Recovery from N Deprivation Is a Transcriptionally andFunctionally 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 Benninga,b,d,4

aDepartment of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824bDepartment of Plant Biology, Michigan State University, East Lansing, Michigan 48824cGenetics Program, Michigan State University, East Lansing, Michigan 48824dDepartment 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 arrestwith drastic changes in metabolism allowing cells to remain viable. Recovering from N deprivation and quiescence is an activeand orderly process as we are showing here for Chlamydomonas reinhardtii. We conducted comparative transcriptomics on thisalga to discern processes relevant to quiescence in the context of N deprivation and recovery following refeeding. A mutant withslow recovery from N deprivation, compromised hydrolysis of triacylglycerols7 (cht7), was included to better define the regulatoryprocesses governing the respective transitions. We identified an ordered set of biological processes with expression patterns thatshowed 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-basedmotility in the mid to late stage of recovery; and (3) recovery phase-specific gene groups cooperating in the rapid replenishmentof 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 concludethat the recovery from N deprivation represents not simply a reversal of processes directly following N deprivation, but adistinct cellular state.

The ability of cells to withdraw temporarily from thecell division cycle is essential for survival during ad-verse conditions in unicellular organisms, and for the

maintenance of tissue homeostasis in multicellular or-ganisms. This nondividing state is termed quiescenceand distinguished from senescence or terminal differ-entiation by its reversibility (Gray et al., 2004; Valcourtet al., 2012). Signals that promote quiescence can varyacross different cell types. For instance, bacteria andyeast enter the stationary phase upon carbon exhaus-tion or in response to the deprivation from a specificnutrient, such as nitrogen (N), sulfur, or phosphate(Thevelein et al., 2000). Quiescence also occurs in thecontext of development. In plant root meristems, stemcells surround a small group of organizing cells, re-ferred to as the quiescent center (Wildwater et al., 2005).In mammals, quiescence is seldom induced by starva-tion; fibroblasts, lymphocytes, and stem cells typicallybecome quiescent unless they are exposed to prolifer-ative signaling molecules (e.g. mitogens and antigens)or situational cues (e.g. tissuewounding; Valcourt et al.,2012). Despite these differences, many quiescence re-sponses appear to be universal, including condensedchromosomes, reduced transcription and translation,reduced synthesis of rRNA and ribosomal proteins, andhigh catabolism versus low anabolism (Gray et al.,2004; Wu et al., 2004; Coller et al., 2006; Miller et al.,2010; Valcourt et al., 2012; Gifford et al., 2013). A re-markable exception in microbial quiescence is the ac-cumulation of carbon storage compounds. Examplesare glycogen, trehalose, and triacylglycerol (TAG) in

1 This work was supported in part by the National Science Foun-dation (grant no. MCB-1515169) and by MSU AgBioResearch. Addi-tional support was provided by a grant from the Chemical Sciences,Geosciences, and Biosciences Division, Office of Basic Energy Sci-ences, Office of Science, U.S. Department of Energy (DE-FG02-98ER2035) and National Science Foundation grants DEB-1655386and IOS-1546617.

2 Current address: Amyris, 5885 Hollis Street, Suite 100, Emery-ville, CA 94608.

3 Current address: Department of Biochemistry, University ofNebraska-Lincoln, Lincoln, NE 68588.

4 Address correspondence to benning@msu.edu.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Christoph Benning (benning@msu.edu).

C.-H.T. designed the experiments, conducted the bench experi-ments, analyzed the RNA-seq data, and wrote the first draft of themanuscript; S.U. analyzed the RNA-seq data and edited the manu-script; R.R. contributed to the experimental design of the study, an-alyzed the data, and edited the manuscript; S.-H.S. analyzed theRNA-seq data and edited the manuscript; C.B. conceived and super-vised the study, designed experiments, analyzed the data, and editedand supervised the writing of the manuscript.

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yeast (Lillie and Pringle, 1980; Hosaka and Yamashita,1984), wax and polyhydroxyalkanoates in bacteria(Daniel et al., 2004; Kadouri et al., 2005; Sirakova et al.,2012), or starch and TAG inmicroalgae (Moellering andBenning, 2010). Notably, there is an inverse relationshipbetween growth and the buildup of carbon stores,which has long hampered the advancement of indus-trial uses of microorganisms as carbon factories. It ap-pears to result from redirection of acetyl-CoA from thetricarboxylic acid cycle to the synthesis of fatty acids(FAs), which are then stored in the form of TAGs (Baeket al., 2011). Yeast mutants unable to produce glycogenor trehalose persist with high tricarboxylic acid fluxesduring stationary phase (Silljé et al., 1999). The pro-pensity of microorganisms to channel acetyl-CoA intoreduced carbon storage compounds after enteringquiescence seems to be an almost universal phenome-non accompanying impaired growth.

During quiescence, a plethora of metabolic adjust-ments has to take place. For example, because quiescentcells do not grow they cannot dilute out reactive oxygenspecies (ROS) as readily as actively growing and di-viding cells. These are toxic to proteins or other mac-romolecules that cannot be replaced by rapidresynthesis during quiescence. Therefore, quiescentcells require specialized ROS-dissipating mechanismsto maintain redox homeostasis. Autophagy under mostconditions is very limited, but is drastically elevatedduring quiescence, allowing for degradation and recy-cling of cellular components (Gray et al., 2004). Forphotosynthetic organisms, there is an additional chal-lenge when entering quiescence: to reduce the highlyredox-susceptible photosynthetic machinery in a waythat it can be restored rapidly as conditions improve.These include transcriptional modifications, such asdown-regulation of photosynthetic genes, and proteindegradation, for example of light-harvesting com-plexes, but also the degradation of photosyntheticmembrane lipids and subsequent storage of acylgroups in TAG. In addition, diverting photosynthate tocomplex carbohydrates is believed to be a necessaryadjustment to avoid harmful ROS production inmicroalgae (Li et al., 2012b; Juergens et al., 2016).Conversely, it is plausible to expect highly coordinatedprocesses that safeguard the successful recovery fromquiescence in photosynthetic organisms. However, ourcurrent understanding of these coordinated recoverymechanisms is lacking.

Chlamydomonas reinhardtii, a unicellular green alga,offers several advantages for researching life cycletransitions in photosynthetic eukaryotes. First, the twostates of interest (i.e. quiescence and cell division) canbe discretely defined and controlled by N availability.Second, the cell cycle arrest caused by N deprivationhas all the hallmarks of quiescence, including revers-ibility (Bölling and Fiehn, 2005; Miller et al., 2010;Moellering and Benning, 2010; Work et al., 2010;Nguyen et al., 2011; Li et al., 2012b; Blaby et al., 2013;Schmollinger et al., 2014). Third, a mutant showing adelay in recovery from N deprivation, compromised

hydrolysis of TAG7 (cht7), has been isolated (Tsai et al.,2014). CHT7 encodes a putative DNA binding protein.In the absence of CHT7, a fraction of transcriptionalchanges that are characteristic for N deprivation-induced quiescence spontaneously occurs underN-replete conditions, pointing toward a possible role ofCHT7 in governing processes relevant during cellularquiescence or during the transition between the regularcell cycle and quiescence and its reverse.

To better understand how photosynthetic cellsreinitiate growth and proliferation as they exit quies-cence, we applied N deprivation and resupply to in-duce the entry and subsequent exit from quiescence,respectively. We undertook comparative tran-scriptomics of the cht7mutant and its parental line (PL),and conducted metabolite measurements to validatesome of the transcriptomics-based findings.

RESULTS

Cytological Parameters for Setting up the RNA-Seq Experiments

Previously, we conducted Illumina RNA sequencing(RNA-seq) on the cht7 mutant and the respective PLgrown under N-replete (midlog phase of a culture instandard Tris-acetate phosphate [TAP] medium) andN-deprived (48 h in TAP lacking N) conditions undercontinuous light (70–80 mmol$m22$s21; Tsai et al.,2014). Here, we expand this study by including twoadditional conditions: 6 and 12 h of N resupply fol-lowing 48 h of N deprivation (abbreviated throughoutas NR6 and NR12, respectively; Supplemental Fig. S1).The NR6 and NR12 RNA samples were harvested,prepared, and sequenced alongside with the N-repleteand N-deprived samples published previously, allow-ing for accurate cross comparisons. The timing ofsampling during N resupply (i.e. NR6 and NR12) wasbased on observations of cytological changes, andtypically falls into the period of key transitions duringthe recovery from N deprivation. It is known thatmRNAs are rapidly degraded in dying cells (Thomaset al., 2015). To be certain that dying cells potentiallyarising during N deprivation would not confoundsubsequent analyses, RNA integrity was examinedwith a 2100 Bioanalyzer (Agilent Technologies), andevery sample had a similar RNA integrity number.7.0, indicating that these RNA samples likely cap-tured the viable cells.

During N resupply, cell number, cell size distribu-tion, and changes in DNAploidyweremonitored usingFACS to test for the uniformity of the cells in cultureand particularly during recovery from N deprivation.At 48 h of N deprivation (0 h of N resupply), PL cellshad a mean volume of 76.4 mm3, which increased to106.3 mm3 after 9 h of N resupply and to a peak value of122.1 mm3 within the first 12 h (Fig. 1A; SupplementalFig. S2). On average, cht7 cells did not enlarge to thesame extent as PL cells. Past 12 h post N resupply, PLcells began to divide as indicated by an increase in

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numbers and concomitant decrease in cell size; after24 h the cell number increased by 161% for PL, but onlyby 25% for cht7 (Fig. 1B; Supplemental Fig. S2).Measurements of DNA content corresponded to the

observation of cell growth. After 48 h of N deprivation,PL and cht7 cells had similar distributions of DNAcontents (Fig. 1C; NR0). Following N resupply, thefraction of 1C (13 chromatin content) PL cells graduallydecreased and the fraction of 2C (23 chromatin con-tent) cells increased; in contrast, within the populationof cht7, a greater fraction of cells remained at 1C duringthe observation period.C. reinhardtii undergoes multiple rapid divisions

bypassing G1 without initially releasing the progeny

cells from the mother cell (Bisova et al., 2005), andhence, the DNA content inC. reinhardtii per particle (cellor mother cell) can be.2C (note that the sum of 1C and2C relative DNA content at any given time point doesnot equal 100%). In the PL, TAG accumulated during Ndeprivation began to be degraded between 6 and 9 hafter N resupply (Fig. 1D). By 12 h, about one-half of theTAG was gone; intriguingly, total FA content per cellremained unchanged. TAG reached a basal steady statein the next 6 h and the FA content stabilized afterward.

N deprivation drastically changes the ultrastructureof C. reinhardtii cells, causing vacuolization, replace-ment of stacked thylakoids by starch granules, and theaccumulation of lipid droplets (Moellering and

Figure 1. Cytological parameters of N-resupplied cells. A and B, Size distribution (A) and cell count (B) of the PL and cht7 cells atdifferent times (h) following N resupply. C, Relative DNA content of the PL and cht7 following N resupply. 1C and 2C, one copyand two copies of chromatin content. Five independent biological repeats were examined for (A–C), all showing a similar patternwith one representative result depicted. Results shown here in A and B are from the same experiment. D, Per cell FA content fortotal 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 darklystained 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); spacebetween thylakoid membranes (K); internal degradation of the unknown vacuole body (L). The scale bar is indicated in eachpanel. ES, Eyespot; LD, lipid droplet; OE, chloroplast outer envelope; P, pyranoid; SG, starch granule; V, vacuole.

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Benning, 2010; Yang et al., 2011; Chapman et al., 2012).Transmission electron micrographs (TEMs) were takenover a time course of N resupply to investigate ultra-structural changes occurring during exit from quies-cence (Fig. 1, E–H; Supplemental Fig. S3, A–H). Duringthe first 12 h after N resupply, lipid droplets decreasedin size while the surface monolayer remained continu-ous with the chloroplast outer envelope membrane(Fig. 1I; Supplemental Fig. S3, I–M). This apparentmembranous continuum between lipid droplet andchloroplast outer envelope membrane forms duringlipid droplet formation (Wang et al., 2009), and mayenable the transport of proteins and polar lipids back tothe chloroplast (Tsai et al., 2015). Nine to twelve hoursafter N resupply smaller lipid droplets appeared tomove toward the vacuole and even entered it (Fig. 1, F,G, and J; Supplemental Fig. S3, N–P). Note that at thisstage, there was no longer a clear delineation betweenlipid droplet and vacuole as compared to the beginningof N resupply (Supplemental Fig. S3Q). This is remi-niscent of lipid droplet degradation in plant seeds andin budding yeast, where lipid droplet interaction withvacuoles in a process that resembles microautophagyhas been observed (Poxleitner et al., 2006; van Zutphenet al., 2014). Starch granules decreased in quantity andsize (Fig. 1, F, G, and K; Supplemental Fig. S3R). Vac-uoles of N-resupplied cells were smaller comparedwiththose of N-deprived cells, which often filled the entirecytoplasm (Supplemental Fig. S3, A and B). Thesevacuoles typically contained darkly stained round-shaped structures (Fig. 1G, yellow arrows;Supplemental Fig. S3, C and D), which strongly re-sembled the protein bodies found in protein storagevacuoles (Herman and Larkins, 1999). As time pro-ceeded, these structures appeared to break down fromthe inside (Fig. 1L; Supplemental Fig. S3, S and T). Fi-nally, cells were observed to divide before fullydegrading lipid droplets and starch granules as thesewere found in new daughter cells (Fig. 1H;Supplemental Fig. S3, G and H).

Based on the observations above, the bulk ofchanges in cell physiology and cell structure of the PLoccurred during the first 12 h following N resupply. AtNR6 changes in cell structure were clearly visible,therefore representing an early stage of N recovery. AtNR12, cells were still undergoing changes, but begin-ning to resume cell divisions at least in the PL. Wetherefore chose NR6 as a representative early stageand NR 12 as a representative of a later stage duringrecovery from N deprivation for subsequent tran-scriptome analyses.

Transcriptome Dynamics across N-Replete, N-Deprived,and N-Resupplied Conditions

Using a sub-data set, we previously showed thatwhen PL cells transitioned from N-replete toN-deprived conditions, 2,647 genes were up-regulatedand 3,346 down-regulated (Tsai et al., 2014). By

comparing transcripts of NR6 PL and NR12 PL withthose of N-deprived PL (for the complete data set, seeSupplemental Data Set S1), we found that in response toN resupply many genes reversed their expression asseen under N deprivation over time. We divided thesegenes into three different phases: early-, mid-, and late-reverse, respectively (Fig. 2A). Taking the 2,647up-regulated genes during N deprivation as an exam-ple, 1.405 of these reversed at least 2-fold (log2 # 21with a P value , 0.05) at NR6. These were defined asearly-reverse with 1,309 of the 1,405 genes still at least2-fold reversed at NR12. Another 519 genes reversedonly at NR12 and were defined as mid-reverse;723 genes had not reversed by NR12 and were as-sumed to reverse at later times andwere defined as late-reverse. Likewise, of the 3,346 genes down-regulatedduring N deprivation, 1,208 (1.136 were still at least2-fold reversed at NR12), 991, and 1,147 genes weredefined as early-, mid-, and late-reverse, respectively(Supplemental Data Set S2).

To take a global view of the pathways most highlyrepresented by these data sets, genes were mapped intofunctional categories based on the MapMan ontology(Thimm et al., 2004; May et al., 2008) and Gene Ontol-ogy (GO), and the enrichment within the up- anddown-regulated groups was assessed (Fig. 2B;Supplemental Data Set S3). The prediction of genefunction by MapMan is considered to be more tailoredto plants than the more general GO terms (Klie andNikoloski, 2012). Indeed, MapMan’s findings por-trayed a clear succession of biological processes.Among the 75 functional categories (q value , 0.05)enriched in the early, mid-, and late-reverse gene sub-sets, 57 were phase-specific (appeared in only one of thesubsets; Fig. 2B). Early-reverse categories are groupedas follows: (1) Lipid degradation (lipases and b-oxida-tion), central metabolism (gluconeogenesis/glyoxylatecycle, glycolysis, and tricarboxylic acid cycle), mito-chondrial electron transport/ATP synthesis, nucleotidemetabolism (pyrophosphatase and adenylate kinase),and photosynthesis are all related to the restoration ofcellular bioenergetics. (2) Transport, amino acid me-tabolism, and tetrapyrrole synthesis (beginning fromGlu), which are processes directly related to N uptakeand assimilation. Nitrate transport (MapMan bin code34.4) was over-represented among the transcripts thatdecreased in abundance, which seems reasonable sincethe N deprivation had ended, and is also consistentwith the fact that ammonium assimilation–the N sourceused in this study–suppresses the expression of nitratereductase genes (Fernández et al., 1989). Notably, genesinvolved in potassium transport (MapMan bin code34.15) were synchronously coordinated even thoughpotassium had never been depleted, suggesting a con-vergent node of nutrient sensing. The nonmevalonatepathway of isoprenoid biosynthesis (MapMan bin code16.1.1) was found among the early-reverseup-regulated categories. One possible explanationcould be to provide the prenyl moiety for chlorophyll,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-, andlate-reverse gene groups in the PL. Numbers represent the transcripts whose abundance changed according to theN status using a2-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 Nresupply, respectively. Numbers associated with the dashed line were inferred. B, Heat map of the overrepresented MapMancategories in the early-,mid-, late-reverse andNR-specific gene groups. The first columnhas the bin code of the categories and the

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plants (Eisenreich et al., 2001). (3) Motility-relatedprocesses (MapMan bin code 31.6 and its subcate-gories). It is still unclearwhy transcripts associatedwithflagellar assembly are reduced during N deprivation,especially because this treatment also induces the for-mation of gametes, which are flagellated (Tsai et al.,2014). However, it has been hypothesized that the as-sembly proteins for a primary cilium of vertebrate cells(similar to a flagellum) can also affect cell cycle pro-gression (Pan and Snell, 2007; Snell and Golemis, 2007).Although motility-related categories are presentthroughout all three phases, there is a clear delineation:genes encoding axonemal outer arm dyneins (MapManbin code 31.6.1.4.1) and radial spoke (MapMan bin code31.6.1.5) were restricted to early-reverse, basal bodies(MapMan bin code 31.6.1.1) and intraflagellar transportcomplex genes (MapMan bin code 31.6.1.3) tomid-reverse, and axonemal inner arm dynein genes(MapMan bin code 31.6.1.4.2) and flagellar associatedprotein genes (MapMan bin code 31.6.1.10) to mid- andlate-reverse. This progression may represent the step-wise formation of a eukaryotic flagellum as was alsopreviously observed during the diurnal cycle (Zoneset al., 2015).

The fact that autophagic protein degradation (Map-Man bin code 29.5.2) was mid-reverse down-regulatedrather than early-reverse is counterintuitive given thatprotein synthesis occurs immediately after N is resup-plied (Tsai et al., 2014), indicating that this process isactive not only duringNdeprivation but also extends toat least the first 6 h following N resupply. It is possiblethat the autophagic machinery is needed for the deg-radation of lipid droplets in vacuoles as suggestedabove (Fig. 1J; Supplemental Fig. S3Q). DNA synthesis(MapMan bin code 28.1) found in mid-reverseup-regulated categories was consistent with the mea-surement of DNA content (Fig. 1B). Finally, [FeFe]-hydrogenase (MapMan bin code 1.1.70.1) was foundin the only non-early-reverse subcategory of photo-synthesis.

Transcriptional Changes Specific to the Recovery Phase

Next, we asked whether there were unique tran-scriptional changes that were only apparent during theN recovery phase, which we called NR-specific. To becounted as an NR-specific gene, its relative RNA a-bundance must not fluctuate when shifting fromN-replete to N-deprived condition (21, log2 , 1), butmust be either up- or down-regulated in both the NRversus N-replete and the NR versus N-deprived

transcriptomes (using a 2-fold cutoff and a P value ,0.05; Fig. 2C). Note that the NR-reverse (Fig. 2A) andNR-specific genes are mutually exclusive by definition,enabling us to further dissect the nature of the tran-scriptomes. Under these criteria, 852 (NR6-specific) and347 (NR12-specific) genes were up-regulated after6 and 12 h of N resupply, respectively, among which197 genes overlapped (Supplemental Data Set S4). Asfor the down-regulated genes, there were 547 (NR6-specific) and 411 (NR12-specific) with 224 over-lapping. A typical example of an NR-specific gene is theone encoding betaine lipid synthase (BTA1), whichsynthesizes diacylglyceryl-trimethylhomo-Ser (DGTS),a major lipid component presumed to replace phos-phatidylcholine in extraplastidic membranes inC. reinhardtii (Riekhof et al., 2005). Transcript levels ofBTA1 remained constant when entering N deprivation,but raised approximately 4-fold after 6 h and 12 h of Nresupply.

In comparison to the NR-reverse data set, the en-richment of the NR-specific gene sets was much lessdiverse. Altogether, 31 of the total 32 categories(30 NR6-specific and 2 NR12-specific) could be attrib-uted to amino acid, RNA, and protein-related processes(Fig. 2B; Supplemental Data Set S5). Only two (Map-Man bin code 13: amino acid metabolism and 13.1:amino acid synthesis) were redundant with theNR-reverse categories (excluding those found in theLate-reverse which were beyond the NR6 and NR12timeline). Apparently, amino acid metabolism is af-fected in different aspects in the two data sets, that is theNR-reverse set covers Ser (MapMan bin code 13.1.5.1)while the NR-specific set covers more the biosynthesisof aromatic amino acids (MapMan bin code 13.1.6) andAsp family amino acids (MapMan bin code 13.1.3). Allthe NR6-specific protein-related categories came fromthe up-regulated gene set (852, Fig. 2C). Particularlynoteworthy are those involved in protein folding(MapMan bin code 29.6.2: chaperones and cochaper-ones, 29.6.2.2/4: HSP60 and HSP90-like proteins),prokaryotic ribosomal protein synthesis (e.g. MapManbin code 29.2.1.1.1.1/2: chloroplast ribosomal 30S/50Ssubunits), and protein targeting (MapMan bin code29.3.3: chloroplast targeting and 29.3.3: mitochondriatargeting). On the contrary, NR12-specific protein-related categories were overrepresented in the down-regulated gene set (411, Fig. 2C). Collectively, thesefindings suggest a unique demand for cells to rapidlyremake, restructure, and transport the proteins lostduring N deprivation, especially those of the chloro-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 fromFisher’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 thegene group name in the x axis of the heatmap. C, Graphic illustration of theNR-specific gene groups in the PL.Numbers representthe transcripts whose abundance did not vary in the –N over +N RNA-seq comparison (21, log2 fold change, 1), but differedover 2-fold (P value , 0.05) in both NR over +N and NR over –N comparisons.

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Acclimation Responses That Failed to Readjust in cht7during the Recovery Phase

CHT7 encodes a putative transcription factor andwithout it cultures are slow to resume growth following

N or phosphate deprivation, or rapamycin addition,treatments which all induce quiescence (Tsai et al.,2014). It would be expected to see differences in tran-script abundance between cells that undergo orderly

Figure 3. Comparative transcriptomics of the PL and the cht7mutant. A and B, Global gene expression analysis of the PL and cht7following N resupply. Blue circles: total number of genes changed in expression in a comparison of PL after 48 h of N deprivationfollowed 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 inexpression 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 Nresupply, respectively. C, Heat map of the overrepresented MapMan categories in the overlapping gene groups as depicted in Aand B. The first column has the bin code of the categories and the second column lists the explanation of each category. The colorin 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). Thenumber of genes in each gene group is given in parentheses near the gene group name in the legend of the heat map.

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progression through the recovery phase and cells thatfail to do so. Therefore, to identify specific mis-regulation of gene expression in cht7 when PL cellswould be recovering from N deprivation, we adoptedan 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 resupplywere compared with those of the N-deprived PL (Fig. 3,A and B, blue circles); transcripts of the PL after 6 h and12 h of N resupply were compared with those of cht7(Fig. 3, A and B, yellow circles). It should be noted thatthe blue circles in Figure 3A contain all the early-reverseand NR6-specific genes mentioned above, and the bluecircles in Figure 3B have all the mid-reverse and NR12-specific genes. Most transcripts responsive to Nresupply 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 incht7 and remained at expression levels that were similarto those of N-deprived PL cells (Fig. 3, A and B, over-laps between the blue and yellow circles; SupplementalData Set S6). It seems possible that these genes areregulated 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 secondaryor compensatory effects resulting from the impairedgrowth of cht7. Closer examination of the overlappinggene sets provided supporting evidence. Overall,60MapMan categories (35 ofNR6 and 25 of NR12)weresignificantly enriched (Fig. 3C; Supplemental Data SetS7), 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 twoMapMan pathways:tetrapyrrole synthesis, which was among theNR-reverse categories, but unlike photosynthesis, itstranscript profile was not affected by the loss of CHT7before the recovery phase (Tsai et al., 2014), and per-oxisomal redox homeostasis (MapMan bin code 21 and21.5), which was neither NR-reverse nor NR-specific.At 6 and 12 h following N resupply, nearly everytetrapyrrole gene showed lower transcript levels incht7 compared with PL (Fig. 4A, top right [includedhere are genes that fall below the 2-fold threshold];

Figure 4. Gene expression and metabolite level for two selectedpathways. A, Overview of the expression of genes involved in the tet-rapyrrole pathway (top) and in peroxisomal redox homeostasis (bot-tom). RNA-seq comparisons of the PL at different N status and thecomparisons between PL and cht7 at each N status are shown in theheat map. +N, N-replete; -N, N-deprived; NR6 andNR12, 6 and 12 h of

N resupply, respectively. For the genes whose expression pattern fol-lowing N resupply has been classified, the respective categories areindicated on the right of the heat map. Early, Early-reverse; Mid, mid-reverse; Late, late-reverse; NR6, NR6-specific; NR12, NR12-specific. B,Chlorophyll content. C1-C4, 4 independent complemented lines. C,TBARS content. For all quantitative data, averages (n = 3) of biologicalreplicates and SD are indicated.

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Supplemental Data Set S8). These genes did not greatlydiffer in expression between cht7 and the PL inN-replete or N-deprived conditions, but upon recoveryfromNdeprivation. The RNA-seq data were confirmedfor representative genes by quantitative PCR (qPCR) inPL and cht7 as well as cht7 complemented lines in thisanalysis (Supplemental Fig. S4A). Genes involved inmaintaining redox homeostasis during high demand ofb-oxidation were down-regulated in cht7 following Nresupply (Fig. 4A, bottom right; Supplemental Fig. S4B;Supplemental Data Set S9). Catalase 1 and 2, ascorbateperoxidase 1 and 2, and monodehydroascorbate re-ductase are enzymes that detoxify the ROS generated asby-product of b-oxidation within the peroxisomes(Eastmond, 2007). Notably, Arabidopsis (Arabidopsisthaliana) genes encoding homologs of HPR1, MAS1,MDH2, MDH4, monodehydroascorbate reductase 1,and PXN1 (all misregulated in cht7 at NR12) causemutant phenotypes when disrupted, affecting seed oilbreakdown and seedling establishment (Graham,2008; Theodoulou and Eastmond, 2012), reminiscentof the defects of cht7 in TAG turnover and regrowth.The changes in transcript abundance were corrobo-rated at the metabolite level. Chlorophyll contentsof the PL and complementation lines increased after6 h of N resupply, and decreased after 12 h likelybecause of cell divisions (Figs. 1C and 4B). In contrast,the chlorophyll content of cht7 remained constantthroughout the same period. TBARS, the cellular me-tabolites reflecting the damage caused by ROS, wereaccumulating in cht7 after transfer to N-replete me-dium (Fig. 4C).

MGDG Is the Sole Polar Lipid Affected in cht7 FollowingN Resupply

Earlier we had shown that TAG turnover took placeafter 6 h of N resupply, but total FAs did not changeuntil 12 h (Fig. 1D). A possible explanation for the dis-crepancy might be that between 6 and 12 h, lipolyticproducts from TAGs were not subjected to the b-oxi-dation cycle but used to reassemble membrane lipids,especially the thylakoid lipids enriched in polyunsatu-rated FAs. Indeed, we found that the absolute quantityof 16:4 (carbons: double bonds) and 18:3v3 (the twomajor FAs of monogalactosyldiacylglycerol [MGDG];Giroud et al., 1988) did not decrease after N resupply,but 18:1D9 (the signature FA of TAG; Liu et al., 2013) andother FAs did (Fig. 5A; Supplemental Fig. S5, A and B).About 25% of 16:4 and 18:3v3 was stored in TAGsduring N deprivation. While in the PL the relative a-bundance of 16:4 and 18:3v3 increased gradually fol-lowing N resupply, the FA profile of total lipids remainedstatic in cht7 (Fig. 5B; Supplemental Fig. S5, C and D). Ac-cordingly, among all the polar lipids being tested includingDGTS, digalactosyldiacylglycerol, phosphatidylethanola-mine, phosphatidylglycerol, phosphatidylinositol, andsulfoquinovosyldiacylglycerol, the cht7 mutant was un-able to restore MGDG following N resupply (Fig. 5C;Supplemental Fig. S6, A–E).

Data obtained at the transcript level are insufficientto provide a cause for the MGDG phenotype.Responding to N resupply, only the genes encodingMGDG-specific desaturases (FAD6, FAD7, and theC16 D4-desaturase) had increasedmRNA abundance,and their expression was normal in cht7 (Fig. 5D;Supplemental Data Set S10). Besides, none of theMGDG synthesis genes was a candidate for CHT7-specific regulation (found in the overlaps betweenthe blue and yellow circles in Fig. 3, A and B). Theinability of cht7 to readjust MGDG may be, at least inpart, a consequence of the delay in TAG turnover,which could normally contribute precursors forchloroplast lipid assembly.

Functional Curation of Lipid Metabolism Genes Based onExpression Patterns

RNA-seq resources generated in this study allow usto filter and classify functionally ambiguous genes.Here we focus on lipidmetabolism (Supplemental DataSet S10). Lipases are a subclass of acyl hydrolases thatdeesterify carboxylic esters, and can affect TAG me-tabolism both positively (e.g. PGD1; Li et al., 2012b) andnegatively (e.g. LIP1; Li et al., 2012a), as illustrated inFigure 6A. Since TAGs increase during N deprivationand decrease following N resupply, we expected thatgenes encoding TAG-hydrolyzing lipases (e.g. LIP1)would be down-regulated during N deprivation andup-regulated after N resupply (NR-reverse) or justup-regulated after N resupply (NR-specific; Fig. 6B, topleft). On the contrary, genes encoding TAG-producinglipases (e.g. PGD1) would respond in an opposite di-rection (Fig. 6B, middle left). Following this principle,we sorted through 131 genes predicted to encode a li-pase, phospholipase, or patatin based on the GXSXGmotif common to hydrolases, and assigned 9 TAG-hydrolyzing lipases and 23 TAG-producing lipaseswith LIP1 and PGD1 defining their respective class.Candidate genes encoding b-oxidation enzymes suchas ATO1 and acyl-CoA oxidases were coordinatedwiththose encoding TAG-hydrolyzing lipases (Fig. 6B, bot-tom left), with the exception of the gene for ECH1, aspecialized enoyl-CoA oxidase/isomerase needed forunsaturated FAs (Goepfert et al., 2008). Notably, Cre17.g707300, Cre06.g265850, Cre03.g195200, and Cre03.g152800 (TAG-hydrolyzing) and PGD1, Cre10.g425100,g9707, and Cre03.g174900 (TAG-producing) were mis-regulated in cht7 in either or both NR6 and NR12 con-ditions in a way that would cause TAGs to be retainedin the cells, making these promising candidates for re-verse genetic studies (Fig. 6B, top and middle right).

For newly synthesized FAs in the form of acyl-ACP(acyl carrier protein) to be exported out of plant chlo-roplasts, the ACP moiety must be removed by the ac-tivity of acyl-ACP thioesterase, and almost instantlylong-chain acyl-CoA synthetase activates the resultingfree FAs to acyl-CoA so they can be incorporated intoglycolipids such as TAG (Li-Beisson et al., 2013). Like-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 timesindicated (h). B, Relative FA compositions of PL and cht7 following N resupply. FAs are designated by the total carbon number followed by the numberof double bonds. The position of specific double bonds is indicated either from the carboxyl end “D” or from themethyl end “v.” C, Polar lipid contentsin 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 ofindividual 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-forms of long-chain acyl-CoA synthetase exist in the C.reinhardtii genome, LACS1, and LACS2. Transcripts ofLACS1 increased in abundance when shifting toN-deprivedmedium and recovered when the conditionwas reversed; those of LACS2 reacted just the opposite(Supplemental Data Set S10). It is thus likely thatLACS1 works in tandem with acyl-ACP thioesterase,and LACS2 channels precursors into b-oxidation. Di-acylglycerol acyltransferase (type 2, DGTT) is a keyenzyme for TAG biosynthesis. Of the five putativecandidates, only the expression of genes encodingDGTT1 andDGTT5 paralleled the accumulation of TAG.Expression of the gene for DGTT4 was NR-specific witha near 4-foldmRNA increase at 6 and 12 h ofN resupply,the time that TAGswere being degraded. The seeminglyconflicting finding may be reconciled by hypothesizingthat during recovery from N deprivation there is a needto fine-tune FA production and sequestration into TAGto avoid the toxicity of free FAs or that nontranscrip-tional regulation comes into play, which is not consid-ered here.

A cAMP-Dependent Protein Kinase Pathway Is Requiredfor Quiescence Exit

Adenylyl cyclase converts ATP to cAMP, and bind-ing of cAMP to the regulatory domains of protein ki-nase A (PKA) facilitates the phosphorylation of diverseenzyme targets. In yeast, the PKA signaling cascadenegatively affects quiescence (Gray et al., 2004).Therefore, we asked whether PKA could have an im-pact on quiescence in C. reinhardtii. Competitive ELISAshowed that concentrations of cAMP responded to thepresence and absence of N (Fig. 7A), a prerequisite for apossible role of PKA activity during recovery from Ndeprivation. To verify this hypothesis, we took apharmacological approach. A 20-amino acid fragmentof a naturally occurring PKA inhibitor (PKI) is knownto bind and inhibit the catalytic domain of PKA(Knighton et al., 1991). Derivatives of this fragmenthave been used to study flagellar assembly in C. rein-hardtii (Howard et al., 1994). When applied simulta-neously with N refeeding, PKI interfered with TAGturnover in thePL in adosage-dependentmanner (Fig. 7B).FA profiles of PL cells treated with PKI resembled thoseof nontreated cht7 (Supplemental Fig. S7). Addition of10 mM of PKI caused severe chlorosis indicative of celldeath, and no degradation of TAG was observed in thePL or cht7. Importantly, within the nontoxic range (0 to5mM), PKI did not exacerbate the lipolytic defect in cht7.PKI treatment also mimicked the slow regrowth of cht7in the PL (Fig. 7C).Adenylyl (and guanylyl) cyclases form one of the

largest families in the genome ofC. reinhardtii (Merchant

et al., 2007), and their activities are counteracted by cyclicnucleotide phosphodiesterases that turn cAMP intoAMP. Stimulating the activity of phosphodiesterase at-tenuates cAMP-mediated lipolysis (Botion and Green,1999). At a glance, many of the candidate genes weredifferentially regulated by N availability and by the lossof CHT7 (Supplemental Data Set S11). Here we assignedpotential adenylyl cyclases and phosphodiesteraseswhose expression profile matched the observed fluctu-ation of cAMP (Fig. 7D).

DISCUSSION

To grow or not is a fundamental decision that everycell has to make in response to developmental, meta-bolic, or environmental stimuli. Based on this decision,cells either progress through the cell division cycle orenter into quiescence. While yeast offers a well-studiedmodel of quiescence, fairly little is known in eukaryotaoutside of fungi and certain mammalian cell lines,photosynthetic eukaryotes in particular. From a bio-logical standpoint, the reversible cessation of growthdepending on nutrient availability provides a facileexperimental system to study quiescence. N depriva-tion is thus far the most effective way to induce theaccumulation of neutral lipids in microalgae, for ex-perimental purposes to study quiescence-related phe-nomena or for practical reasons in developing algae as arenewable energy source (Hu et al., 2008). Cellular re-sponses toNdeprivation have been studied onmultiple-omic levels, drawing an integrated picture of N econ-omy (Miller et al., 2010; Blaby et al., 2013; Schmollingeret al., 2014; Wase et al., 2014). In contrast, research onthe recovery from N deprivation has lagged behind.Here, we used a systems biology approach to addressthe question of how photosynthetic cells recover fromN deprivation to begin to understand mechanism in-volved in quiescence exit, using C. reinhardtii as a ref-erence model. The use of the cht7 mutant incomparative transcriptomics helped to reduce noiseand unravel the transcriptional patterns potentiallyrelevant to the resumption of growth and proliferation.While our understanding of how CHT7 affects cell vi-ability and proliferation in response to different N re-gimes is only in its infancy, these data provide additionalinsights into the function of this potential regulator ofquiescence-relevant transcriptional programs.

Quiescence Exit Is Not Simply the Reverse ofQuiescence Entry

Ultimately, cells recovering from N deprivation-induced quiescence return to the G1 phase of the cellcycle. However, recovery from N deprivation is hardly

Figure 5. (Continued.)responsible forMGDG synthesis. RNA-seq comparisons of PL at differentN status and the comparisons between PL and cht7 at eachN status are shownin 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 whilecells become N deprived. We categorized genes thatreversed their expression in response to N resupply intoearly-, mid-, and late-reverse groups, implicating pri-orities of transcriptional reprogramming. Early- andmid-reverse groups aremore likely to be responsible forrestarting the cell cycle, as they coincided with the timethat cells began to proliferate. These unique temporalpatterns of expression suggest that the recovery fromNdeprivation including the transition to the resumptionof the cell cycle is subject to an ordered set of sequentialevents. In an emerging model of microbial quiescence,growth-limiting conditions appear to trigger a commonpathway that reduces growth by redirecting the carbonfluxes away from the central metabolic pathways andtoward storage depots (Rittershaus et al., 2013). This isespecially the case for microalgae. Nutrient starvation(e.g. N, phosphate, sulfur, zinc, and iron), high salt, heatshock, and oxidative stress are all able to cause TAGaccumulation (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, andof course, uncovered many that had reversed expres-sion patterns (Fig. 6). Importantly, we also identifiedgenes whose transcript abundance only fluctuatedduring the time of N recovery, termed NR6- and NR12-specific groups. This discovery provides direct evi-dence that the expression profile of N-resupplied cellsexiting quiescence, is distinct from that of N-deprivedquiescent cells or N-replete cells, which are mostly inthe G1 phase of the cell cycle. TEM also captured keymorphological distinctions between newly dividingN-resupplied and N-replete cells, showing that grow-ing cells after N resupply retained small lipid dropletsand starch granules, and their thylakoids were not fullystacked (Fig. 1H; Supplemental Fig. S3). However,what happens in cells during recovery from N depri-vation that causes a delay before they can undergogenome replication andmitosis comparedwith the cellsthat actively traverse the cell division cycle? A 6- to 8-hdoubling time for regular cycling cells was lengthenedto 12 to 15 h counting from the moment that N wasrefed (Fig. 1B), or even longer if cells were N-deprivedfor long periods (Tsai et al., 2015). Aside from a re-duction in viability during prolonged N deprivation, itseems likely based on the current data that this delayrelates to the reorganization of metabolism. Indeed,much of the provided ontology analysis detected met-abolic processes related to the synthesis of macromol-ecules (nucleotides, amino acids, and proteins), cellularbioenergetics (central metabolism, photosynthesis, mi-tochondrial electron transport, and ATP synthesis),cellular components (lipids and chlorophylls), nutrientassimilation (nitrogen and phosphate), and redox ho-meostasis. Perhaps the most intriguing findingwas thatthe MapMan categories enriched in NR-specific genesappeared to center on the replenishment of chloroplastproteins. This finding fits nicely with reports thatchloroplast ribosomes, specific photosynthetic electron

Figure 6. Gene expression of putative lipase and b-oxidation genes. A,An example depicting how lipases might positively or negatively in-fluence TAG content. MAG, monoacylglycerol; PGD1 and LIP1, li-pases. B, Transcript profiles of genes encoding lipases possibly involvedin the degradation of TAG (top), in the production of TAG (middle), or inb-oxidation (bottom). RNA-seq comparisons of the PL at different Nstatus and the comparisons between PL and cht7 at each N status areshown in the heat map. +N, N-replete; 2N, N-deprived; NR6 andNR12, 6 and 12 h of N resupply. For the geneswhose expression patternfollowing N resupply has been classified, the respective categories areindicated on the right of the heat map. Early, Early-reverse; Mid, mid-reverse; Late, late-reverse; NR6, NR6-specific; NR12, NR12-specific.

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transfer complexes, plastid ATPase, and Calvin-Bensoncycle enzymes especially Rubisco, are more activelytargeted by N-sparing mechanism during N depriva-tion in C. reinhardtii (Gray et al., 2004; Schmollingeret al., 2014). Note that elevated protein abundance ofmitochondrial ATP synthase and the mitochondrial bc1complex was apparent. Measurements of MGDG and

chlorophylls, both confined to the chloroplast, alsoreflected a scenario of rebuilding the chloroplast (Figs.4C and 5C). Thus, the time delay required by recoveringcells might be to restore chloroplast integrity, whichrepresents an important distinction between photo-synthetic eukaryotes on one hand and yeast andmammalian cells on the other.

Figure 7. The effect of the cAMP-PKA pathway on the recovery fromN deprivation. A, ELISA assay to quantify the cellular contentof 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, TAGdegradation of PL and cht7 in the presence of PKI. The TAG content (depictedas 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 contentsof cells treatedwith different concentrations of PKI (as indicated in the x axis) were quantified at 24 h of N resupply (NR24). C, Theregrowth of PL and cht7 in the presence of PKI. The fold change of regrowth is calculated by dividing the cell count measured at24 h of N resupply by that at 0 h of N resupply (NR24/NR0). PKI treatments in B and Cwere done simultaneously with N resupply.D, Transcript profiles of genes encoding putative adenylyl cyclase and phosphodiesterase. RNA-seq comparisons of PL at differentN status and the comparisons between PL and cht7 at each N status are shown in the heat map. For the genes whose expressionpattern 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.

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How Does CHT7 Facilitate the Recovery fromN Deprivation?

On one hand, 34 NR-reverse categories (MapMan)failed to revert back to the state prior to N deprivationin N-resupplied cht7. This seems to support the notionthat CHT7 contributes to the reversal of the quiescentstate by acting as its suppressor. On the other hand, thefinding of 14 NR-specific gene expression categoriesthat appeared in N-resupplied cht7 as if cells were stillunder N deprivation suggests that CHT7 has addi-tional, specific functions during the recovery from Ndeprivation. The discussed examples of misregulatedpathways (i.e. tetrapyrrole synthesis and peroxisomalredox homeostasis) represent just a fraction of genesaffected in their expression by CHT7. Particularlyimportant is that the expression of genes affectingthese pathways was completely normal in the cht7mutant at any other stage outside of N recovery. Wehypothesize that when cells receive signals to recoverfrom N deprivation, CHT7 governs some transcrip-tional programs, directly or indirectly, that allow theresumption of growth and proliferation. This hy-pothesis can be addressed through the identification ofthe in vivo chromatin binding sites for CHT7 or CHT7-containing complexes under different growth condi-tions. A future integrative analysis of the tran-scriptome and chromatin binding data will helpdistinguish the genes that are primary or secondarytargets of CHT7, as well as clarify the potential feed-back regulations by metabolite levels.

Interaction between CHT7 and Other Regulatory Modulesof Quiescence

In yeast, the cAMP-PKA pathway is active duringabundance of nutrients and represses aspects ofnutrient deprivation-induced quiescence by targetingnutrient-sensitive transcription factors MSN2 andMSN4 (Smith et al., 1998; Beck and Hall, 1999). ThecAMP signal is also required for a timely recovery, asmutants unable to transiently elevate cAMP levels fol-lowing the addition of Glc to starved cells show ex-tended delays in resuming growth (Jiang et al., 1998).This is somewhat similar to the observed increase ofcAMP following N resupply (Fig. 7A). Furthermore, weshowed that PKI-treated PL cells recapitulated both theregrowth and TAG phenotypes of cht7, presumablydue to the deactivation of PKA (Fig. 7, B and C). To oursurprise, when cht7 cells were treated with nontoxicconcentrations of PKI we did not observe deterioration,suggesting some level of functional redundancybetween the CHT7 and cAMP-PKA pathways. Inadipocytes, adenylyl cyclase and PKA transducesignals between hormone binding to cells and lipo-lytic responses. Upon activation, PKA phosphorylateshormone-sensitive lipase and perilipin 1 (Guo et al.,2009). This raises the question, whether during the

response to N resupply the cAMP-PKA pathway trig-gers TAG breakdown by phosphorylating protein an-alogs found in adipocytes, which in turn fuels therecovery or acts on nuclear targets such as CHT7 tostimulate gene expression that promotes growth. Bio-chemical studies have confirmed the presence of PKAcatalytic subunits in C. reinhardtii although the respec-tive genes remain uncertain (Howard et al., 1994).Continued study of cAMP-PKA pathway genes (e.g.the listed candidates in Fig. 7D) will shed light on therole of this pathway in C. reinhardtii quiescence exit.

For the bioindustry using microorganisms to pro-duce drugs, biofuels, nutritional supplements, flavorsand fragrances, control of nutrient deprivation-inducedquiescence is at the core of research that strives to gaininsights into the inverse relationship between biomassproduction and production of the target compound.Thus, the information gathered here has practical im-plications for the engineering and cultivation of pho-tosynthetic algae, and potentially more broadly to cropplants. Due to their sessile nature, plants are continu-ously exposed to biotic and abiotic stresses that promptcells to arrest growth and division to spare resources forrespective defense responses. Understanding how tomanipulate the balance between growth versus defensebased on insights into the regulation of cellular quies-cence may one day help in improving crop yields inagricultural settings.

MATERIALS AND METHODS

Strains and Growth Conditions

The Chlamydomonas reinhardtii dw15.1 (cw15, nit1, mt+) or CC-4619 (http://chlamycollection.org/strain/cc-4619-cw15-nit1-mt-dw15-1/) strain wasobtained fromArthur Grossman and is referred to as the wild type (with regardto CHT7) PL throughout. A cell-walled strain CC-125 obtained from Chlamy-domonas Resource Center (http://www.chlamycollection.org) was used forTEM. The four independent complemented lines of cht7 were generated aspreviously described (Tsai et al., 2014). Growth conditions and media were aspreviously described (Tsai et al., 2014). For N deprivation, mid-log-phase cellsgrown in TAP were collected by centrifugation (2,000g, 4°C, 2 min), washedtwice with TAP-N (NH4Cl omitted from TAP), and resuspended in TAP-N at0.3 OD550. N was resupplied by adding 1% culture volume of 1 M NH4Cl (1003)to the N-deprived culture. The size and concentration of cells in all assays wasmonitored using a Z2 Coulter Counter.

Lipid Analysis

Lipid extraction, TLC, fatty acid methyl ester preparation, and gas chro-matography were conducted as previously described (Tsai et al., 2015) withmodifications. For neutral lipids, 5mL of cell culture was pelleted and extractedinto 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-ration at 3,000g centrifugation for 3 min. For polar lipids, 10 mL of culture wasextracted with methanol-chloroform-88% formic acid (2:1:0.1 v/v/v) followedby phase separationwith 1 MKCl and 0.2 MH3PO4. Lipid species were separatedby TLC on Silica G60 plates (EMD Chemicals) developed in petroleum ether-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-ter brief exposure to iodine vapor for visualization of lipids, fatty acid methylesters of each lipid or total cellular lipid were processed and quantified by gaschromatography as previously described (Rossak et al., 1997).

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TEM

For electron microscopy, walled strains were fixed and processed as pre-viously described (Harris, 1989), except that TAP medium with or without Nwas used as diluents for fixatives. Transmission electron micrographs werecaptured using a JEOL100 CXII instrument (Japan Electron Optics Laborato-ries).

Metabolite Measurements

Chlorophylls were extracted from fresh cell pellets using 80% acetone, andconcentrations were calculated from the absorbance values at 647 and 664 nmaccording to Zieger and Egle (1965). For the TBARS assay, 5 mL of culture wascentrifuged and analyzed immediately. Cell pellets were resuspended in 1 mLof thiobarbituric acid/trichloroacetic acid solution (0.3 and 3.9%, respectively)and heated at 95°C for 15min. The solution alonewas also heated to serve as theblank for spectrophotometric measurements. Samples and blank were mea-sured after no further gas bubbles were released. TBARS were determined byabsorbance at 532 and 600 nm as previously described (Baroli et al., 2003). Theextinction coefficient used was 155 mM

21 cm21. Quantification of cellular cAMPwas conducted using a cAMP Competitive ELISA kit (Thermo Scientific)according to the manufacturer’s instructions. For some experiments, proteinkinase A inhibitor (P9115; Sigma-Aldrich) was added to the culture at the timewhen N was resupplied.

Illumina RNA-Seq and Analysis

The raw data for Illumina RNA-seq were generated in our previous study(Tsai et al., 2014). In each of the experiments, three replicates were taken in-dependently to ensure the reproducibility of the data. RNA abundance in thesamples was computed as previously described (Tsai et al., 2014). C. reinhardtiigenome sequence and annotations version 5.3.1 were downloaded from JGI(www.phytozome.net/chlamy.php). Differential expression was determinedby using the numbers of mapped reads overlapping with annotated C. rein-hardtii genes as inputs to DESeq, version 1.10.1 (Anders and Huber, 2010). Thehierarchical clustering was conducted and heat maps were generated usingQlucore Omics Explorer (qlucore.com). The quality of the RNA-seq data wasalso validated by qPCR on select metabolic pathways. To assess which pathwaygenes tend to be differentially regulated, Fishers exact test was used to deter-mine overrepresentation. Each gene set was compared to each MapManpathway gene group (http://mapman.gabipd.org/web/guest/mapmanstore,mapmen_Creinhardtii_236, retrieved on 12/11/2014). P values obtained fromFishers exact test were corrected for multiple testing to obtain q-values (Storeyand Tibshirani, 2003). Significant overrepresentation was reported for thepathways with q, 0.05. We also repeated the overrepresentation analyses withGO terms. To get GO annotation, we compared Creinhardtii_v5.3_223 peptidesequences available from Phytozome (http://phytozome.jgi.doe.gov/pz/portal.html) against the NCBI nonredundant database using blast (BLASTP2.2.25+). The BLAST results were imported into Blast2Go (version 2.7) andpeptides were mapped to GO terms. The resulting overrepresented terms wereplotted as heat maps in R using the gplots package (https://cran.r-project.org/web/packages/gplots/index.html).

qPCR

qPCR was done as previously described (Tsai et al., 2014) following theMinimum Information for Publication of Quantitative Real-Time PCR Experi-ments guidelines (Bustin et al., 2009). All experiments contained at least twobiological replicates and each reaction was run with technical repeats. Primerscan be found in Supplemental Table S1.

Fluorescence-Activated Cell Sorting

To monitor the DNA content in synchronized cells, fluorescence-activatedcell sorting (FACS)byflowcytometrywas carriedout aspreviouslydescribedbyFang et al. (2006) with modifications. For this purpose, 10 mL of cells wascollected and the cells were fixed by resuspension in 10 mL of 70% ethanol for1 h at room temperature. Fixed cells were washed once with FACS buffer (0.2 M

Tris, pH7.5, 20 mM EDTA, and 5 mM NaN3), resuspended in 1 mL of FACSbuffer, and stored at 4°C. Prior to flow cytometry, 2 3 106 of these cells werepelleted, and resuspended in 1mL of FACS bufferwith 100mg/mLRNase A for

2 h in the dark. Cells were washed with 1mL of PBS and stainedwith 1mL of PIsolution (PBS supplemented with 50 mg/mL propidium iodine [Sigma-AldrichP4864]) overnight in the dark. The samples were then analyzed at the FlowCytometry Core Facility at theMI State University (http://rtsf.natsci.msu.edu/flow-cytometry/).

Accession Numbers

Thefiltered sequencedata sets have beendeposited at theNationalCenter forBiotechnology Information Sequence Read Archive (www.ncbi.nlm.nih.gov/Traces/sra/) with the BioProject ID PRJNA241455.

Supplemental Data

The following supplemental materials are available.

Supplemental Table S1. Oligonucleotide primers used in this study.

Supplemental Figure S1. Experimental setup of RNA-seq experiments.

Supplemental Figure S2. Cell count and size distribution following Nresupply.

Supplemental Figure S3. Cellular ultrastructure following N resupply.

Supplemental Figure S4. Confirmation of transcriptional changes byqPCR.

Supplemental Figure S5. Fatty acid composition of cells following Nresupply.

Supplemental Figure S6. Polar lipid analysis of cells following N resupply.

Supplemental Figure S7. Fatty acid composition of PKI-treated cells fol-lowing N resupply.

Supplemental Data Sets. Combined File of Supplemental Data Sets 1 to 11.

ACKNOWLEDGMENTS

We thank Dr. Barbara B. Sears and Dr. Ben Lucker for critically reading themanuscript.

Received October 26, 2017; accepted December 26, 2017; published December29, 2017.

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