Figure 1.
Nuclear and chloroplastic transcription is differentially regulated during prolonged darkness.
qRT-PCR normalization factors were estimated by comparing the levels of the nuclear-encoded DLST transcript (A) or the chloroplast-encoded atpB transcript (B) in three biological replicas of dark-treated cells (D48) and cells re-exposed to WL for 0.5, 6 and 24 h to the levels found in CWL cells. Equal amounts of total RNA was used as starting material for the cDNA synthesis. Bars represent the average DLST or atpB normalization factor as calculated by REST2009 from two (A) or three (B) different qRT-PCR runs.
Figure 2.
Schematic gene expression profiles of photosynthesis-associated nuclear genes and photoreceptors.
Expression patterns describing the mRNA expression levels at continuous white light (CWL), after 48 h dark treatment (D48) and re-exposure to white light (WL) for 0.5, 6, and 24 h. A) Light-responsive expression patterns common for category 1 genes representing the majority of the PhANGs characterized by low transcriptional activity in darkness, modest induction by 0.5 h re-exposure to WL and generally high expression when exposed to WL for longer periods of time. I: Gene expression peaking after 6 h of re-exposure to WL. II: Gene expression stabilizing at CWL level after only 6 h of re-exposure to WL. III: Gene expression steadily increasing during 24 h of re-exposure to WL, but never reaching CWL levels. B) Expression pattern representative for category 2 genes including several genes belonging to the LHC family encoding proteins believed to have a photoprotective function, as well as for the POR4 gene and two photoreceptor genes. Genes are characterized by being strongly, but transiently induced by WL exposure for 0.5 h. C) Expression pattern representative for the category 3 genes including a few PhANGs and a photoreceptor gene characterized by high transcriptional activity in darkness and a gradual reduction towards CWL levels during 24 h of WL re-exposure.
Table 1.
mRNA recovery from total RNA.
Figure 3.
Gene expression responses in the hypothesized chlorophyll a and non-mevalonate pathways in P. tricornutum.
Colored squares indicate the regulation pattern of genes encoding putative enzymes functioning in the two pathways after dark treatment for 48 h (D48) and re-exposure to white light (WL) for 0.5, 6 and 24 h. Continous white light (CWL) were control conditions. Squares with a diagonal line inside indicate genes with an expression ratio greater than +/−0.5 that are not significantly regulated. The asterisk marking the expression pattern of subunit I of Mg-chelatase (MgCh) indicates that the gene is chloroplast encoded, and that the expression ratios was assessed by qRT-PCR instead of microarray analysis. The scale on the right represents gene expression ratio values, log2 transformed. The gene encoding Mg-protoporphyrin IX monomethyl ester cyclase responsible for converting Mg-protoporphyrin IX monomethyl ester to divinyl protochlorophyllide a in higher plants [108] is absent in P. tricornutum, and this step is marked with a question mark in the figure. The abbreviations used are: GLURS: glutamyl-tRNA synthetase; HEMA: glutamyl-tRNA reductase; HEML: glutamate-1-semialdehyde 2,1-aminomutase, HEMB: porphobilinogen synthase; HEMC: hydroxymethylbilane synthase; HEMD: uroporphyrinogen-III synthase; HEME: uroporphyrinogen decarboxylase; HEMF: coproporphyrinogen III oxidase; PPO: protoporphyrinogen oxidase; MgCh: magnesium chelatase (comprising of subunits H, D and I); CHLM: Mg-protoporphyrin IX methyl transferase; POR: protochlorophyllide oxidoreductase; DVR: divinyl protochlorophyllide a 8-vinyl reductase; CHLG: chlorophyll synthase; DXS: deoxyxylulose-5-phosphate synthase; DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase; ISPD: 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; ISPF: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS: 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase; HDR: 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; FDPS: farnesyl diphosphate synthase; GGPS: geranylgeranyl pyrophosphate synthase; IDI: isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerise; CHLP: geranylgeranyl reductase.
Figure 4.
Cladogram of the antenna proteins of P. tricornutum.
Phylogenetic analysis of all predicted P. tricornutum antenna proteins belonging to the light-harvesting complex (LHC) superfamily, together with other Stramenopile LHC proteins sequences as well as sequences from cryptophytes, haptophytes, rhodophytes, viridiplantae, rhizaria and an alveolate. LHCs from P. tricornutum are indicated in green. The two main clades are separated by a dashed red line. The abbreviations used are Aa: Aureococcus anophagefferens; Bn: Bigelowiella natans; Cc: Cyclotella cryptica; Cf: Cylindrotheca fusiformis; Cn: Chaetoceros neogracile; Cr: Chl amydomonas reinhardtii; Eh: Emiliania huxleyi; Es: Ectocarpus siliculosus; Fracy1: Fragilariopsis cylindrus; Gc: Gracilaria changii; Gs: Galdieria sulphuraria; Gt: Guillardia theta; Ht: Heterocapsa triquetra; Ig: Isochrysis galbana; Mp: Macrocystis pyrifera; Sl: Saccharina latissima; Pf: Pseudochattonella farcimen; Pl: Pavlova lutheri; Pp: Physcomitrella patens subsp. Patens; Pt: Phaeodactylum tricornutum; Rh: Rhodomonas sp. CS24; Tp: Thalassiosira pseudonana; Ul = Ulva linza; Vc: Volvox carteri f. nagariensis. The accession numbers corresponding to the protein sequences used in the analysis are listed in Table S3.
Figure 5.
Expression patterns of genes encoding photosynthesis-associated proteins and photoreceptors after dark treatment and re-exposure to white light.
The differentially regulated genes include antenna proteins, components involved in oxidative photosynthesis and photoreceptors after dark treatment for 48 h (D48) and re-exposure to white light (WL) for 0.5, 6 and 24 h. Continuous white light (CWL) were control conditions. The color code indicates expression values, and squares with a diagonal line inside indicate genes with an expression ratio (log2 transformed) greater than +/−0.5 that are not significantly regulated. Genes where at least one of the two probes used for calculation of the expression ratios were significantly regulated by >2-fold at least at one time point during the experimental period were included in the figure. The expression patterns of genes marked with an asterisk indicate that they are chloroplast-encoded, and that the expression ratios was assessed by qRT-PCR instead of microarray analyses. The nuclear-encoded LHC48798 gene was not represented on the microarray, and were therefore also assessed by qRT-PCR analyses. The scale on the right represents gene expression ratio values, log2 transformed. The abbreviations used are LHCF: major fucoxanthin Chl a/c proteins; LHCR: red algal-like proteins; LHCX: LI818-like proteins; LHCZ: unknown function antenna protein, LHC#: unclassified light harvesting proteins, numbers refer to protein ID in JGI; LHL: Lhc-like; Psa: PSI proteins; Psb: PSII proteins; PETB-C: cytochrome b6f complex proteins; PETH: Ferredoxin-NADP reductase; PETJ: cytochrome c6; Atp: F-ATPase proteins; AUR: aureochrome; PAS: PER-ARNT-SIM domain, probably aureochrome; CRYL: cryptochrome-like protein; CPF: cryptochrome/photolyase family protein; SKP3: Sensor Kinase Protein 3.
Figure 6.
Changes in cell pigment concentrations after dark treatment and re-exposure to white light.
Total cell pigment and cellular Chl a, Chl c, Fuco and Diadino concentrations (mol cell−1) for the samples from continuous white light (CWL), 48 h of darkness (D48) and 0.5, 6 and 24 h (WL 0.5–24 h) of re-exposure to CWL. The CWL and D48 sample values are a mean of 12 biological replicates. The WL 0.5–24 h values are the mean of three biological replicas. Values are averages of three parallel HPLC samples, and values are presented with ±SD bars.
Figure 7.
Photo-physiological dark-light responses by in vivo chlorophyll a fluorescence kinetics (PAM).
A) Maximum quantum yield of charge separation in PSII (ΦPSII_max), B) maximum light utilization coefficient (α), and C) maximum rETR after exposure to continuous white light (CWL), 48 h of darkness (D48) and 0.5, 6 and 24 h of re-exposure to white light after darkness (WL 0.5–24 h). ΦPSII_max was measured subsequently to a 3 min dark acclimation period and is a measure of the maximum efficiency of photosynthetic electron yield per mol photons absorbed in PSII. Bars are S.D. (n = 3).
Figure 8.
Changes in fluorescence emission during re-exposure of dark-treated cells.
Time series of scaled quantum corrected fluorescence emission in P. tricornutum exposed to 48 h of darkness (D48) and subsequent 1–30 minutes of re-exposure to light. Fluorescence emission was measured at 730 nm to ensure origin from charge separation in reaction center II Chl a (P680).
Figure 9.
Intracellular changes during dark treatment.
Intracellular differences in dark-treated P. tricornutum cells and cells re-exposed to moderate white light (E = 100 µmol m−2 s−1) of different duration. A) cells grown in continuous white light, B) cells exposed to 48 h of darkness (D48), C) cells exposed to 8 days of complete darkness, D–F) cells after white light (WL) re-exposure to 0.5, 6 and 24 h respectively. Inset in B and C: green arrows; vacuole-like structures, black arrows; cytoplasm. Scale bar, 25 µm.
Table 2.
Initial responses to dark-light shift compared to light intensity shift.