began to divide as indicated by an increase in
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Figure 1. Cytological parameters of N-resupplied cells. A and B, Size distribution (A) and cell count (B) of the PL and cht7 cells at
different times (h) following N resupply. C, Relative DNA content of the PL and cht7 following N resupply. 1C and 2C, one copy
and two copies of chromatin content. Five independent biological repeats were examined for (A–C), all showing a similar pattern
with one representative result depicted. Results shown here in A and B are from the same experiment. D, Per cell FA content for
total lipid and TAG following N resupply of the PL. Averages (n = 3) and SD are indicated. E to H, Transmission electron mi-
crographs showing an overview of wild-type CC-125 cells at the indicated time points (h) during N resupply (NR). The darkly
stained vacuole bodies are marked by yellow arrows. I to L, Closer details of subcellular organization of NR12 CC-125 cells.
Physical contact of lipid droplets with the chloroplast outer envelope (I); small lipid droplet fused with the vacuole (J); space
between thylakoid membranes (K); internal degradation of the unknown vacuole body (L). The scale bar is indicated in each
panel. ES, Eyespot; LD, lipid droplet; OE, chloroplast outer envelope; P, pyranoid; SG, starch granule; V, vacuole.
numbers and concomitant decrease in cell size; after cells from the mother cell (Bisova et al., 2005), and
24 h the cell number increased by 161% for PL, but only hence, the DNA content in C. reinhardtii per particle (cell
by 25% for cht7 (Fig. 1B; Supplemental Fig. S2). or mother cell) can be .2C (note that the sum of 1C and
Measurements of DNA content corresponded to the 2C relative DNA content at any given time point does
observation of cell growth. After 48 h of N deprivation, not equal 100%). In the PL, TAG accumulated during N
PL and cht7 cells had similar distributions of DNA deprivation began to be degraded between 6 and 9 h
contents (Fig. 1C; NR0). Following N resupply, the after N resupply (Fig. 1D). By 12 h, about one-half of the
fraction of 1C (13 chromatin content) PL cells gradually TAG was gone; intriguingly, total FA content per cell
decreased and the fraction of 2C (23 chromatin con- remained unchanged. TAG reached a basal steady state
tent) cells increased; in contrast, within the population in the next 6 h and the FA content stabilized afterward.
of cht7, a greater fraction of cells remained at 1C during N deprivation drastically changes the ultrastructure
the observation period. of C. reinhardtii cells, causing vacuolization, replace-
C. reinhardtii undergoes multiple rapid divisions ment of stacked thylakoids by starch granules, and the
bypassing G1 without initially releasing the progeny accumulation of lipid droplets (Moellering and
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Benning, 2010; Yang et al., 2011; Chapman et al., 2012). comparing transcripts of NR6 PL and NR12 PL with
Transmission electron micrographs (TEMs) were taken those of N-deprived PL (for the complete data set, see
over a time course of N resupply to investigate ultra- Supplemental Data Set S1), we found that in response to
structural changes occurring during exit from quies- N resupply many genes reversed their expression as
cence (Fig. 1, E–H; Supplemental Fig. S3, A–H). During seen under N deprivation over time. We divided these
the first 12 h after N resupply, lipid droplets decreased genes into three different phases: early-, mid-, and late-
in size while the surface monolayer remained continu- reverse, respectively (Fig. 2A). Taking the 2,647
ous with the chloroplast outer envelope membrane up-regulated genes during N deprivation as an exam-
(Fig. 1I; Supplemental Fig. S3, I–M). This apparent ple, 1.405 of these reversed at least 2-fold (log2 # 21
membranous continuum between lipid droplet and with a P value , 0.05) at NR6. These were defined as
chloroplast outer envelope membrane forms during early-reverse with 1,309 of the 1,405 genes still at least
lipid droplet formation (Wang et al., 2009), and may 2-fold reversed at NR12. Another 519 genes reversed
enable the transport of proteins and polar lipids back to only at NR12 and were defined as mid-reverse;
the chloroplast (Tsai et al., 2015). Nine to twelve hours 723 genes had not reversed by NR12 and were as-
after N resupply smaller lipid droplets appeared to sumed to reverse at later times and were defined as late-
move toward the vacuole and even entered it (Fig. 1, F, reverse. Likewise, of the 3,346 genes down-regulated
G, and J; Supplemental Fig. S3, N–P). Note that at this during N deprivation, 1,208 (1.136 were still at least
stage, there was no longer a clear delineation between 2-fold reversed at NR12), 991, and 1,147 genes were
lipid droplet and vacuole as compared to the beginning defined as early-, mid-, and late-reverse, respectively
of N resupply (Supplemental Fig. S3Q). This is remi- (Supplemental Data Set S2).
niscent of lipid droplet degradation in plant seeds and To take a global view of the pathways most highly
in budding yeast, where lipid droplet interaction with represented by these data sets, genes were mapped into
vacuoles in a process that resembles microautophagy functional categories based on the MapMan ontology
has been observed (Poxleitner et al., 2006; van Zutphen (Thimm et al., 2004; May et al., 2008) and Gene Ontol-
et al., 2014). Starch granules decreased in quantity and ogy (GO), and the enrichment within the up- and
size (Fig. 1, F, G, and K; Supplemental Fig. S3R). Vac- down-regulated groups was assessed (Fig. 2B;
uoles of N-resupplied cells were smaller compared with Supplemental Data Set S3). The prediction of gene
those of N-deprived cells, which often filled the entire function by MapMan is considered to be more tailored
cytoplasm (Supplemental Fig. S3, A and B). These to plants than the more general GO terms (Klie and
vacuoles typically contained darkly stained round- Nikoloski, 2012). Indeed, MapMan’s findings por-
shaped structures (Fig. 1G, yellow arrows; trayed a clear succession of biological processes.
Supplemental Fig. S3, C and D), which strongly re- Among the 75 functional categories (q value , 0.05)
sembled the protein bodies found in protein storage enriched in the early, mid-, and late-reverse gene sub-
vacuoles (Herman and Larkins, 1999). As time pro- sets, 57 were phase-specific (appeared in only one of the
ceeded, these structures appeared to break down from subsets; Fig. 2B). Early-reverse categories are grouped
the inside (Fig. 1L; Supplemental Fig. S3, S and T). Fi- as follows: (1) Lipid degradation (lipases and b-oxida-
nally, cells were observed to divide before fully tion), central metabolism (gluconeogenesis/glyoxylate
degrading lipid droplets and starch granules as these cycle, glycolysis, and tricarboxylic acid cycle), mito-
were found in new daughter cells (Fig. 1H; chondrial electron transport/ATP synthesis, nucleotide
Supplemental Fig. S3, G and H). metabolism (pyrophosphatase and adenylate kinase),
Based on the observations above, the bulk of and photosynthesis are all related to the restoration of
changes in cell physiology and cell structure of the PL cellular bioenergetics. (2) Transport, amino acid me-
occurred during the first 12 h following N resupply. At tabolism, and tetrapyrrole synthesis (beginning from
NR6 changes in cell structure were clearly visible, Glu), which are processes directly related to N uptake
therefore representing an early stage of N recovery. At and assimilation. Nitrate transport (MapMan bin code
NR12, cells were still undergoing changes, but begin- 34.4) was over-represented among the transcripts that
ning to resume cell divisions at least in the PL. We decreased in abundance, which seems reasonable since
therefore chose NR6 as a representative early stage the N deprivation had ended, and is also consistent
and NR 12 as a representative of a later stage during with the fact that ammonium assimilation–the N source
recovery from N deprivation for subsequent tran- used in this study–suppresses the expression of nitrate
scriptome analyses. reductase genes (Fernández et al., 1989). Notably, genes
involved in potassium transport (MapMan bin code
34.15) were synchronously coordinated even though
Transcriptome Dynamics across N-Replete, N-Deprived, potassium had never been depleted, suggesting a con-
and N-Resupplied Conditions vergent node of nutrient sensing. The nonmevalonate
pathway of isoprenoid biosynthesis (MapMan bin code
Using a sub-data set, we previously showed that 16.1.1) was found among the early-reverse
when PL cells transitioned from N-replete to up-regulated categories. One possible explanation
N-deprived conditions, 2,647 genes were up-regulated could be to provide the prenyl moiety for chlorophyll,
and 3,346 down-regulated (Tsai et al., 2014). By the primary end-product of the tetrapyrrole pathway in
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Figure 2. Summary scheme of transcriptomic analyses across different N regimes. A, Graphic illustration of the early-, mid-, and
late-reverse gene groups in the PL. Numbers represent the transcripts whose abundance changed according to the N status using a
2-fold cutoff (log2 fold change equals to 1) and a P value , 0.05. +N, N-replete; -N, N-deprived; NR6 and NR12, 6 and 12 h of N
resupply, respectively. Numbers associated with the dashed line were inferred. B, Heat map of the overrepresented MapMan
categories in the early-, mid-, late-reverse and NR-specific gene groups. The first column has the bin code of the categories and the
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plants (Eisenreich et al., 2001). (3) Motility-related transcriptomes (using a 2-fold cutoff and a P value ,
processes (MapMan bin code 31.6 and its subcate- 0.05; Fig. 2C). Note that the NR-reverse (Fig. 2A) and
gories). It is still unclear why transcripts associated with NR-specific genes are mutually exclusive by definition,
flagellar assembly are reduced during N deprivation, enabling us to further dissect the nature of the tran-
especially because this treatment also induces the for- scriptomes. Under these criteria, 852 (NR6-specific) and
mation of gametes, which are flagellated (Tsai et al., 347 (NR12-specific) genes were up-regulated after
2014). However, it has been hypothesized that the as- 6 and 12 h of N resupply, respectively, among which
sembly proteins for a primary cilium of vertebrate cells 197 genes overlapped (Supplemental Data Set S4). As
(similar to a flagellum) can also affect cell cycle pro- for the down-regulated genes, there were 547 (NR6-
gression (Pan and Snell, 2007; Snell and Golemis, 2007). specific) and 411 (NR12-specific) with 224 over-
Although motility-related categories are present lapping. A typical example of an NR-specific gene is the
throughout all three phases, there is a clear delineation: one encoding betaine lipid synthase (BTA1), which
genes encoding axonemal outer arm dyneins (MapMan synthesizes diacylglyceryl-trimethylhomo-Ser (DGTS),
bin code 31.6.1.4.1) and radial spoke (MapMan bin code a major lipid component presumed to replace phos-
31.6.1.5) were restricted to early-reverse, basal bodies phatidylcholine in extraplastidic membranes in
(MapMan bin code 31.6.1.1) and intraflagellar transport C. reinhardtii (Riekhof et al., 2005). Transcript levels of
complex genes (MapMan bin code 31.6.1.3) to BTA1 remained constant when entering N deprivation,
mid-reverse, and axonemal inner arm dynein genes but raised approximately 4-fold after 6 h and 12 h of N
(MapMan bin code 31.6.1.4.2) and flagellar associated resupply.
protein genes (MapMan bin code 31.6.1.10) to mid- and In comparison to the NR-reverse data set, the en-
late-reverse. This progression may represent the step- richment of the NR-specific gene sets was much less
wise formation of a eukaryotic flagellum as was also diverse. Altogether, 31 of the total 32 categories
previously observed during the diurnal cycle (Zones (30 NR6-specific and 2 NR12-specific) could be attrib-
et al., 2015). uted to amino acid, RNA, and protein-related processes
The fact that autophagic protein degradation (Map- (Fig. 2B; Supplemental Data Set S5). Only two (Map-
Man bin code 29.5.2) was mid-reverse down-regulated Man bin code 13: amino acid metabolism and 13.1:
rather than early-reverse is counterintuitive given that amino acid synthesis) were redundant with the
protein synthesis occurs immediately after N is resup- NR-reverse categories (excluding those found in the
plied (Tsai et al., 2014), indicating that this process is Late-reverse which were beyond the NR6 and NR12
active not only during N deprivation but also extends to timeline). Apparently, amino acid metabolism is af-
at least the first 6 h following N resupply. It is possible fected in different aspects in the two data sets, that is the
that the autophagic machinery is needed for the deg- NR-reverse set covers Ser (MapMan bin code 13.1.5.1)
radation of lipid droplets in vacuoles as suggested while the NR-specific set covers more the biosynthesis
above (Fig. 1J; Supplemental Fig. S3Q). DNA synthesis of aromatic amino acids (MapMan bin code 13.1.6) and
(MapMan bin code 28.1) found in mid-reverse Asp family amino acids (MapMan bin code 13.1.3). All
up-regulated categories was consistent with the mea- the NR6-specific protein-related categories came from
surement of DNA content (Fig. 1B). Finally, [FeFe]- the up-regulated gene set (852, Fig. 2C). Particularly
hydrogenase (MapMan bin code 1.1.70.1) was found noteworthy are those involved in protein folding
in the only non-early-reverse subcategory of photo- (MapMan bin code 29.6.2: chaperones and cochaper-
synthesis. ones, 29.6.2.2/4: HSP60 and HSP90-like proteins),
prokaryotic ribosomal protein synthesis (e.g. MapMan
Transcriptional Changes Specific to the Recovery Phase bin code 29.2.1.1.1.1/2: chloroplast ribosomal 30S/50S
subunits), and protein targeting (MapMan bin code
Next, we asked whether there were unique tran- 29.3.3: chloroplast targeting and 29.3.3: mitochondria
scriptional changes that were only apparent during the targeting). On the contrary, NR12-specific protein-
N recovery phase, which we called NR-specific. To be related categories were overrepresented in the down-
counted as an NR-specific gene, its relative RNA a- regulated gene set (411, Fig. 2C). Collectively, these
bundance must not fluctuate when shifting from findings suggest a unique demand for cells to rapidly
N-replete to N-deprived condition (21 , log2 , 1), but remake, restructure, and transport the proteins lost
must be either up- or down-regulated in both the NR during N deprivation, especially those of the chloro-
versus N-replete and the NR versus N-deprived plast, to complete the recovery.
Figure 2. (Continued.)
second column lists the explanation of each category. The color in the heat map represents the -log10 of the q-value obtained from
Fisher’s exact test ranging from 1.3 to 10 (0.05–1E-10). The number of genes in each gene group is given in parentheses near the
gene group name in the x axis of the heat map. C, Graphic illustration of the NR-specific gene groups in the PL. Numbers represent
the transcripts whose abundance did not vary in the –N over +N RNA-seq comparison (21 , log2 fold change , 1), but differed
over 2-fold (P value , 0.05) in both NR over +N and NR over –N comparisons.
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Figure 3. Comparative transcriptomics of the PL and the cht7 mutant. A and B, Global gene expression analysis of the PL and cht7
following N resupply. Blue circles: total number of genes changed in expression in a comparison of PL after 48 h of N deprivation
followed by 6 h (A) or 12 h (B) of N resupply over PL N-deprived for 48 h. Yellow circles: total number of genes changed in
expression in a comparison of PL NR6 over cht7 NR6 (A) or PL NR12 over cht7 NR12 (B). NR6 and NR12, 6 and 12 h of N
resupply, respectively. C, Heat map of the overrepresented MapMan categories in the overlapping gene groups as depicted in A
and B. The first column has the bin code of the categories and the second column lists the explanation of each category. The color
in the heat map represents the -log10 of the q-value obtained from Fishers exact test ranging from 1.3 to 10 (0.05–1E-10). The
number of genes in each gene group is given in parentheses near the gene group name in the legend of the heat map.
Acclimation Responses That Failed to Readjust in cht7 N or phosphate deprivation, or rapamycin addition,
during the Recovery Phase treatments which all induce quiescence (Tsai et al.,
CHT7 encodes a putative transcription factor and 2014). It would be expected to see differences in tran-
without it cultures are slow to resume growth following script abundance between cells that undergo orderly
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progression through the recovery phase and cells that
fail to do so. Therefore, to identify specific mis-
regulation of gene expression in cht7 when PL cells
would be recovering from N deprivation, we adopted
an integrated pairwise comparative approach
(González-Ballester et al., 2010; Castruita et al., 2011).
Transcripts of the PL after 6 h and 12 h of N resupply
were compared with those of the N-deprived PL (Fig. 3,
A and B, blue circles); transcripts of the PL after 6 h and
12 h of N resupply were compared with those of cht7
(Fig. 3, A and B, yellow circles). It should be noted that
the blue circles in Figure 3A contain all the early-reverse
and NR6-specific genes mentioned above, and the blue
circles in Figure 3B have all the mid-reverse and NR12-
specific genes. Most transcripts responsive to N
resupply in the PL were readjusted normally in cht7
(Fig. 3, A and B, blue circles outside the overlap).
However, a specific subset of genes did not respond in
cht7 and remained at expression levels that were similar
to those of N-deprived PL cells (Fig. 3, A and B, over-
laps between the blue and yellow circles; Supplemental
Data Set S6). It seems possible that these genes are
regulated by CHT7 and that a subset of them are spe-
cifically needed for the recovery from N deprivation,
whereas the changes in expression of other genes (yel-
low circles outside the overlap) likely reflect secondary
or compensatory effects resulting from the impaired
growth of cht7. Closer examination of the overlapping
gene sets provided supporting evidence. Overall,
60 MapMan categories (35 of NR6 and 25 of NR12) were
significantly enriched (Fig. 3C; Supplemental Data Set
S7), among which 34 have been defined as NR-reverse
(e.g. MapMan bin code 1: photosynthesis and its subcat-
egories, 19: tetrapyrrole synthesis) and 14 as NR-specific
(e.g. MapMan bin code 13.1.6: aromatic amino acid,
29.2.1.1: prokaryotic ribosomal protein synthesis).
As examples, we focused on two MapMan pathways:
tetrapyrrole synthesis, which was among the
NR-reverse categories, but unlike photosynthesis, its
transcript profile was not affected by the loss of CHT7
before the recovery phase (Tsai et al., 2014), and per-
oxisomal redox homeostasis (MapMan bin code 21 and
21.5), which was neither NR-reverse nor NR-specific.
At 6 and 12 h following N resupply, nearly every
tetrapyrrole gene showed lower transcript levels in
cht7 compared with PL (Fig. 4A, top right [included
here are genes that fall below the 2-fold threshold];
N resupply, respectively. For the genes whose expression pattern fol-
Figure 4. Gene expression and metabolite level for two selected lowing N resupply has been classified, the respective categories are
pathways. A, Overview of the expression of genes involved in the tet- indicated on the right of the heat map. Early, Early-reverse; Mid, mid-
rapyrrole pathway (top) and in peroxisomal redox homeostasis (bot- reverse; Late, late-reverse; NR6, NR6-specific; NR12, NR12-specific. B,
tom). RNA-seq comparisons of the PL at different N status and the Chlorophyll content. C1-C4, 4 independent complemented lines. C,
comparisons between PL and cht7 at each N status are shown in the TBARS content. For all quantitative data, averages (n = 3) of biological
heat map. +N, N-replete; -N, N-deprived; NR6 and NR12, 6 and 12 h of replicates and SD are indicated.
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Supplemental Data Set S8). These genes did not greatly Data obtained at the transcript level are insufficient
differ in expression between cht7 and the PL in to provide a cause for the MGDG phenotype.
N-replete or N-deprived conditions, but upon recovery Responding to N resupply, only the genes encoding
from N deprivation. The RNA-seq data were confirmed MGDG-specific desaturases (FAD6, FAD7, and the
for representative genes by quantitative PCR (qPCR) in C16 D4-desaturase) had increased mRNA abundance,
PL and cht7 as well as cht7 complemented lines in this and their expression was normal in cht7 (Fig. 5D;
analysis (Supplemental Fig. S4A). Genes involved in Supplemental Data Set S10). Besides, none of the
maintaining redox homeostasis during high demand of MGDG synthesis genes was a candidate for CHT7-
b-oxidation were down-regulated in cht7 following N specific regulation (found in the overlaps between
resupply (Fig. 4A, bottom right; Supplemental Fig. S4B; the blue and yellow circles in Fig. 3, A and B). The
Supplemental Data Set S9). Catalase 1 and 2, ascorbate inability of cht7 to readjust MGDG may be, at least in
peroxidase 1 and 2, and monodehydroascorbate re- part, a consequence of the delay in TAG turnover,
ductase are enzymes that detoxify the ROS generated as which could normally contribute precursors for
by-product of b-oxidation within the peroxisomes chloroplast lipid assembly.
(Eastmond, 2007). Notably, Arabidopsis (Arabidopsis
thaliana) genes encoding homologs of HPR1, MAS1, Functional Curation of Lipid Metabolism Genes Based on
MDH2, MDH4, monodehydroascorbate reductase 1, Expression Patterns
and PXN1 (all misregulated in cht7 at NR12) cause
mutant phenotypes when disrupted, affecting seed oil RNA-seq resources generated in this study allow us
breakdown and seedling establishment (Graham, to filter and classify functionally ambiguous genes.
2008; Theodoulou and Eastmond, 2012), reminiscent Here we focus on lipid metabolism (Supplemental Data
of the defects of cht7 in TAG turnover and regrowth. Set S10). Lipases are a subclass of acyl hydrolases that
The changes in transcript abundance were corrobo- deesterify carboxylic esters, and can affect TAG me-
rated at the metabolite level. Chlorophyll contents tabolism both positively (e.g. PGD1; Li et al., 2012b) and
of the PL and complementation lines increased after negatively (e.g. LIP1; Li et al., 2012a), as illustrated in
6 h of N resupply, and decreased after 12 h likely Figure 6A. Since TAGs increase during N deprivation
because of cell divisions (Figs. 1C and 4B). In contrast, and decrease following N resupply, we expected that
the chlorophyll content of cht7 remained constant genes encoding TAG-hydrolyzing lipases (e.g. LIP1)
throughout the same period. TBARS, the cellular me- would be down-regulated during N deprivation and
tabolites reflecting the damage caused by ROS, were up-regulated after N resupply (NR-reverse) or just
accumulating in cht7 after transfer to N-replete me- up-regulated after N resupply (NR-specific; Fig. 6B, top
dium (Fig. 4C). left). On the contrary, genes encoding TAG-producing
lipases (e.g. PGD1) would respond in an opposite di-
MGDG Is the Sole Polar Lipid Affected in cht7 Following rection (Fig. 6B, middle left). Following this principle,
N Resupply we sorted through 131 genes predicted to encode a li-
pase, phospholipase, or patatin based on the GXSXG
Earlier we had shown that TAG turnover took place motif common to hydrolases, and assigned 9 TAG-
after 6 h of N resupply, but total FAs did not change hydrolyzing lipases and 23 TAG-producing lipases
until 12 h (Fig. 1D). A possible explanation for the dis- with LIP1 and PGD1 defining their respective class.
crepancy might be that between 6 and 12 h, lipolytic Candidate genes encoding b-oxidation enzymes such
products from TAGs were not subjected to the b-oxi- as ATO1 and acyl-CoA oxidases were coordinated with
dation cycle but used to reassemble membrane lipids, those encoding TAG-hydrolyzing lipases (Fig. 6B, bot-
especially the thylakoid lipids enriched in polyunsatu- tom left), with the exception of the gene for ECH1, a
rated FAs. Indeed, we found that the absolute quantity specialized enoyl-CoA oxidase/isomerase needed for
of 16:4 (carbons: double bonds) and 18:3v3 (the two unsaturated FAs (Goepfert et al., 2008). Notably, Cre17.
major FAs of monogalactosyldiacylglycerol [MGDG]; g707300, Cre06.g265850, Cre03.g195200, and Cre03.
Giroud et al., 1988) did not decrease after N resupply, g152800 (TAG-hydrolyzing) and PGD1, Cre10.g425100,
but 18:1D9 (the signature FA of TAG; Liu et al., 2013) and g9707, and Cre03.g174900 (TAG-producing) were mis-
other FAs did (Fig. 5A; Supplemental Fig. S5, A and B). regulated in cht7 in either or both NR6 and NR12 con-
About 25% of 16:4 and 18:3v3 was stored in TAGs ditions in a way that would cause TAGs to be retained
during N deprivation. While in the PL the relative a- in the cells, making these promising candidates for re-
bundance of 16:4 and 18:3v3 increased gradually fol- verse genetic studies (Fig. 6B, top and middle right).
lowing N resupply, the FA profile of total lipids remained For newly synthesized FAs in the form of acyl-ACP
static in cht7 (Fig. 5B; Supplemental Fig. S5, C and D). Ac- (acyl carrier protein) to be exported out of plant chlo-
cordingly, among all the polar lipids being tested including roplasts, the ACP moiety must be removed by the ac-
DGTS, digalactosyldiacylglycerol, phosphatidylethanola- tivity of acyl-ACP thioesterase, and almost instantly
mine, phosphatidylglycerol, phosphatidylinositol, and long-chain acyl-CoA synthetase activates the resulting
sulfoquinovosyldiacylglycerol, the cht7 mutant was un- free FAs to acyl-CoA so they can be incorporated into
able to restore MGDG following N resupply (Fig. 5C; glycolipids such as TAG (Li-Beisson et al., 2013). Like-
Supplemental Fig. S6, A–E). wise, FAs hydrolyzed from TAGs also need to be
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Figure 5. Lipid analysis and the expression profile of MGDG synthesis genes. A, FA content of total lipid and TAG in the PL following NR at times
indicated (h). B, Relative FA compositions of PL and cht7 following N resupply. FAs are designated by the total carbon number followed by the number
of double bonds. The position of specific double bonds is indicated either from the carboxyl end “D” or from the methyl end “v.” C, Polar lipid contents
in the presence (+N, N-replete) or absence (2N, N-deprived) of N, or following N resupply at times indicated. The y axis is depicted as the ratio of
individual polar lipid FAs over total FAs. Averages (n = 4) of biological replicates and SD are indicated. D, Overview of the expression of genes
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converted to acyl-CoA prior to b-oxidation. Two iso- et al., 2007), and their activities are counteracted by cyclic
forms of long-chain acyl-CoA synthetase exist in the C. nucleotide phosphodiesterases that turn cAMP into
reinhardtii genome, LACS1, and LACS2. Transcripts of AMP. Stimulating the activity of phosphodiesterase at-
LACS1 increased in abundance when shifting to tenuates cAMP-mediated lipolysis (Botion and Green,
N-deprived medium and recovered when the condition 1999). At a glance, many of the candidate genes were
was reversed; those of LACS2 reacted just the opposite differentially regulated by N availability and by the loss
(Supplemental Data Set S10). It is thus likely that of CHT7 (Supplemental Data Set S11). Here we assigned
LACS1 works in tandem with acyl-ACP thioesterase, potential adenylyl cyclases and phosphodiesterases
and LACS2 channels precursors into b-oxidation. Di- whose expression profile matched the observed fluctu-
acylglycerol acyltransferase (type 2, DGTT) is a key ation of cAMP (Fig. 7D).
enzyme for TAG biosynthesis. Of the five putative
candidates, only the expression of genes encoding
DGTT1 and DGTT5 paralleled the accumulation of TAG. DISCUSSION
Expression of the gene for DGTT4 was NR-specific with
a near 4-fold mRNA increase at 6 and 12 h of N resupply, To grow or not is a fundamental decision that every
the time that TAGs were being degraded. The seemingly cell has to make in response to developmental, meta-
conflicting finding may be reconciled by hypothesizing bolic, or environmental stimuli. Based on this decision,
that during recovery from N deprivation there is a need cells either progress through the cell division cycle or
to fine-tune FA production and sequestration into TAG enter into quiescence. While yeast offers a well-studied
to avoid the toxicity of free FAs or that nontranscrip- model of quiescence, fairly little is known in eukaryota
tional regulation comes into play, which is not consid- outside of fungi and certain mammalian cell lines,
ered here. photosynthetic eukaryotes in particular. From a bio-
logical standpoint, the reversible cessation of growth
A cAMP-Dependent Protein Kinase Pathway Is Required depending on nutrient availability provides a facile
for Quiescence Exit experimental system to study quiescence. N depriva-
tion is thus far the most effective way to induce the
Adenylyl cyclase converts ATP to cAMP, and bind- accumulation of neutral lipids in microalgae, for ex-
ing of cAMP to the regulatory domains of protein ki- perimental purposes to study quiescence-related phe-
nase A (PKA) facilitates the phosphorylation of diverse nomena or for practical reasons in developing algae as a
enzyme targets. In yeast, the PKA signaling cascade renewable energy source (Hu et al., 2008). Cellular re-
negatively affects quiescence (Gray et al., 2004). sponses to N deprivation have been studied on multiple
Therefore, we asked whether PKA could have an im- -omic levels, drawing an integrated picture of N econ-
pact on quiescence in C. reinhardtii. Competitive ELISA omy (Miller et al., 2010; Blaby et al., 2013; Schmollinger
showed that concentrations of cAMP responded to the et al., 2014; Wase et al., 2014). In contrast, research on
presence and absence of N (Fig. 7A), a prerequisite for a the recovery from N deprivation has lagged behind.
possible role of PKA activity during recovery from N Here, we used a systems biology approach to address
deprivation. To verify this hypothesis, we took a the question of how photosynthetic cells recover from
pharmacological approach. A 20-amino acid fragment N deprivation to begin to understand mechanism in-
of a naturally occurring PKA inhibitor (PKI) is known volved in quiescence exit, using C. reinhardtii as a ref-
to bind and inhibit the catalytic domain of PKA erence model. The use of the cht7 mutant in
(Knighton et al., 1991). Derivatives of this fragment comparative transcriptomics helped to reduce noise
have been used to study flagellar assembly in C. rein- and unravel the transcriptional patterns potentially
hardtii (Howard et al., 1994). When applied simulta- relevant to the resumption of growth and proliferation.
neously with N refeeding, PKI interfered with TAG While our understanding of how CHT7 affects cell vi-
turnover in the PL in a dosage-dependent manner (Fig. 7B). ability and proliferation in response to different N re-
FA profiles of PL cells treated with PKI resembled those gimes is only in its infancy, these data provide additional
of nontreated cht7 (Supplemental Fig. S7). Addition of insights into the function of this potential regulator of
10 mM of PKI caused severe chlorosis indicative of cell quiescence-relevant transcriptional programs.
death, and no degradation of TAG was observed in the
PL or cht7. Importantly, within the nontoxic range (0 to Quiescence Exit Is Not Simply the Reverse of
5 mM), PKI did not exacerbate the lipolytic defect in cht7. Quiescence Entry
PKI treatment also mimicked the slow regrowth of cht7
in the PL (Fig. 7C). Ultimately, cells recovering from N deprivation-
Adenylyl (and guanylyl) cyclases form one of the induced quiescence return to the G1 phase of the cell
largest families in the genome of C. reinhardtii (Merchant cycle. However, recovery from N deprivation is hardly
Figure 5. (Continued.)
responsible for MGDG synthesis. RNA-seq comparisons of PL at different N status and the comparisons between PL and cht7 at each N status are shown
in the heat map. Arrows indicate the sequence of reactants. FAs at the sn-1/sn-2 position of diacylglycerol (DAG) or MGDG are shown.
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the exact reversal of the processes encountered while
cells become N deprived. We categorized genes that
reversed their expression in response to N resupply into
early-, mid-, and late-reverse groups, implicating pri-
orities of transcriptional reprogramming. Early- and
mid-reverse groups are more likely to be responsible for
restarting the cell cycle, as they coincided with the time
that cells began to proliferate. These unique temporal
patterns of expression suggest that the recovery from N
deprivation including the transition to the resumption
of the cell cycle is subject to an ordered set of sequential
events. In an emerging model of microbial quiescence,
growth-limiting conditions appear to trigger a common
pathway that reduces growth by redirecting the carbon
fluxes away from the central metabolic pathways and
toward storage depots (Rittershaus et al., 2013). This is
especially the case for microalgae. Nutrient starvation
(e.g. N, phosphate, sulfur, zinc, and iron), high salt, heat
shock, and oxidative stress are all able to cause TAG
accumulation (Hu et al., 2008; Matthew et al., 2009;
Kropat et al., 2011; Siaut et al., 2011; Hemme et al.,
2014), and TAG utilization is required for regrowth
(Tsai et al., 2014). We curated every putative lipase, and
of course, uncovered many that had reversed expres-
sion patterns (Fig. 6). Importantly, we also identified
genes whose transcript abundance only fluctuated
during the time of N recovery, termed NR6- and NR12-
specific groups. This discovery provides direct evi-
dence that the expression profile of N-resupplied cells
exiting quiescence, is distinct from that of N-deprived
quiescent cells or N-replete cells, which are mostly in
the G1 phase of the cell cycle. TEM also captured key
morphological distinctions between newly dividing
N-resupplied and N-replete cells, showing that grow-
ing cells after N resupply retained small lipid droplets
and starch granules, and their thylakoids were not fully
stacked (Fig. 1H; Supplemental Fig. S3). However,
what happens in cells during recovery from N depri-
vation that causes a delay before they can undergo
genome replication and mitosis compared with the cells
that actively traverse the cell division cycle? A 6- to 8-h
doubling time for regular cycling cells was lengthened
to 12 to 15 h counting from the moment that N was
refed (Fig. 1B), or even longer if cells were N-deprived
for long periods (Tsai et al., 2015). Aside from a re-
duction in viability during prolonged N deprivation, it
seems likely based on the current data that this delay
relates to the reorganization of metabolism. Indeed,
much of the provided ontology analysis detected met-
Figure 6. Gene expression of putative lipase and b-oxidation genes. A,
An example depicting how lipases might positively or negatively in-
abolic processes related to the synthesis of macromol-
fluence TAG content. MAG, monoacylglycerol; PGD1 and LIP1, li- ecules (nucleotides, amino acids, and proteins), cellular
pases. B, Transcript profiles of genes encoding lipases possibly involved bioenergetics (central metabolism, photosynthesis, mi-
in the degradation of TAG (top), in the production of TAG (middle), or in tochondrial electron transport, and ATP synthesis),
b-oxidation (bottom). RNA-seq comparisons of the PL at different N cellular components (lipids and chlorophylls), nutrient
status and the comparisons between PL and cht7 at each N status are assimilation (nitrogen and phosphate), and redox ho-
shown in the heat map. +N, N-replete; 2N, N-deprived; NR6 and meostasis. Perhaps the most intriguing finding was that
NR12, 6 and 12 h of N resupply. For the genes whose expression pattern the MapMan categories enriched in NR-specific genes
following N resupply has been classified, the respective categories are appeared to center on the replenishment of chloroplast
indicated on the right of the heat map. Early, Early-reverse; Mid, mid-
proteins. This finding fits nicely with reports that
reverse; Late, late-reverse; NR6, NR6-specific; NR12, NR12-specific.
chloroplast ribosomes, specific photosynthetic electron
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Figure 7. The effect of the cAMP-PKA pathway on the recovery from N deprivation. A, ELISA assay to quantify the cellular content
of cAMP in the PL. +N, N-replete; 2N, N-deprived; NR, N resupply at times indicated (h). Asterisks indicate a statistically sig-
nificant difference (unpaired t test, P , 0.05). B, TAG degradation of PL and cht7 in the presence of PKI. The TAG content (depicted
as the ratio of TAG FA over total FA) of cells just before N resupply (designated here as NR0) is shown on the far left. TAG contents
of cells treated with different concentrations of PKI (as indicated in the x axis) were quantified at 24 h of N resupply (NR24). C, The
regrowth of PL and cht7 in the presence of PKI. The fold change of regrowth is calculated by dividing the cell count measured at
24 h of N resupply by that at 0 h of N resupply (NR24/NR0). PKI treatments in B and C were done simultaneously with N resupply.
D, Transcript profiles of genes encoding putative adenylyl cyclase and phosphodiesterase. RNA-seq comparisons of PL at different
N status and the comparisons between PL and cht7 at each N status are shown in the heat map. For the genes whose expression
pattern following N resupply has been classified, the respective categories are indicated on the right of the heat map. Early, Early-
reverse; Mid, mid-reverse; Late, late-reverse; NR6, NR6-specific; NR12, NR12-specific. For all quantitative data, averages (n = 3)
and SD are indicated.
transfer complexes, plastid ATPase, and Calvin-Benson chlorophylls, both confined to the chloroplast, also
cycle enzymes especially Rubisco, are more actively reflected a scenario of rebuilding the chloroplast (Figs.
targeted by N-sparing mechanism during N depriva- 4C and 5C). Thus, the time delay required by recovering
tion in C. reinhardtii (Gray et al., 2004; Schmollinger cells might be to restore chloroplast integrity, which
et al., 2014). Note that elevated protein abundance of represents an important distinction between photo-
mitochondrial ATP synthase and the mitochondrial bc1 synthetic eukaryotes on one hand and yeast and
complex was apparent. Measurements of MGDG and mammalian cells on the other.
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How Does CHT7 Facilitate the Recovery from response to N resupply the cAMP-PKA pathway trig-
N Deprivation? gers TAG breakdown by phosphorylating protein an-
alogs found in adipocytes, which in turn fuels the
On one hand, 34 NR-reverse categories (MapMan) recovery or acts on nuclear targets such as CHT7 to
failed to revert back to the state prior to N deprivation stimulate gene expression that promotes growth. Bio-
in N-resupplied cht7. This seems to support the notion chemical studies have confirmed the presence of PKA
that CHT7 contributes to the reversal of the quiescent catalytic subunits in C. reinhardtii although the respec-
state by acting as its suppressor. On the other hand, the tive genes remain uncertain (Howard et al., 1994).
finding of 14 NR-specific gene expression categories Continued study of cAMP-PKA pathway genes (e.g.
that appeared in N-resupplied cht7 as if cells were still the listed candidates in Fig. 7D) will shed light on the
under N deprivation suggests that CHT7 has addi- role of this pathway in C. reinhardtii quiescence exit.
tional, specific functions during the recovery from N For the bioindustry using microorganisms to pro-
deprivation. The discussed examples of misregulated duce drugs, biofuels, nutritional supplements, flavors
pathways (i.e. tetrapyrrole synthesis and peroxisomal and fragrances, control of nutrient deprivation-induced
redox homeostasis) represent just a fraction of genes quiescence is at the core of research that strives to gain
affected in their expression by CHT7. Particularly insights into the inverse relationship between biomass
important is that the expression of genes affecting production and production of the target compound.
these pathways was completely normal in the cht7 Thus, the information gathered here has practical im-
mutant at any other stage outside of N recovery. We plications for the engineering and cultivation of pho-
hypothesize that when cells receive signals to recover tosynthetic algae, and potentially more broadly to crop
from N deprivation, CHT7 governs some transcrip- plants. Due to their sessile nature, plants are continu-
tional programs, directly or indirectly, that allow the ously exposed to biotic and abiotic stresses that prompt
resumption of growth and proliferation. This hy- cells to arrest growth and division to spare resources for
pothesis can be addressed through the identification of respective defense responses. Understanding how to
the in vivo chromatin binding sites for CHT7 or CHT7- manipulate the balance between growth versus defense
containing complexes under different growth condi- based on insights into the regulation of cellular quies-
tions. A future integrative analysis of the tran- cence may one day help in improving crop yields in
scriptome and chromatin binding data will help agricultural settings.
distinguish the genes that are primary or secondary
targets of CHT7, as well as clarify the potential feed-
back regulations by metabolite levels.
MATERIALS AND METHODS
Strains and Growth Conditions
Interaction between CHT7 and Other Regulatory Modules The Chlamydomonas reinhardtii dw15.1 (cw15, nit1, mt+) or CC-4619 (http://
of Quiescence chlamycollection.org/strain/cc-4619-cw15-nit1-mt-dw15-1/) strain was
obtained from Arthur Grossman and is referred to as the wild type (with regard
In yeast, the cAMP-PKA pathway is active during to CHT7) PL throughout. A cell-walled strain CC-125 obtained from Chlamy-
abundance of nutrients and represses aspects of domonas Resource Center (http://www.chlamycollection.org) was used for
TEM. The four independent complemented lines of cht7 were generated as
nutrient deprivation-induced quiescence by targeting previously described (Tsai et al., 2014). Growth conditions and media were as
nutrient-sensitive transcription factors MSN2 and previously described (Tsai et al., 2014). For N deprivation, mid-log-phase cells
MSN4 (Smith et al., 1998; Beck and Hall, 1999). The grown in TAP were collected by centrifugation (2,000g, 4°C, 2 min), washed
cAMP signal is also required for a timely recovery, as twice with TAP-N (NH4Cl omitted from TAP), and resuspended in TAP-N at
0.3 OD550. N was resupplied by adding 1% culture volume of 1 M NH4Cl (1003)
mutants unable to transiently elevate cAMP levels fol-
to the N-deprived culture. The size and concentration of cells in all assays was
lowing the addition of Glc to starved cells show ex- monitored using a Z2 Coulter Counter.
tended delays in resuming growth (Jiang et al., 1998).
This is somewhat similar to the observed increase of
cAMP following N resupply (Fig. 7A). Furthermore, we Lipid Analysis
showed that PKI-treated PL cells recapitulated both the Lipid extraction, TLC, fatty acid methyl ester preparation, and gas chro-
regrowth and TAG phenotypes of cht7, presumably matography were conducted as previously described (Tsai et al., 2015) with
due to the deactivation of PKA (Fig. 7, B and C). To our modifications. For neutral lipids, 5 mL of cell culture was pelleted and extracted
surprise, when cht7 cells were treated with nontoxic into 1 mL of methanol and chloroform (2:1 v/v). To this extract 0.5 mL 0.9%
KCL were added and the suspension was vortexed, followed by phase sepa-
concentrations of PKI we did not observe deterioration, ration at 3,000g centrifugation for 3 min. For polar lipids, 10 mL of culture was
suggesting some level of functional redundancy extracted with methanol-chloroform-88% formic acid (2:1:0.1 v/v/v) followed
between the CHT7 and cAMP-PKA pathways. In by phase separation with 1 M KCl and 0.2 M H3PO4. Lipid species were separated
adipocytes, adenylyl cyclase and PKA transduce by TLC on Silica G60 plates (EMD Chemicals) developed in petroleum ether-
signals between hormone binding to cells and lipo- diethyl ether-acetic acid (80:20:1 v/v/v, for neutral lipids) or chloroform-
methanol-acetic acid-distilled water (75:13:9:3 v/v/v/v, for polar lipids). Af-
lytic responses. Upon activation, PKA phosphorylates ter brief exposure to iodine vapor for visualization of lipids, fatty acid methyl
hormone-sensitive lipase and perilipin 1 (Guo et al., esters of each lipid or total cellular lipid were processed and quantified by gas
2009). This raises the question, whether during the chromatography as previously described (Rossak et al., 1997).
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