EUKARYOTIC CELL, Nov. 2008, p. 1965–1979
1535-9778/08/$08.00⫹0 doi:10.1128/EC.00418-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 7, No. 11
Transcriptome for Photobiological Hydrogen Production Induced by
Sulfur Deprivation in the Green Alga Chlamydomonas reinhardtii䌤†
Anh Vu Nguyen,1,3§ Skye R. Thomas-Hall,1§ Alizée Malnoë,2 Matthew Timmins,1 Jan H. Mussgnug,2,3
Jens Rupprecht,2 Olaf Kruse,3 Ben Hankamer,1,2 and Peer M. Schenk1*
School of Integrative Biology1 and Institute for Molecular Bioscience,2 University of Queensland, St. Lucia, Queensland 4072,
Australia, and Department of Biology/AlgaeBioTech Group, University of Bielefeld, Bielefeld, Germany3
Received 15 November 2007/Accepted 5 August 2008
Photobiological hydrogen production using microalgae is being developed into a promising clean fuel stream
for the future. In this study, microarray analyses were used to obtain global expression profiles of mRNA
abundance in the green alga Chlamydomonas reinhardtii at different time points before the onset and during the
course of sulfur-depleted hydrogen production. These studies were followed by real-time quantitative reverse
transcription-PCR and protein analyses. The present work provides new insights into photosynthesis, sulfur
acquisition strategies, and carbon metabolism-related gene expression during sulfur-induced hydrogen production. A general trend toward repression of transcripts encoding photosynthetic genes was observed. In
contrast to all other LHCBM genes, the abundance of the LHCBM9 transcript (encoding a major lightharvesting polypeptide) and its protein was strongly elevated throughout the experiment. This suggests a major
remodeling of the photosystem II light-harvesting complex as well as an important function of LHCBM9 under
sulfur starvation and photobiological hydrogen production. This paper presents the first global transcriptional
analysis of C. reinhardtii before, during, and after photobiological hydrogen production under sulfur
deprivation.
The development of new systems to produce zero CO2 emission fuels for the future is of major importance to combat the
effects of climate change. Consequently, biofuel production
using conventional crops has increased substantially in recent
years with the aim of reducing our CO2 footprint. A downside
of this approach has been the increased competition between
food and fuel production. Microalgae offer a promising alternative and likely a higher efficiency route (6) to the production
of a wide range of biofuels, as they can be cultivated in PBRs
sited on nonarable land in which nutrient, light, and temperature levels can be carefully regulated.
In 2000, Melis and coworkers (30) reported a two-phase microalgal photobiological H2 production process. This process consists of an aerobic and an anaerobic phase and is summarized by
the following two reactions: aerobic phase ⫽ H2O 3 2H⫹ ⫹
2e⫺ ⫹ 1/2O2; anaerobic phase ⫽ 2H⫹ ⫹ 2e⫺ 3 H2.
Photosystem II (PSII) drives the first stage of the process by
splitting H2O into protons (H⫹), electrons (e⫺), and O2. Normally, the photosynthetic light reactions and the Calvin cycle
produce carbohydrates that fuel mitochondrial respiration and
cell growth (Fig. 1). However, under anaerobic conditions,
mitochondrial oxidative phosphorylation is largely inhibited.
Under these conditions, some organisms (e.g., Chlamydomonas reinhardtii) reroute the energy stored in carbohydrates to a
chloroplast hydrogenase, likely using an NAD(P)H-plastoquinone (PQ) e⫺ transfer mechanism (29), to facilitate ATP production via photophosphorylation (Fig. 1). Thus, hydrogenase
essentially acts as an H⫹/e⫺ release valve by recombining H⫹
(from the medium) and e⫺ (from reduced ferredoxin) to produce H2 gas that is excreted from the cell (30). C. reinhardtii
and potentially other green algae could therefore provide the
basis for solar-driven biological hydrogen production. The
combustion of the evolved H2 yields only H2O and thereby
completes the clean energy cycle.
During the aerobic phase, the microalgae can be grown
either photoautotrophically or photoheterotrophically to increase the cell density of the culture. The H⫹ and e⫺ extracted
from water by PSII (and under heterotrophic growth conditions, potentially from added exogenous substrates) during this
phase are stored in a range of metabolic products, including
starch and protein, the latter reported to be an important
potential H⫹ and e⫺ source for H2 production (30).
To induce H2 production, Melis and coworkers (30) depleted the cultures of S to inhibit the repair of the methioninecontaining PSII reaction center protein (D1) after photodamage. When PSII functions below normal capacity, the culture
turns anaerobic. Anaerobiosis is a strict requirement for algal
photobiological H2 production, since O2 blocks the activity of
hydrogenases by binding to the reaction center of the already
assembled enzyme, preventing it from catalyzing the reduction
of protons (12, 15). Oxygen also competes with hydrogenase as
an electron acceptor, making hydrogen production even more
oxygen sensitive (25). Consequently, Melis and coworkers (30)
included acetate in the medium to maintain a high level of
respiration during the early stage of the S depletion phase and
so assist in the consumption of residual O2. Melis and coworkers concluded that “acetate is consumed by respiration for as
* Corresponding author. Mailing address: School of Integrative Biology, University of Queensland, St. Lucia, Queensland 4072, Australia. Phone: 61-7-33658817. Fax: 61-7-33651699. E-mail: p.schenk@uq
.edu.au.
† Supplemental material for this article may be found at http://ec
.asm.org/.
§ These authors contributed equally.
䌤
Published ahead of print on 15 August 2008.
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NGUYEN ET AL.
EUKARYOT. CELL
quirement for high hydrogenase activity. A recent study using
NAD(P)H dehydrogenase inhibitors strongly suggested that a
NAD(P)H dehydrogenase is involved in this pathway (34).
Given the metabolic complexity of inducing and producing
H2 under S-deprived conditions, further studies are required.
Here, detailed transcriptional analyses of wild-type C. reinhardtii cultures sampled at different time points during the
aerobic and anaerobic phase of the photobiological H2 production process are presented to provide new insights into the
complex interplay between these biochemical pathways.
MATERIALS AND METHODS
FIG. 1. Photobiological hydrogen production in Chlamydomonas reinhardtii. Under aerobic conditions, electrons derived from the watersplitting reaction of PSII are passed along the photosynthetic ETC via PQ,
cytochrome b6f (Cyt b6f), PSI, and ferrodoxin (Fd) before being used in
the production of NADPH and starch, which can be used for mitochondrial respiration. H⫹ released into the thylakoid lumen by PSII and the
PQ/PQH2 cycle (H⫹ flow; dashed lines), generates an H⫹ gradient, which
drives ATP production via ATP synthase. Under anaerobic conditions,
mitochondrial respiration is not functional, H⫹/e⫺ is stored in starch, and
NADPH is used by hydrogenase for bio-H2 production.
long as there is O2 in the culture medium (0 to 30 h)” for
wild-type cells, but that “it does not contribute significantly to
the source of electrons in the H2-production process (30 to
120 h)” (30).
Although Chlamydomonas reinhardtii cultures can also
evolve H2 under dark and anaerobic conditions, the yield is
very much lower than that observed in illuminated anaerobic
cultures (16, 20). The photobiological hydrogen production
process is reported to source H⫹ and e⫺ directly from the
water-splitting reaction or via an indirect route in which they
are first stored in starch/protein (22, 30). The possibility that
some H⫹ and e⫺ are derived from acetate must also be considered. The precise contribution from the water-splitting reaction in PSII, from starch degradation and from acetate, remains a debated issue. PSII has been suggested to be the main
source of electrons for hydrogen production, since hydrogen
production was reduced to 20% when the PSII inhibitor 3-(3,4dichlorophenyl)-1,1-dimethylurea (DCMU) was added (13,
22). In addition, mutants defective in performing water photolysis or starch accumulation were also reported to have reduced H2 production capacities (38, 46). Furthermore, mutant
strains that have increased starch reserves (stm6) or the ability
to take up externally supplied glucose via an introduced glucose transporter (stm6glc4) have shown significant improvements in H2 production (8, 24). The degree of starch contribution to increased H2 production in stm6 cannot be easily
quantified because stm6 is also inhibited in cyclic electron flow
around photosystem I (PSI) (arguably reducing electron competition with the hydrogenases) as well as its respiratory metabolism. The matter is further complicated by the fact that
starch degradation not only provides H⫹ and e⫺ for photobiological H2 production but is also thought to be able to contribute to the establishment of anaerobiosis, which is the re-
Strains and growth conditions. Chlamydomonas reinhardtii wild-type strain
CC124 was obtained from the Chlamydomonas Genetics Center. The alga was
cultured in Tris-acetate-phosphate (TAP) medium (pH 7.3) (23) in sterilized
conical 2-liter Erlenmeyer flasks under continuous white fluorescent illumination
at 200 mol photons m⫺2 s⫺1 on an orbital shaker at 80 rpm. For the microarray
experiment, 3 liters of CC124 culture was inoculated from the same starter
culture which was first grown to mid-logarithmic growth phase (an optical density
at 750 nm [OD750] of 1.5 and a chlorophyll concentration of 30 mg/liter). For the
control sample, 100 ml of culture from each flask was harvested from the cultures
described above by centrifugation at 3,000 ⫻ g for 1.5 min at 4°C. The resulting
cell pellets were immediately frozen in liquid nitrogen and stored at ⫺80°C for
later RNA extractions. The remaining cultures were harvested for S deprivation
and H2 production. The cultures were washed three times to remove residual
sulfur by centrifugation at 3,000 ⫻ g for 5 min at 4°C before resuspending the
pellets in 100 ml of sulfur-free TAP medium. The washed cells were pelleted
again under the same centrifugation conditions, resuspended, and then pooled
together in a total of 2.7 liters of sulfur-free TAP medium, resulting in a sulfurfree suspension with an OD750 of 1.2 and a chlorophyll concentration of ⬃22.8
mg/liter. The suspension was poured into four custom-built 650-ml photobioreactors (PBRs) for H2 production and sample collection at later time points. The
OD of the cultures stayed relatively constant after S deprivation, and no significant growth was observed. This experimental design allows direct comparisons
between the samples taken after S depletion (time points 1 to 6 [T1 to T6]; see
details described below) and the sample just prior to S deprivation (T0).
H2 production conditions. The PBRs were tightly sealed and connected to
custom-built gas collection cylinders placed on corner positions of a six-spot HP
magnetic stirrer and illuminated from opposite sides of the stirrer unit with white
fluorescent light. In all cases, the light intensity measured at the surfaces of the
PBRs ranged from 250 E m⫺2 s⫺1 (on the darker side of the two PBRs) to 450
E m⫺2 s⫺1 (on the side closer to the light source). The cultures were stirred
continuously with magnetic bars at 150 rpm. The temperature, pH, and dissolved
O2 (DO) levels in the PBRs were recorded every 5 min by a D130 data logger
(Consort, Belgium). The volume of gas produced by the cultures was constantly
monitored once the DO level reached zero. The hydrogen gas content was
analyzed as described previously (24). The samples were collected from the
PBRs through a gas-tight septum attached to a sidearm by a syringe to ensure no
O2 was introduced into the system. Cells were immediately centrifuged (3,000 ⫻
g for 90 s at 4°C), and pellets were flash-frozen in liquid nitrogen and stored at
⫺80°C before RNA isolations. When the gas volume in the collection cylinders
was lower than 20 ml, nitrogen was used to flush the gas-collecting cylinders
before sample collections to avoid diluting the cultures with the incoming distilled water used to submerge the cylinders’ outlet tubes. Samples were taken at
six time points (T1 to T6). These correspond to the peak DO level (“peak oxygen”
production; T1), the DO level at approximately half of its peak (“mid-oxygen”
production; T2), the DO level at zero (“zero oxygen” production; T3), the hydrogen
production rate at approximately half its peak (“mid-hydrogen” production; T4), the
H2 production rate at its maximum (“peak hydrogen” production; T5), and the H2
production rate at near zero (“zero hydrogen” production; T6) (Fig. 2). The exact
times for the T1 to T6 sampling were 6 h, 16 h, 21 h, 37 h, 52 h, and 86 h after the
start of S deprivation, respectively (Fig. 2). The volume of culture collected was 20
ml each for T1, T2, T3, T4, and T5 and 50 ml for T6. The control/reference sample
taken just prior to S deprivation allowed direct comparison between samples at
different time points while minimizing the volume of culture taken from the PBRs.
RNA preparation. All samples were collected directly from the active PBRs
using a syringe through an airtight sampling port. Samples were immediately
transferred to 50-ml Falcon tubes and centrifuged at 3,000 ⫻ g for 90 s, and
the supernatant was poured off and then transferred to liquid nitrogen. This
sampling process was generally completed within 5 min; it was designed to
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TRANSCRIPTOME FOR PHOTOBIOLOGICAL HYDROGEN PRODUCTION
FIG. 2. Changes in the DO level and rate of H2 production during
the course of S deprivation-induced photobiological hydrogen production. Standard error values are shown from three independent PBRs;
arrows show sampling time points.
minimize the impact on the active PBRs and limit changes within the RNA.
Frozen cells were stored at ⫺80°C before RNA extraction. RNA was extracted following the centrifugation protocol for plant tissues using the SV
total RNA isolation system (Promega) without sample grinding. Purified
RNA from each sample was eluted in 100 l of Milli-Q H2O. RNA was
quantified using a NanoDrop ND-1000 spectrophotometer. RNA integrity
was checked by gel electrophoresis and quantitative reverse transcriptionPCR (qRT-PCR). All the control RNA samples were pooled together. Purified RNA was further purified by using the RNeasy MinElute cleanup kit
(Qiagen) before preparation of microarray probes. The RNA extracted from
each time point was used for both microarray and qRT-PCR analyses.
Preparation of fluorescent microarray probes using the indirect labeling
method. Fluorescent microarray probes were prepared by using the indirect
labeling method. Total RNA (30 to 40 g) was denatured at 70°C for 10 min
with 1 g of oligonucleotide deoxyribosylthymine-12 in a reaction mixture
volume of 15.5 l. Master mix 2 was then added to the ice-chilled reaction
mixture, containing 6 l of 5⫻ SuperScript III reverse transcriptase buffer,
1.5 l of 0.1 M dithiothreitol, 5 l of deoxynucleoside triphosphate mix (0.9
mM dTTP, 3 mM dATP, 3 mM dGTP, 3 mM dCTP, and 1.8 mM aminoallyldUTP), and 2 l of SuperScript III reverse transcriptase (200 U/l; Invitrogen). The 30-l reaction mixture was incubated at 50°C in a Thermocycler for
3 h and was then stopped by being heated to 95°C for 5 min. RNA was
hydrolyzed by the addition of 15 l of 1 M NaOH and incubation at 70°C for
15 min before the pH was neutralized by the addition of 15 l of 1 M HCl.
The amine-modified cDNA was purified using the Labeled Star purification
kit (Qiagen) according to the manufacturer’s directions and then concentrated to a 5-l volume using a vacuum centrifuge. The concentrated cDNA
was then labeled with either Alexa Fluor dye 555 or 647 (Invitrogen), 5 l of
cDNA with 3 l of 0.3 M NaHCO3, and 2 l of the Alexa Fluor succinimidyl
ester dye in dimethyl sulfoxide and was incubated at room temperature for 1 h
in the dark. The labeled cDNA from each time point following the initiation
of S deprivation was combined with the labeled control cDNA. The labeled
sample/control combined cDNA was purified with a Qiagen Labeled Star
purification kit. Purified cDNA was recovered in 25 l of Milli-Q Ultrapure
H2O. The incorporation of the fluorescent dyes into cDNA was quantified
with a NanoDrop ND-1000 spectrophotometer. A direct labeling method
using Alexa Flour 555 5-aminohexylacrylamido-dUTP (aha-dUTP) or Alexa
Flour 647 aha-dUTP (Invitrogen) was trialed at T2 (oxygen at approximately
half its peak) but was found to be less efficient than the indirect method. The
cDNA synthesis method was similar to the indirect labeling method described
above but with the following changes. Master mix 2 consisted of the following:
6 l of 5⫻ buffer, 3 l of 0.1 M dithiothreitol, 0.5 l of deoxynucleoside
triphosphates (10 mM dTTP, 25 mM dATP, 25 mM dGTP, and 25 mM
dCTP), 2 l of SuperScript II reverse transcriptase (200 U/l), and 5 l of 2
mM Alexa Fluor 555 aha-dUTP or Alexa Fluor 647 aha-dUTP. Hydrolysis,
purification, and quantification were performed as described above.
Preparation, hybridization, washing, and scanning of the microarrays.
Chlamydomonas microarray slides version 2 (9) were obtained from Arthur
Grossman (Stanford, CA). The microarray slides were immobilized, prehybrid-
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ized, hybridized, and washed as instructed in the accompanying protocol. The
slides were scanned at 532 nm and 635 nm in an Axon GenePix 4000B scanner
at a 10-m resolution. Photomultiplier tube voltages were adjusted to minimize
the background signal and the number of saturated spots. Images of the fluorescence at 532 nm (for Alexa Fluor 555) and 635 nm (for Alexa Fluor 647) were
recorded and analyzed.
Microarray data analysis. Microarray images from scanned slides were imported into the GenePix Pro 6.0 program (Axon Instruments). Spots were automatically identified using the built-in spot alignment algorithm based on the
information of spot positions provided in the GenePix array list file. All spots
were visually checked and adjusted (if necessary) manually. Spots with signals
distorted by dusts or high local background as well as spots that were marked
absent by the GenePix program were not included in the analyses. The spot
intensity medians were normalized by GeneSpring GX using the Lowess algorithm. Induction/repression ratios were obtained by dividing the sample’s median
intensity by the control’s median intensity. Spots with signal intensities more than
threefold away from the average signal intensity from all valid spots of the same
gene at one time point were not included in further analyses. At each time point,
for each gene, a paired two-way Student’s t test was conducted to determine if a
gene showed significant change (either an increase or decrease in its relative
transcript compared to the control). The null hypothesis was that the log2 of the
unaveraged ratio for a gene was 0 (sample/control ratio ⫽ 1): for H0,
log2(sample/control) ⫽ 0 (sample/control ⫽ 1); for H1, log2(sample/control) ⬍⬎
0 (sample/control ⬍⬎ 1). Further paired one-way t tests were conducted to assess
the significance of the relative transcript abundance levels, with the thresholds
being twofold changes. For this, the null hypothesis was either that the log2 of the
unaveraged ratio for a gene was not higher than 1 or was not lower than ⫺1: for
H0, log2(sample/control) ⱖ ⫺1 (sample/control ⱖ 0.5); for H1, log2(sample/
control) ⬍ ⫺1 (sample/control ⬍ 0.5); and for H0, log2(sample/control) ⱕ 1
(sample/control ⱕ 2); for H1: log2(sample/control) ⬎ 1 (sample/control ⬎ 2).
The degrees of freedom used to determine the significance of the test equaled
the number of spots that gave valid intensities for that gene minus one. In this
case, duplicate spots on the same slide were also considered pseudoindependent
samples. This approach has been used in other microarray experiments where
the number of true independent samples (biological replicates) was limited (51).
On the Chlamydomonas microarray slide v2.0, there are duplicate spots for each
gene. Therefore, in the data analysis, genes that have less than three valid spots
were excluded to ensure that at least two independent biological samples were
present in the statistical analysis. A total of 12 microarray slides were used, with
two slides for each time point and three slides for T3 and T5. The microarray
analysis for T5 (peak H2) was prepared from samples taken from three PBRs in
a separate experiment. The culturing conditions and RNA isolation were as
described previously. Labeled cDNA was prepared from total RNA using the
ChipShot indirect labeling and clean-up system (Promega), following the supplier’s protocol. The labeled cDNA was resuspended in DIG EasyHyb Solution
(Roche) and hybridized to prehybridized microarray slides in a Tecan HS
4800 hybridization station at 42°C for 2 h and washed as described previously.
Slides were scanned with a resolution of 10 m using the ScanArray 4000
(PerkinElmer), and images were processed using the ImaGene 6.0 software
(BioDiscovery). Microarray slides used for T5 analysis are from a slightly different version of the C. reinhardtii version 2 slides. These slides contain two extra
rows of spots in each subgrid, giving the well-characterized genes three replica
spots on each slide. The layout of this version can be found in the NCBI Gene
Expression Omnibus platform GPL6589.
Real-time qRT-PCR. Real-time qRT-PCR and analysis were carried out as
previously described (27, 35). A list of all primers used for qRT-PCR is shown
in Table S2 in the supplemental material. Names of the 57 genes used for
qRT-PCR (as shown in Fig. 3) encoding for proteins and corresponding to
GenBank accession numbers or annotations as reported by Eberhard et al.
(9) are as follows: LHCBM1 (AF495473); LHCBM2 (XM_001693935);
LHCBM3 (XM_001703647); LHCBM5 (XM_001697474); LHCBM6 (AF495472);
LHCBM8 (XM_001695415); LHCBM9 (AF479778); LHCBM11 (CHLRE3.0:scaffold_26: 641372:644769); TBC2, TBC2 translation factor (contig 114.36.1.0); LOX,
lipoxygenase (scaffold_11 1392588 1392986 [399 bp]); PETC, cytochrome b6f Rieske
subunit (D32003.1); PETG, cytochrome b6f complex subunit 5 (X66250); LHCA,
LHCI-4/LHCA8 (XM_001696150); LHCI, LHCA7 (XM_001691907); LHC1-6,
LHCA1 (AB122119); LHCB4 (AB051211); PSB1, oxygen-evolving enhancer 1
(OEE1; X13826); PSB3, OEE3 (X13832); FDX, ferredoxin (L10349.1); LHCSR1,
chlorophyll a/b binding protein (XM_001696086); CPX1, coproporphyrinogen oxidase (AF133672.1); OXR, putative oxidoreductase (contig 15.82.2.11); HYDA1, hydrogenase 1 (AY055755); HYDA2, hydrogenase 2 (AY090770); NPK, novel protein
kinase (U36196); RBP, RNA binding protein (contig 53.14.3.11); GPAT, putative
glycerol-3-phosphate acyltransferase (XM_001694926); VFL, variable flagellar num-
FIG. 3. Real-time qRT-PCR analysis of photosynthetic genes and other genes of interest that were identified by microarray analysis. Shown are
averages ⫾ the standard error of induction ratios of 57 genes from three independent biological replicates from six different time points before,
during, and after hydrogen production. Full gene names and accession numbers/annotations are provided in Materials and Methods.
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TRANSCRIPTOME FOR PHOTOBIOLOGICAL HYDROGEN PRODUCTION
ber protein (XM_001697461); TPR, tetratricopeptide repeat (TPR) domain-containing protein (contig 81.50.2.11); PFL, pyruvate formate lyase (PFL) (AJ620191);
ICL, isocitrate lyase (U18765.1); NAB1, nucleic acid binding protein 1 (AY157846);
SDB, scaffold attachment region DNA binding protein (contig 15.105.2.51); VSP3,
VSP3 protein (L29029); AOX1, alternative oxidase 1 (AF047832); COX2A, cytochrome c oxidase subunit 2a (XM_001697494); GLPV/PHOA, starch phosphorylase
(XM_001700039); AMYA2, alpha amylase 2 (XM_001695962); GBP, GTP binding
protein (contig 11.120.1.5); NAD, NADH ubiquinone oxidoreductase (AY347479);
Nucleolin (contig 7.42.1.5); HPP3, hypothetical protein 3 (XM_001692849); NadMDH, cytoplasmic malate dehydrogenase (AJ250844.1); NADP-MDH, chloroplast
NADP-malate dehydrogenase (AJ277281.2); ARS2, arylsulfatase 2 (X52304);
ECP88, extracellular protein 88 (AF359252); CAMK4, calcium/calmodulin-dependent protein kinase 4 (contig 25.9.2.51); SRP, hypothetical serine-rich protein
(XM_001703235); HPP1, hypothetical protein 1 (XM_001693665); HYDEF, iron
hydrogenase assembly protein (AY582739); Unknown 1 (contig 116.19.1.5);
Unknown 2 (contig 85.6.1.0); Unknown 4 (contig 8.45.1.0); Unknown 5
(XM_001698756); Unknown 7 (contig 134.13.3.11); Unknown 8 (contig
400.2.1.5); and Unknown 9 (XM_001703534).
Isolation of thylakoid membranes. For the analysis of differentially expressed
LHCBM proteins during sulfur starvation, thylakoid membranes were isolated
from a photobiological H2 production experiment identical in design to that
described above. All protein isolation procedures were performed under lowlight conditions to prevent photodamage to the isolated thylakoid membranes.
Cells were harvested by centrifugation (10 min at 2,200 ⫻ g for 4°C) and washed
in 30 ml of buffer A (25 mM HEPES [pH 7.5], 1 mM MgCl2, 0.3 M sucrose). The
washed cells were again pelleted by the same centrifugation procedure and then
resuspended in 8 ml of buffer A and broken by two passes through a French press
(2,000 lb/in2 at 4°C). The sample volume was then increased to 30 ml with buffer
A. Thylakoid membranes were precipitated by centrifugation (45 min at
20,000 ⫻ g) at 4°C. The pellet was resuspended in 30 ml of buffer B (5 mM
HEPES [pH 7.5], 10 mM EDTA, 0.3 M sucrose), centrifuged (45 min at 4°C and
48,000 ⫻ g), and finally resuspended in 2.65 ml of buffer B and 1 ml of buffer C
(5 mM HEPES [pH 7.5], 10 mM EDTA, 2.2 M sucrose) to adjust the final
sucrose concentration (not including the volume of the pellet) to 1.82 M. The
suspension was dispensed into a Beckman SW 32 Ti centrifugation tube overlaid
with 3 ml of 1.75 M sucrose solution and a further 5-ml layer of buffer D (5 mM
HEPES, [pH 7.5], 0.5 M sucrose) before centrifugation (60 min at 100,000 ⫻ g
and 4°C). The centrifuged thylakoid membrane sample formed a dense green
band at the position of the sucrose cushion step. Upon harvesting, the sample
was diluted with 5 volumes of buffer E (20 mM MES [morpholineethanesulfonic
acid], pH 6.3, 5 mM MgCl2, 15 mM NaCl, 10% [vol/vol] glycerol) to facilitate
pelleting of the purified thylakoid membranes upon centrifugation (20 min at
40,000 ⫻ g for 4°C). The thylakoid membranes were then resuspended in a
minimal volume (⬃1 ml) of MMNB buffer (25 mM MES, pH 6.0, 5 mM MgCl2,
10 mM NaCl, 2 M betaine) and flash-frozen in liquid N2 prior to storage at
⫺80°C.
Pigment measurements. Pigments were extracted from C. reinhardtii cells in
80% acetone, and the insoluble fraction was precipitated by centrifugation (1
min at 17,000 ⫻ g) before measuring the chlorophyll concentration and the
chlorophyll a/b ratio according to Arnon (2).
SDS gel electrophoresis. Polypeptide fractions from the sucrose density gradient were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% Tris-tricine gel. The stacking gel was made of 5%
acrylamide (Bio-Rad), 0.7 M Tris, pH 8.45, 0.07% SDS, 0.11% AMPS (ammonium persulfate), 3.8 l TEMED (N,N,N⬘,N⬘-tetramethylethylenediamine). The
resolving gel was made of 10% acrylamide (Bio-Rad), 0.9 M Tris, pH 8.45, 0.09%
SDS, 12.5% glycerol, 0.05% AMPS, 3.8 l TEMED. Samples from sucrose
gradient fractions were concentrated from 2 ml to ⬃30 l with Vivaspin 500 l.
Pigments were extracted from the protein samples with acetone in excess, dried
with N2 gas, and resuspended in H2O. Solubilization was conducted in a buffer
containing 0.2 M Tris, pH 6.8, 5 mM EDTA, 1 M sucrose, 0.4% dithiothreitol,
3.1% SDS, 0.8% glycerol, and bromophenol blue for approximately 15 min. Gel
lanes were loaded with an equal amount of chlorophyll (10 g). Gels were
stained with 0.1% Coomassie blue R250 and G250 for protein visualization.
Identification of proteins by MS, MALDI-TOF, and MALDI-TOF-TOF. All
bands from protein gels were excised and digested with trypsin (Promega) at 4
g/ml. The standard preparation of the calibration mixture for matrix-assisted
laser desorption ionization–time of flight (MALDI-TOF) contained des-Argbradykinin (1.0 pmol/liter), angiotensin I (2.0 pmol/liter), Glu-fibrino-peptide B
(1.3 pmol/liter), adrenocorticotropin (ACTH) 1-17 clip (2.0 pmol/liter), ACTH
18-39 clip (1.5 pmol/liter), and ACTH 7-38 clip (3.0 pmol/liter). A 10-fold
dilution of the standard calibration mixture was used for calibration of the
MALDI plates on which the tryptic digest samples were spotted. The sample
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matrix used was ␣CHCA (␣-cyano-4-hydroxycinnamic acid) at a concentration of
(5 mg/ml) in 50% acetonitrile in 5 mM ammonium phosphate and 1% formic
acid (aqueous). Samples were analyzed using a 4700 Proteomics analyzer
MALDI-tandem TOF (MALDI-TOF-TOF) (Applied Biosystems, CA). The
peptides were resuspended directly with 5 mg/ml of ␣CHCA in 60% acotinase/
0.1% formic acid (aqueous) onto a MALDI target plate. All mass spectrometry
(MS) spectra were recorded in the positive reflector mode at a laser energy
setting of 4800. For MS data, 1,000 shots were accumulated for each spectrum
using an MS-positive ion reflectron mode acquisition method. All tandem MS
(MS-MS) data from the TOF-TOF were acquired using the default positive ion,
1 kV collision energy, reflectron mode, MS-MS method at a laser energy of 5500.
MS-MS data acquisition was performed in a four-step process. First, MS spectra
were recorded from each of the six calibration spots, and the default calibration
parameters of the instrument and the plate model for that plate were updated.
Second, MS spectra were recorded for all sample spots on the plate. Each
spectrum was generated by accumulating the data from 1,000 laser shots, using
the newly updated default calibration settings. Third, the TOF MS spectra were
analyzed using the Peak Picker software supplied with the instrument. The 10
most abundant spectral peaks that met the threshold (a signal/noise ratio of ⬎20)
criteria and were not on the exclusion list were included in the acquisition list for
the TOF-TOF, MS-MS portion of the experiment. The threshold criteria were
set as follows: a mass range of 850 to 4,000 Da, a minimum cluster area of 500,
a minimum signal/noise ratio of 20, and a maximum number of MS-MS spectra
per spot of 10. A mass filter excluding matrix cluster ions and trypsin autolysis
peaks was applied. An XML file was generated that contains the list of the
precursor masses selected for MS-MS. Database searching of noninterpreted
TOF-MS and TOF-TOF MS-MS data was carried out using the Mascot search
engine (Matrix Science) (44) and the MSDB 20040630 containing 1,501,893
sequences and 480,537,669 residues with the Viridiplantae taxonomy. Proteins
were identified from the MS-MS data with confidence of at least 95%. Confidence was based on the number of peptides found in MS and the ion score from
MS-MS.
Microarray data accession numbers. The complete microarray data set has
been submitted to Gene Expression Omnibus and can be accessed at http://www
.ncbi.nlm.nih.gov/geo/query/acc.cgi?token⫽prszzawmwoakcty&acc⫽GSE9165/.
RESULTS
Global expression profiling in Chlamydomonas reinhardtii
before, during, and after hydrogen production. Cultures of C.
reinhardtii (CC124) were grown as independent biological replicates and induced for hydrogen production by sulfur deprivation. Samples for RNA extraction were taken from the PBRs
at T1 to T6 corresponding to 6 h, 16 h, 21 h, 37 h, 52 h, and 86 h
after the initiation of S depletion. The first three time points
correspond with different DO levels in the PBRs, referred as
peak oxygen, mid-oxygen, and zero oxygen production rates
(Fig. 2). The last three time points represent mid-hydrogen,
peak hydrogen, and zero hydrogen production rates (Fig. 2).
Quality control of all RNA samples was carried out by gel
electrophoresis and real-time qRT-PCR to ensure that the
RNA samples did not show any signs of degradation. Only a
limited amount of quality RNA could be obtained from T6
samples due to cell death at this late time point. Consequently,
microarray hybridizations were only conducted on samples collected during the first five time points (T1 to T5). All samples
were compared to the control taken immediately prior to sulfur deprivation (T0). Stringent analyses of the data identified
166 genes that showed at least a twofold change in one or more
of the five time points consistently across technical (duplicate
spots on the array) and biological replicates of the experiment
and that had shown statistical significance (Table 1). Based on
their closest match to GenBank entries, genes were assigned
to different groups according to their putative functions.
These include photosynthesis (22 genes), sulfur metabolism
(8 genes), carbon metabolism (4 genes), proteolysis (5
1970
NGUYEN ET AL.
EUKARYOT. CELL
TABLE 1. Induction and repression ratios of 166 C. reinhardtii genes that showed at least a twofold change in transcript abundance in one or
more time points (T1 to T5) before and during hydrogen productiona
Gene function and
GenBank accession no. or
contig (no. of genes)
GAL
file ID
Photosynthesis (22)
AB122117.1
257.A
AY171231
X15166
XM_001691032
AB050007
AF244524
AB051211
262.A
250.A
8094.D
217.A
158.A
159.A
AF170026.1
XM_001701652
J05524
X04472.1
426.A
4465.C
463.A
45.A
D32003
155.A
X92488
84.A
L10349
X13826
243.A
54.A
M15187
96.A
X13832
83.A
M36123
AF107303
352.A
458.A
XM_001696073
251.A
XM_001696086
8770.D
AF479778
350.A
Gene annotation description (closest match)
Peak O2
Mid O2
Zero O2
Mid H2
Peak H2
LhcI-4 gene for light-harvesting chlorophyll a/b
protein of PSI
Light-harvesting complex I protein
PsaK mRNA for 8.4-kDa subunit of PSI
PSI reaction center subunit XI, chloroplast precursor
CP26
Chlorophyll a/b binding protein
Light-harvesting chlorophyll a/b binding protein
Lhcb4
PSII reaction center W protein, chloroplast precursor
4.1-kDa PSII subunit (PSBX)
Plastocyanin, chloroplast precursor
RbcS2 for ribulose bisphosphate
carboxylase/oxygenase
Cytochrome B6-F complex iron-sulfur subunit,
chloroplast precursor
Cytochrome B6-F complex 4-kDa subunit, chloroplast
precursor
Ferredoxin, chloroplast precursor
Oxygen-evolving enhancer protein 1, chloroplast
precursor
Oxygen-evolving enhancer protein 2, chloroplast
precursor
Oxygen-evolving enhancer protein 3, chloroplast
precursor
Phosphoribulokinase, chloroplast precursor (PRK)
Open reading frame of intron 2 from chloroplast
psbA gene
Chlorophyll a/b binding protein homolog LHCSR1
precursor
Chlorophyll a/b binding protein homolog LHCSR3
precursor
Major light-harvesting complex II protein m9
(LHCBM9)
0.39
0.20
0.19
0.22
NA
0.36
0.31
0.47
NA
NA
0.52
NA
NA
NA
NA
NA
0.50
0.29
0.28
NA
NA
0.20
0.27
0.25
0.32
0.45
0.43
0.56
0.14
NA
NA
NA
NA
NA
0.46
NA
NA
0.27
NA
NA
NA
NA
NA
0.50
0.30
0.11
0.12
0.34
0.34
0.22
0.13
0.50
NA
0.29
NA
0.53
NA
0.30
0.36
NA
NA
NA
NA
0.90
0.27
0.21
0.56
0.44
0.77
0.11
0.42
0.12
0.42
0.27
NA
0.44
NA
NA
0.31
NA
NA
NA
0.39
0.39
NA
NA
NA
NA
NA
1.68
1.48
3.37
1.91
NA
2.84
1.93
1.30
NA
4.55
3.07
4.89
NA
NA
1.29
NA
NA
11.26
17.41
109.03
22.30
15.39
3.61
NA
NA
NA
2.19
2.96
3.25
NA
2.90
NA
NA
NA
NA
1.31
NA
7.62
2.68
NA
NA
NA
NA
2.75
2.40
18.10
3.16
0.93
NA
2.86
4.27
2.15
18.90
5.63
NA
3.96
7.25
2.22
NA
NA
2.33
Sulfur assimilation (8)
X52304
XM_001697170
XM_001699252
X16179.1
U57088
AF359251
199.8.2.31
AF359252
73.A
7778.D
8577.D
382.A
468.A
432.A
7885.D
167.A
Arylsulfatase 2 precursor (ARS2)
Sulfate transporter (SULTR2)
Sulfite reductase
Arylsulfatase 2 precursor
Probable sulfate adenylyl transferase ATS1
Extracellular polypeptide Ecp76
Extracellular polypeptide Ecp88
Extracellular polypeptide Ecp88
Carbon metabolism (4)
AF394513
U18765
X66410
U42979.1
337.A
40.A
254.A
252.A
6-Phosphogluconate dehydrogenase
Probable isocitrate lyase
Pyruvate formate lyase
Malate dehydrogenase, sodium acetate induced
2.65
0.14
NA
0.64
NA
0.11
NA
NA
1.66
0.17
3.54
NA
2.73
0.14
1.82
0.42
NA
NA
NA
0.6
Proteolysis (5)
X60826
XM_001696123
67.A
3750.C
UBI3 mRNA for ubiquitin fusion protein
26S proteasome regulatory complex, lid subcomplex,
non-ATPase subunit RPN3 (subunit 3) (PSD3)
(PSMD3)
F15M7.13, ubiquitin carboxyl-terminal hydrolase,
Arabidopsis
Ubiquitin-conjugating enzyme E2 isoform
Putative serine carboxypeptidase precursor
NA
NA
NA
3.63
1.47
2.77
3.18
0.76
NA
NA
NA
4.01
NA
2.98
NA
2.09
NA
NA
NA
6.00
2.48
NA
NA
NA
0.38
0.29
NA
72.37.1.5
7281.C
AY062935
XM_001695251
348.A
4464.C
Amino acid synthesis (4)
U36197
135.A
5-Methyltetrahydropteroyltriglutamate-homocysteine
methyltransferase
NA
NA
Continued on following page
VOL. 7, 2008
TRANSCRIPTOME FOR PHOTOBIOLOGICAL HYDROGEN PRODUCTION
1971
TABLE 1—Continued
Gene function and
GenBank accession no. or
contig (no. of genes)
GAL
file ID
Gene annotation description (closest match)
Peak O2
Mid O2
Zero O2
Mid H2
Peak H2
AF078693
U46207
XM_001695007
447.A
112.A
9339.E
Putative O-acetylserine lyase precursor
Glutamine synthetase, cytosolic isozyme
Aspartate-semialdehyde dehydrogenase (ASSD1)
NA
NA
NA
1.89
NA
0.80
3.00
0.37
0.74
2.89
0.56
0.40
10.93
NA
1.17
Transcription and
translation (10)
XM_001696696
XM_001691400
XM_001691255
AY337612
X95313
2019.1.1.5
5869.C
9120.E
4469.C
4147.C
110.A
8710.D
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
5.31
NA
NA
2.78
NA
NA
NA
2.53
2.84
4.11
NA
2.55
0.56
NA
1.45
NA
2.49
0.67
NA
XM_001696564
XM_001692618
XM_001689821
AY177616
819.C
3219.C
9565.E
312.A
PRP3, pre-mRNA processing factor 3
Eukaryotic initiation factor 4A-10-like protein
SF3A3 splicing factor 3a, subunit 3 (SPL3)
Chloroplast 30S ribosomal protein S11 (rps11)
60S ribosomal protein L11
60S ribosomal protein L22 关Drosophila melanogaster兴,
54.2% identity
60S ribosomal protein L37A
60S ribosomal protein L13
60S ribosomal protein L6
41-kDa ribosome-associated protein precursor
0.55
NA
NA
0.60
NA
NA
NA
NA
NA
NA
NA
0.42
0.33
0.43
0.35
0.48
0.82
0.53
NA
0.7
Redox cycling (3)
XM_001693558
15.82.2.11
9401.E
9635.E
Similar to vanadium chloroperoxidase
Putative oxidoreductase 关Oryza sativa subsp.
japonica兴, 82.3% identity
Thioredoxin H-type
2.72
0.67
1.28
NA
NA
3.11
3.22
1.90
7.71
NA
NA
NA
NA
0.46
NA
Protein kinase, 48K
WNK protein kinase (WNK1)
CDK inhibitor kinase (WEE1)
Rhodanese domain phosphatase (RDP3)
G protein-coupled seven transmembrane receptor
PWR motif protein (PWR1)
Selenium binding protein (SBD1)
Conserved protein similar to EMP70/nonaspanin
Ezy2
VSP-3
Alanine aminotransferase
Hydroxylamine reductase, hybrid cluster protein
HCP4
NOP58 structural component of C/D snoRNPs
Type II NADH dehydrogenase (NDA7)
Dehydrogenase with pyrroloquinoline quinone as a
cofactor
Endoplasmic reticulum-located HSP110/SSE-like
protein
Flagellar-associated protein
Coproporphyrinogen III oxidase precursor
Mu1-adaptin (AP1M1)
1-Deoxy-d-xylulose 5-phosphate reductoisomerase
(DXR1)
Zygote-specific protein
THI4 regulatory protein
Acyl carrier protein (ACP2)
Chlamyopsin 2
Cell wall protein pherophorin C2 (PHC2)
Flagellar-associated protein (FAP218)
Cell wall protein pherophorin C3 (PHC3)
0.45
NA
NA
5.99
NA
NA
5.17
NA
NA
NA
NA
2.18
NA
NA
NA
NA
3.31
3.15
NA
3.51
2.91
3.33
3.46
3.42
NA
2.86
NA
2.96
NA
2.85
2.48
NA
NA
NA
NA
NA
0.46
1.37
3.98
1.94
0.64
6.72
2.39
0.55
0.60
0.39
0.67
1.32
NA
0.74
NA
2.32
NA
NA
4.54
NA
NA
0.58
NA
NA
NA
2.42
NA
4.38
NA
NA
NA
NA
3.84
0.46
NA
1.34
NA
NA
NA
NA
NA
2.67
1.46
NA
NA
NA
NA
NA
NA
NA
NA
NA
3.04
NA
1.85
NA
NA
3.53
2.44
2.98
NA
2.40
NA
NA
0.40
0.38
0.25
0.53
NA
NA
NA
0.55
0.37
NA
NA
NA
NA
NA
0.46
NA
0.47
0.48
0.25
NA
0.52
0.55
0.28
0.44
0.28
0.36
0.43
0.35
NA
NA
NA
NA
NA
1.58
NA
Low-CO2-inducible protein (LCI30)
Low-CO2-inducible protein (LCI3)
Phenylcoumaran benzylic ether reductase homolog
TH6
CAMK4, calcium/calmodulin-dependent kinase IV
关Homo sapiens兴, 62.2% identity
NA
0.71
NA
2.31
NA
NA
NA
NA
NA
1.35
0.35
NA
0.39
0.20
6.82
NA
NA
3.21
NA
0.68
X78822
17.A
Other pathways and
processes (27)
U36196
XM_001691558
XM_001702027
XM_001692562
XM_001696853
XM_001703235
XM_001703306
XM_001698919
AF399653.1
L29029
U31975
XM_001694402
161.A
4567.C
516.B
506.B
645.C
9249.E
3414.C
4207.C
389.A
186.A
188.A
9383.E
XM_001690300
XM_001703004
XM_001702995
6995.C
8375.D
8352.D
XM_001690491
4875.C
XM_001701229
AF133672
XM_001696259
XM_001693906
2968.C
311.A
2161.C
1657.C
X76117
XM_001698620
XM_001693730
AF295371
XM_001690256
XM_001697461
XM_001697963
212.A
9223.E
166.A
63.A
9591.E
6272.C
4463.C
Unknown (83)
XM_001690783
AF015661
141.1.1.0
1650.C
477.A
5077.C
25.9.2.51
6103.C
Continued on following page
1972
NGUYEN ET AL.
EUKARYOT. CELL
TABLE 1—Continued
Gene function and
GenBank accession no. or
contig (no. of genes)
GAL
file ID
44.35.1.5
6796.C
688.1.1.0
9070.E
13.36.2.11
7462.C
26.39.1.0
2318.C
X96877
XM_001695499
483.A
1422.C
XM_001700193
668.C
3.43.1.0
4962.C
16.97.1.5
6593.C
121.14.1.5
6312.C
XM_001694590
111.18.4.11
7.42.1.5
3153.C
724.C
3362.C
XM_001697504
152.14.1.5
58.41.1.5
9998.E
1904.C
1097.C
576.2.1.0
356.1.3.11
46.52.2.11
XM_001689556
9678.E
9243.E
2626.C
3709.C
XM_001696388
116.19.1.5
XM_001700674
9338.E
8373.D
4539.C
XM_001694976
Contig 85.6.1.0
XM_001693665
XM_001689728
1.8.3.11
400.2.1.5
8.83.1.5
XM_001698598
XM_001699124
117.7.1.0
30.47.1.5
205.4.1.5
XM_001691394
103.28.2.31
667.1.1.0
17.108.1.0
XM_001699240
26.58.1.5
26.55.1.5
3.101.2.11
XM_001698756
8.45.1.0
XM_001692849
6697.C
2154.C
9725.E
5855.C
8427.D
7404.C
4854.C
8873.D
5272.C
3343.C
3534.C
1902.C
4410.C
8178.D
4037.C
6106.C
1640.C
7656.D
4842.C
6615.C
5644.C
2919.C
4703.C
XM_001695799
XM_001692679
1.59.2.11
10.23.2.11
12.107.1.5
2172.C
1212.C
3917.C
4728.C
8247.D
Gene annotation description (closest match)
Peak O2
Mid O2
Zero O2
Mid H2
Peak H2
Fibroin heavy chain precursor 关Bombyx mori兴, 6.8%
identity
Large tegument protein 关Herpes simplex virus兴, 10.7%
identity
SRP40, suppressor protein SRP40 关Saccharomyces
cerevisiae兴, 68.7% identity
EBNA-1 nuclear protein 关Human herpesvirus 4兴,
27.0% identity
Hypothetical luminal protein precursor, chloroplast
Predicted protein (SSA15) with TPR, ANK, and
zf-MYND domains
Predicted protein CAP_ED, effector domain of CAP
transcription factor
Putative glucan synthase 关Arabidopsis thaliana兴,
72.8% identity
Y25C1A.3 protein 关Caenorhabditis elegans兴, 57.9%
identity
Vegetative cell wall protein gp1 precursor
关C. reinhardtii兴, 30% identity
Hypothetical protein
Cullin 1B (Nicotiana tabacum), 99.7% identity
Nucleolin (protein C23) 关Mesocricetus auratus兴, 67.8%
identity
Hypothetical protein
CreA protein (Shigella flexneri), 55.4% identity
Fibroin heavy chain precursor (Fib-H) (H-fibroin)
关B. mori兴, 10.5% identity
NA
NA
3.75
NA
NA
2.49
NA
NA
1.19
NA
NA
4.62
NA
0.56
NA
NA
NA
NA
0.38
NA
0.57
NA
0.54
NA
0.34
3.72
0.40
1.38
0.72
NA
NA
NA
2.56
1.44
0.63
NA
NA
2.76
1.49
NA
NA
NA
3.96
1.68
NA
NA
NA
NA
2.27
NA
NA
NA
NA
2.30
2.87
6.30
NA
NA
NA
NA
NA
0.14
NA
NA
0.50
NA
NA
NA
NA
NA
NA
0.49
2.74
2.20
0.39
1.29
0.89
0.33
NA
0.61
0.56
NA
NA
NA
NA
3.73
NA
0.52
0.52
0.86
1.35
0.33
0.12
0.79
2.16
1.81
NA
NA
NA
NA
2.53
NA
NA
2.55
NA
1.39
1.56
1.62
2.23
0.44
2.68
1.57
NA
NA
NA
0.85
5.31
NA
1.73
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
6.61
NA
3.79
NA
NA
NA
NA
NA
NA
NA
NA
NA
6.32
4.04
4.90
4.17
4.40
6.04
4.03
3.91
4.64
3.88
2.14
2.42
2.64
2.39
5.22
0.47
2.39
2.85
2.63
3.64
3.26
4.02
3.46
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
3.64
0.10
3.94
1.31
4.16
0.69
0.70
1.10
1.11
1.25
1.32
1.41
1.73
0.47
0.51
0.61
0.78
0.81
0.12
0.38
0.33
0.20
0.45
2.48
NA
3.02
NA
NA
NA
NA
NA
NA
NA
NA
1.40
1.33
NA
NA
NA
NA
NA
NA
NA
NA
NA
0.74
1.76
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
2.64
2.18
2.70
2.68
0.42
1.37
NA
1.28
NA
NA
Hypothetical protein
CG2839 protein
Hypothetical protein, homology to Phytophthora
infestans CRN1
Hypothetical protein
C_1160006 关158018:161045兴
Hypothetical protein 45% identity to A. thaliana
UDP glucosyl transferase
Hypothetical protein
CAS_like, clavaminic acid synthetase-like
Hypothetical protein with ring finger domain
HC_10364 关926397:929841兴
Tax_Id⫽9606 splice isoform 1 of Q15149 plectin 1
Hypothetical protein
Hypothetical protein
Y25C1A.3 protein
Hypothetical protein
Hypothetical protein
C_1740018 关20231:20880兴
TPR repeat protein
MGC33630, hypothetical protein MGC33630
Hypothetical protein
Hypothetical protein, 26% identity to DNAdependent RNA polymerase E⬘ (ISS)
关Ostreococcus tauri兴
Hypothetical protein
Hypothetical protein
Tax_Id⫽10090 Ensembl_locations:None RAD54-like
Continued on following page
VOL. 7, 2008
TRANSCRIPTOME FOR PHOTOBIOLOGICAL HYDROGEN PRODUCTION
1973
TABLE 1—Continued
Gene function and
GenBank accession no. or
contig (no. of genes)
GAL
file ID
XM_001702460
8328.D
XM_001694296
5751.C
145.2.2.11
XM_001695405
5700.C
4344.C
XM_001702135
XM_001691387
XM_001701095
XM_001699679
XM_001702888
53.15.3.11
59.52.1.0
6.97.1.0
XM_001703534
1572.C
2256.C
5542.C
2712.C
6018.C
8244.D
1456.C
9930.E
4240.C
93.32.2.31
117.16.2.11
XM_001696892
XM_001696875
69.8.2.31
3.168.1.0
XM_001701276
XM_001701229
3081.C
9185.E
5915.C
8243.D
632.C
5052.C
9373.E
2968.C
5.123.2.31
329.4.1.5
XM_001699578
XM_001699290
341.1.1.5
XM_001703283
5749.C
4269.C
4127.C
5393.C
6015.C
2353.C
81.50.2.11
9274.E
Gene annotation description (closest match)
44% identity to molecular cochaperone STI1 (ISS)
关O. tauri兴
45% identity to PBCV-1 prolyl 4-hydroxylase (ISS)
关O. tauri兴
37% identity to PEX22 (peroxin 22), protein binding
关A. thaliana兴
Hypothetical protein
Tax_Id⫽9606
LETM1-like protein
CG10555 protein
CG1517 protein
Tax_Id⫽10090, Ensembl_locations:7-33233606
C_60084 关691340:694926兴
Hypothetical protein, 31% identity to ERD4-related
membrane protein
C_1170030 关93583:99018兴
SPRY superfamily
Hypothetical protein
Hypothetical protein
Hypothetical 38.7-kDa protein, 65.6% identity
Hypothetical protein
Flagellar-associated protein 关Chlamydomonas
reinhardtii兴
C_3290004 关33926:36211兴
Hypothetical protein
31% identity to 2OG-Fe(II) oxygenase
关Frankia sp. strain EAN1pec兴
Hypothetical protein containing the TPR domain
Peak O2
Mid O2
Zero O2
Mid H2
Peak H2
NA
NA
NA
2.24
NA
NA
NA
NA
0.17
NA
NA
NA
NA
NA
NA
NA
2.40
2.51
1.38
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
2.18
2.33
3.31
3.12
2.24
2.16
0.47
3.75
0.38
NA
NA
1.40
NA
NA
NA
NA
1.74
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
3.75
2.46
2.43
4.76
2.51
6.36
3.04
2.20
1.51
1.56
1.62
1.63
1.65
5.95
NA
NA
0.77
NA
NA
NA
NA
6.53
NA
2.94
NA
NA
0.79
NA
1.92
1.21
1.63
1.77
NA
NA
2.79
NA
NA
NA
NA
1.22
NA
NA
0.33
2.16
2.03
2.08
NA
NA
NA
1.17
3.39
3.40
NA
NA
NA
1.48
2.70
2.44
a
Shown are averages obtained from microarray data using two to three independent biological replicates. Ratios of at least twofold induction or repression that
passed all stringent criteria for data analysis, including Student’s t test (P ⬍ 0.05), are bold. Student’s t test was carried out on normalized microarray signals against
control signals to establish significant differences. A complete list, including results for all genes used for microarray analysis, is shown in Table S1 in the supplemental
material. NA, data points that did not pass the background cutoff or other stringent analysis criteria; GAL file ID, GenePix array list file identification number.
genes), amino acid synthesis (4 genes), transcription and
translation (10 genes), redox cycling (3 genes), other pathways and processes (27 genes), and a large number of genes
with unknown functions (83 genes).
Photosynthesis genes. The majority of transcripts encoding
proteins involved in photosynthesis declined soon after S deprivation and were consistently suppressed throughout the experiment (Table 1, photosynthesis). These included transcripts
encoding a ferredoxin; cytochrome b6f; plastocyanin; an oxygenevolving enhancer (OEE1/PSB1) 1, 2, and 3; subunits of PSI and
PSII; and light-harvesting complex proteins (LHCA, LHCB4, and
LHCB5). Surprisingly, there was an increase in transcript abundance of the genes encoding the major light-harvesting complex 9
(LHCBM9) and two different stress-related chlorophyll a/b binding proteins (LHCSR1 and LHCSR3) (39, 40). In addition, the
transcripts of PRK encoding phosphoribulokinase and the open
reading frame containing psbA intron 2 increased more than
twofold during mid-hydrogen production. Together with the increase of LHCBM9 and LHCSR transcripts, this result indicates
that photosynthetic activity and its regulation did not undergo a
simple decrease but underwent a more complex adjustment dur-
ing photobiological hydrogen production (PBHP) (see Discussion).
Sulfur assimilation and carbon metabolism genes. As expected, the microarray data showed strong and consistent responses to S deprivation for genes involved in S assimilation.
Expression was strongly induced at all four time points for genes
encoding arylsulfatase (ARS2) and the sulfate transporter
(SULTR2). Genes encoding sulfite reductase and extracellular
proteins ECP76 and two different ECP88 proteins were induced
in at least one time point. It was apparent that major changes also
occurred in the carbon metabolic pathways after the initiation of
S depletion. There was a marked and consistent decrease in the
transcript levels of genes encoding isocitrate lyase and malate
dehydrogenase, two enzymes in the glyoxylate cycle and the citric
acid cycle (Table 1, carbon metabolism). On the other hand,
transcript levels of the genes encoding 6-phosphogluconase dehydrogenase (an enzyme in the pentose phosphate pathway) and
PFL (belonging to the fermentative PFL pathway) increased during the H2 production phase.
Proteolysis and amino acid synthesis. There were also indications of increased protein degradations in C. reinhardtii cells
1974
NGUYEN ET AL.
during PBHP. Genes involved in protein degradation that
showed an increase in transcript abundance encode a 26S proteasome subunit S3, a putative serine carboxypeptidase, a putative ubiquitin, an E2 ubiquitin-conjugating enzyme and a
ubiquitin carboxyl-terminal hydrolase (Table 1, proteolysis).
Furthermore, transcript levels of genes involved in amino acid
synthesis, such as those encoding methionine synthase and
aspartate -semialdehyde dehydrogenase, were consistently
decreased (two- to threefold), especially at mid-H2 production
(T4). This is consistent with a previous report by Melis et al.
(30), demonstrating that significant protein degradation occurred during H2 production. The only exception was the induction of a gene encoding the putative cysteine-synthesizing
O-acetylserine lyase precursor which could also be classed to
the sulfur assimilation category.
Genes involved in transcription, translation, and regulation. Among the more than twofold differentially expressed
genes, 10 were classified as having a putative role in transcription or translation. Four of these were upregulated during the
hydrogen production phase, including the genes encoding premRNA processing factor 3 (PRP3), the eukaryotic initiation
factor 4A-10 (eIF4A-10), splicing factor 3a subunit 3 (SP3a3),
and the chloroplast 30S ribosomal protein L11 (rps11). The
genes encoding the remaining ribosomal proteins were mostly
repressed throughout the course of the experiment. Other
genes also potentially involved in regulation or signal transduction include, for example, genes encoding protein kinases
(WNK1 and WEE1), phosphatase (RDP3), or the G proteincoupled transmembrane receptor (Table 1, other pathways and
processes).
Genes involved in other pathways and processes and unspecified genes with unknown functions. A total of 27 genes
were identified with differential expression during hydrogen
production that are likely to be involved in other general cell
maintenance, regulation, and developmental processes not
specified above (Table 1). Interestingly, these include a relatively large number of genes (11) that were induced at early
time points (T1 or T2). A large number of genes (83 genes)
could not be assigned to any putative function. Although their
sequences were used for BLAST searches to check against
GenBank entries (3), none returned any significant match to
any other gene or protein with known functions.
Expression profiles using real-time qRT-PCR. Real-time
qRT-PCR was carried out on cDNA from all C. reinhardtii
CC124 samples (three independent biological replicates) and
using all six time points (T1 to T6). Among the 166 genes that
were found by microarray analysis to be significantly (P ⬍ 0.05)
differentially expressed by more than twofold, 30 were chosen
for further expression profiling by qRT-PCR using specific
primers. In addition, another 27 genes of particular interest
were subjected to further qRT-PCR analysis (Fig. 3). These
genes were grouped according to their functions (e.g., photosynthesis) or sorted by similar expression patterns. As shown in
Fig. 3A and C and in agreement with the microarray data, most
photosynthesis genes analyzed by qRT-PCR were consistently
downregulated, with the exception of LHCSR1 (encoding a
light-induced chlorophyll a/b binding protein), LHCBM9 (both
strongly upregulated in all time points), and the gene encoding
the chloroplast Rieske Fe-S precursor protein (PETC; T6
only). The upregulation of LHCBM9 was further investigated
EUKARYOT. CELL
(see below). Other repressed genes include isocitrate lyase
(ICL), light-harvesting gene translation suppressor (NAB1),
and the scaffold attachment region DNA binding protein
(SDB), as shown in Fig. 3H. Genes that were notably induced
include, as expected, the hydrogenase-encoding genes HYDA1,
HYDA2, and HYDEF as well as genes encoding a putative
glycerol-3-phosphate acyltransferase (GPAT) involved in glycerol lipid biosynthesis, the first step in phospholipid biosynthesis. Also induced were TPR, PFL, the gene encoding the variable flagellar number protein VFL, and an unknown gene
termed “Unknown 2” (Fig. 3E to G). Apart from LHCBM9,
strong induction (up to several thousand-fold) throughout
the time course experiment was found for the genes encoding arylsulfatase ARS2, the extracellular 88-kDa polypeptide
ECP88, and calcium-dependent calmodulin kinase CAMK4
(not in T1), suggesting that these genes may play important
roles during S starvation and PBHP (Fig. 3F). For the same
genes, the microarray results display the same induction trend
although at a much lower magnitude (Table 1, sulfur assimilation). A gene termed “Unknown 7” and genes encoding the
coproporphyrinogen III oxidase precursor CPX1 and a putative NADPH oxidoreductase (OXR) showed a strong induction
from T3 onwards when anaerobiosis started (Fig. 3D), making
them potential targets for hydrogen production improvements.
Genes that were differentially regulated in an antagonistic
fashion during the early S starvation (T1 and T2) and the
late-hydrogen-production phase (T5 and T6) include a lipoxygenase (LOX), cytochrome b6f complex small subunit PETG
and PETC as well as an unknown gene (“Unknown 4”); these
were generally repressed in T1 to T4 and then induced in T5
and/or T6 (Fig. 3B and G). The opposite expression pattern
(early induction followed by repression during H2 production)
was observed for a range of genes, notably those encoding
alternative oxidase 1 (AOX1), cytochrome c oxidase subunit II
(COX2A), glycogen/starch phosphorylase (GLPV), and the
gene encoding alpha amylase 2 (AMYA2) (repressed during
hydrogen production but induced in earlier time points) (Fig.
3I). Modification of these genes, together with genes listed
under transcription and translation in Table 1, including
regulatory genes, may also be suitable for further improvements of PBHP.
PSII light-harvesting complex is remodeled during S deprivation and PBHP. Our microarray analyses showed a strong
increase of LHCBM9 transcripts before (T2 and T3) and
during PBHP with induction ratios up to 109-fold (mid-H2
production; T4). To further investigate this, we carried out
detailed expression profiling of LHCBM-encoding genes,
followed by biochemical analyses of LHCBM isoforms. The
Lhc family of light-harvesting proteins in C. reinhardtii, which
capture the light required for normal photosynthesis and
PBHP, consists of 11 highly conserved proteins (LHCBM1 to
LHCBM6, -8, -9, and -11; LHCBM4 and LHCBM5) that are
predominantly associated with PSII (10), and nine proteins
(LHCA1 to LHCA9) that are more closely associated with PSI
(43). Although the LHC genes and proteins share a high degree of sequence similarity, using specific primers designed in
the 5⬘ untranslated region, qRT-PCR allowed us to distinguish
individual members of this gene family (35). Results obtained
by qRT-PCR confirmed that LHCBM9 was massively induced
(up to 3,000-fold) immediately after S deprivation (T1) and
VOL. 7, 2008
TRANSCRIPTOME FOR PHOTOBIOLOGICAL HYDROGEN PRODUCTION
FIG. 4. Induction ratios of LHCBM9 in C. reinhardtii CC124 following anaerobiosis by N2 bubbling in comparison to sulfur depletion
under aerobic or anaerobic conditions. N2 bubbling, transcripts measured at zero O2 production and under anaerobic H2 production conditions; S deplete aerobic, S-depleted cultures bubbled with air to
maintain aerobic conditions; S deplete anaerobic, anaerobiosis (microoxic conditions) induced by S depletion. Transcript levels were
determined by qRT-PCR and compared to T0 from two to three
independent biological replicates (shown are averages ⫾ the standard
error; hours show time after S depletion).
then decreased but maintained high levels along the course of
PBHP (Fig. 3A). While LHCBM9 was highly induced throughout the experiment, there was strong suppression of the other
genes encoding LHCBM isoforms (Fig. 3A). Interestingly, under normal conditions, LHCBM9 transcript abundance was
very low (⬃0.25% of that of LHCBM1). However, during S
deprivation, its abundance rose to as high as twice the combined abundance of all the other LHCBM-encoding genes
under normal conditions (data not shown).
To test whether remodeling of the antenna system of PSII
was linked not only to S deprivation but also to anaerobiosis or
PBHP, we induced PBHP solely by anaerobiosis using N2 bubbling (“microoxic H2 production”) rather than by S starvation.
In addition, we carried out S deprivation under aerobic and
anaerobic conditions. In this experiment, transcriptional regulation of LHCBM9 in cells of C. reinhardtii wild-type strain
CC124 was analyzed under the following three conditions: (i)
anaerobicity was induced by purging the culture with N2; (ii)
cells were deprived of sulfur but compressed air was bubbled to
keep the culture oxygenated; and (iii) cells were deprived of
sulfur, the culture became anaerobic, and H2 production was
induced. As shown in Fig. 4, LHCBM9 induction (sixfold) was
also observed during PBHP, following anaerobiosis independently of S starvation. It was also about 45-fold induced at 72 h
following aerobic sulfur deprivation and up to 470-fold during
anaerobic sulfur deprivation. This suggests that LHCBM9 regulation depends on both sulfur deprivation and the microoxic
H2 production process. Although LHCBM9 transcript levels
increased during anaerobic H2 production conditions induced
by N2 bubbling, S deprivation had a more marked effect. The
strongest upregulation was observed during anaerobic conditions induced by S depletion at all time points analyzed (Fig.
3A and 4). This result therefore suggests that although
LHCBM9 is upregulated during H2 production, its induction is
regulated mainly by sulfur depletion. In an independent time
course experiment under aerobic conditions, LHCBM9 showed
initial induction at 12 h, as expected, as a response to sulfur
1975
FIG. 5. Identification of C. reinhardtii CC124 thylakoid proteins
before (⫹ S) and after (⫺ S) induction of photobiological hydrogen
production by sulfur deprivation. Shown are protein bands with corresponding names after size separation by 10% SDS-PAGE. All proteins were identified in both fractions (indicated by the connecting
lines between both lanes) except for LhcbM9 and Ecp88, which were
identified only under conditions of S deprivation. Note that LhcbM9
contributed to the band labeled with the white dot. Proteins were
identified by MS-MS or by selective extraction from thylakoid membranes (17-kDa and 33-kDa extrinsic proteins).
deprivation; however, this was followed by a steady decrease in
transcript abundance until 96 h (data not shown).
To directly analyze the protein composition of the C. reinhardtii Lhc family of light-harvesting proteins under normal
and under S starvation-induced PBHP conditions, we isolated
thylakoid membrane fractions (same samples as those shown
in Fig. 4) and subjected the thylakoid proteins to one-dimensional SDS-PAGE. As shown in Fig. 5, a difference in intensity
could be observed for proteins around 27 kDa. To further
identify the proteins, bands corresponding to C. reinhardtii
before/after S thylakoid membrane proteins were excised, digested with trypsin, and analyzed by MS-MS. The proteins that
were identified with a confidence greater than 95% are listed in
Fig. 5, most notably LHCBM9 and ECP88, which were identified only in thylakoid fractions after S induction. This confirms the marked upregulation of expression for both genes
observed in the microarray and qRT-PCR studies. Although
the homology between LHCBM proteins is very high (66 to
95% amino acid sequence identity, with an average of 77%
(43), the differentiation between LHCBM9 and other LHCBM
proteins was possible thanks to a specific precursor ion which
is unique to LHCBM9, which has an observed mass of 2,540.25
Da. Among the SDS-PAGE-resolved proteins, LHCBM isoforms were by far the most dominant, followed by ␣ and 
subunits of ATP synthase.
DISCUSSION
Following microarray experiments and stringent data analysis, we were able to confidently measure the regulation of
7,124 transcripts from C. reinhardtii CC124 during sulfur de-
1976
NGUYEN ET AL.
privation at five time points: peak O2 production, mid-O2 production (consumption), zero O2 production, mid-H2 production, and peak H2 production. Among the characterized genes,
166 showed more than a twofold change in transcript abundance. Out of these, 18 genes were confirmed by qRT-PCR, 4
were not differentially regulated, and 8 did not confirm the
microarray results (Fig. 3; Table 1; see Table S1 in the supplemental material). Different results obtained by both methods
are likely to be caused by weak signals and cross-hybridization
events to other genes, a common problem with microarray
hybridizations that are carried out at relatively low temperatures (5, 14, 18, 23, 41), suggesting that further confirmations
by qRT-PCR may be required for certain genes of interest.
Nevertheless, our microarray data from three biological replicates that passed all stringent criteria for data analysis, including Student’s t test, will provide a useful platform for gene
discovery in C. reinhardtii during PBHP. The Blast search homology matches in GenBank provide a guide for putative functions of C. reinhardtii genes (listed in this paper after their
closest match), and the following paragraphs discuss the possible roles of genes during PBHP, keeping in mind that further
experimentation may be required to determine the exact functions.
Expression patterns in C. reinhardtii cells during initial
phases of PBHP reveal commonalities to responses to sulfur
stress. A large portion of the expression signature presented
here shows overlaps to results from S starvation experiments
(51), suggesting that S starvation presents a main factor for
gene expression changes in our experiment. PBHP in C. reinhardtii using sulfur depletion is a complex process. From the
start of the experiment to peak H2 production, C. reinhardtii
cells undergo two major physiological changes, S starvation
and then anaerobiosis. In the first two phases (oxygen evolution phase and oxygen consumption phase), both the cell’s
structure and its metabolic system must adapt to the lack of
sulfur supply. Consequently, the cell has to conserve S by
minimizing usage and maximizing scavenging efforts. Such adaptation has been studied in detail (37, 42, 51). Evidence at the
transcript level and some evidence at the protein level have
shown that during this phase, major cellular activities such as
photosynthesis and protein synthesis decrease, while a significant increase in the abundance of transcripts encoding stressresponsive proteins was observed. A steep decline in photosynthetic activity at 20 h after sulfur deprivation was confirmed
by pulse amplitude modulation measurements in four independent PBRs (data not shown). The following three phases (anaerobic phase, hydrogen production phase, and termination
phase) have not been analyzed for transcriptional changes until
now. During these phases, in addition to S starvation, cells also
suffer a lack of oxygen. As a result, carbon metabolism shifts
from respiration to fermentation and reduced hydrogen is released as H2 gas to circumvent NAD(P)⫹ reduction (17).
Therefore, a large number of sulfur starvation and anoxiaresponsive genes mask the process of hydrogen production.
The results showed that photosynthetic activities were reduced
under S starvation. With a few exceptions, the transcript level
of most photosynthesis genes decreased significantly throughout the first 40 h (Fig. 3A and C). Previous studies showed that
the concentration of functional PSII (QA), cytochrome b6f, and
PSI (P700) as well as the chlorophyll concentration in cultures
EUKARYOT. CELL
undergoing PBHP decreased steadily after the initiation of S
deprivation (30). In contrast to those of most photosynthesis
genes, transcripts of C. reinhardtii sulfur assimilation genes
increased significantly during PBHP in response to the lack of
sulfur in the medium to increase the capacity for S scavenging
(37, 42, 51). All transcripts encoding sulfur assimilation genes
presented in this study have been described as sulfur acclimation-specific genes (37). Our data show that cells undergoing
PBHP also display a downregulation of genes required for
amino acid synthesis and an upregulation of genes involved in
protein degradation, as expected for cells that do not multiply
but maintain only their basic functions for survival. This is in
agreement with Melis et al. (30), who reported that under
PBHP conditions, the protein concentration of the culture
decreased steadily over time.
Changes in carbon metabolism. From the beginning of S
deprivation, carbon metabolism in C. reinhardtii changed
markedly. Our results indicate a strong suppression of the
glyoxylate and Calvin cycles. Both cycles are coordinately regulated at the enzyme level to balance the energy-yielding metabolism and glucogenesis from acetate. In one glyoxylate cycle, one succinate and one NADH are produced for every two
acetyl coenzyme A (acetyl-CoA) and one NAD⫹. When
enough energy is produced through the glycolytic and citric
acid cycles, acetate is funneled into the glyoxylate cycle for the
production of glucose, amino acids, or nucleotides. Although
reducing power is generated as NADH, probably the process is
not favorable due to the lack of energy for synthesizing acetylCoA and due to the decreased demand for succinate under
S-depleted conditions. It is expected that both cycles are downregulated during PBHP, since energy supply from respiration is
very limited toward the hydrogen production phase of PBHP
(29). In contrast, the pentose phosphate pathway was induced.
Such changes are expected because the glyoxylate cycle functions only when the cell has enough energy from glycolysis and
respiration, which is not the case under microoxic or anaerobic
conditions due to S starvation. Although acetate is ubiquitous
in the S-deprived culture, acetyl-CoA is not readily available
for the glyoxylate cycle. The following conversion of acetate to
acetyl-CoA catalyzed by acetyl-CoA synthase enzyme requires
ATP: acetate ⫹ ATP ⫹ CoA 3 acetyl-CoA ⫹ ADP.
The pentose phosphate pathway produces two NADPH, one
CO2, and one ribose-5-phosphate from one glucose-6-phosphate. The five-carbon sugar can be used for nucleotide synthesis or recycled into glucose-6-phosphate. In the latter case,
the pentose phosphate can provide protection from oxidative
damage to the cell by recycling the five-carbon-sugar product
ribose-5-phosphate. This generates primarily NADPH, whose
oxidative damage protection role is rather well known (e.g., in
erythrocytes). S starvation studies have shown that cells suffer
from oxidative stress under these conditions (51). Moreover,
the NADPH produced from the pentose phosphate pathway
potentially can feed electrons into the chloroplast electron
transport chain (ETC) via one of the putative NAD(P)H dehydrogenases for hydrogen production and therefore maintains ATP production (34). An increase in transcript abundance of phosphogluconate dehydrogenase was also reported
in S-deprived culture (51). The increase in the pentose phosphate pathway indicates that C. reinhardtii cells already suffer
from redox stress at the beginning of S starvation. Although no
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TRANSCRIPTOME FOR PHOTOBIOLOGICAL HYDROGEN PRODUCTION
microarray data were available for the alternative oxidase
genes, qRT-PCR data showed a substantial increase in the
transcript abundance of AOX1 at peak and mid-O2 production
levels before gradually decreasing to below that of the control
level at mid-H2 and zero H2 production, indicating that C.
reinhardtii cells were under redox stress during the initial
phases of the experiment (Fig. 3I). Zhang et al. (51) reported
that AOX1 was repressed after 24 h of S deprivation (their first
point of measurement), which is earlier than that in our experiment. This may be attributed to slightly different conditions used in our experimental setup that led to more severe
stress (gas-tight conditions and sulfur depletion under highlight conditions). During the oxygen evolution phase, the rate
of photosynthetic O2 evolution rapidly slows down. At peak O2
levels where sample T1 was harvested, the respiration rate
began to exceed that of O2 evolution, causing O2 shortage and
redox stress in the photosynthetic ETC (Fig. 2). More evidence
came from the increase in the transcript abundance of a gene
similar to the Yersinia pestis hydroxylamine reductase (HCP)encoding gene soon after the initiation of S deprivation (Table
1). HCP has been reported to increase under redox stress (1),
and more recently, HCP was shown to be strongly induced
shortly after the initiation of dark anaerobiosis in C. reinhardtii
(33). There was also evidence that fermentative pathways increased from the start of anaerobiosis. Our data show that the
transcript encoding formate acetyltransferase (PFL) increased
substantially at T0 O2 and mid-H2 production. At those points,
the culture had used up all the DO available for respiration.
Consequently, fermentation was the only way to generate ATP
at the substrate level, with an additional mechanism being
through cyclic electron transport. By catalyzing the conversion
of pyruvate to formate, ethanol, and acetate with the production of ATP, PFL helps the cell to generate energy without the
requirement of O2. This result agrees with previous studies of
the role of the PFL pathway in C. reinhardtii carbon metabolism under anoxic conditions (3, 17).
Surprisingly, our qRT-PCR data showed that transcripts encoding starch degradation enzymes starch phosphorylase
(GLPV) and alpha amylase (AMY2) were upregulated very
early after S depletion, when starch accumulation patterns are
normally observed. Prevailing evidence at the substrate level
demonstrated that C. reinhardtii cells accumulate starch in the
early phase (20 to 30 h) of both S starvation only and in
combination with closed conditions for H2 production (4, 11,
21, 30, 44, 49, 50). Similar results to ours were also observed in
at least two other shorter time course studies of sulfur (24 h)
and phosphate (48 h) deprivation (32, 51). In these studies,
genes encoding starch phosphorylase and alpha amylase were
significantly upregulated at the same time as the gene encoding
the granule-bound starch synthesizing enzyme starch synthase
(STA2). Our data further show that GLPV (PHOA) and
AMYA2 were suppressed later during H2 production (Fig. 3I)
when starch is consumed (21). While transcriptional regulation
may not always be linked to enzymatic regulation, Moseley et
al. (32) suggested that starch degradation was required for
starch synthesis. Another possible explanation for the spatial
separation of simultaneous degradation and synthesis of starch
can also be excluded, since starch-degrading activities in C.
reinhardtii are located solely in the chloroplast (19, 26), unlike
in many higher plants where it also occurs in the cytosol. This
1977
may suggest that GLPV and AMYA2 are involved in starch
accumulation rather than degradation. Supporting evidence
from Yu et al. (48) showed that alpha amylase was not required for transitory starch breakdown in Arabidopsis spp.
leaves, while a more recent study of starch phosphorylase postulated that PHOA, which is identical to GLPV, also has starch
synthase activities (7).
The trigger for HYDA transcription is not just anaerobiosis.
Although anaerobiosis is a requirement for hydrogenase activity, it is not the only trigger for HYDA transcription. Our
qRT-PCR results clearly show that HYDA transcription was
induced soon after S starvation, long before anaerobiosis in the
medium was established, although anaerobiosis did enhance
the transcription of HYDA. After the first 16 h of our experiment (peak O2 and mid-O2 production), the DO levels were at
⬃9 mg liter⫺1, which is far above the induction level for hydrogenases reported by others, although fully aerobic sulfurdeprived culture showed repression of HYDA transcription
during the first 24 h (51). Therefore, it is possible that the
induction of HYDA1 and HYDA2 was influenced by the effect
of higher levels of light in our experiment (250 to 450 E
compared to only 100 E used by Zhang et al. [51]).
Remodeling of PSII during S-deprived PBHP. It is not surprising that a large number of differentially expressed genes
can be classified as S starvation responsive. This group of genes
is either constitutively induced or suppressed throughout the
experiment. Belonging to this group are most of the photosynthesis, sulfur assimilation, and acquiring genes and some of the
genes involved in carbon metabolism, protein synthesis, and
degradation (Table 1; Fig. 3). To this end, our data confirm
previous findings about the impacts of S starvation on the
activities of photosynthesis, S scavenging, protein degradation,
and synthesis in C. reinhardtii (51). Furthermore, we provide
strong evidence of the remodeling of PSII-LHC where
LHCBM9 and LHCSR-encoding genes were constitutively induced up to thousands-fold in spite of the dramatic repression
of genes encoding other LHCBM isoforms. This was also confirmed by our biochemical analyses of LHCBM isoforms (Fig.
5). The induction of different LHCSR isoforms is in agreement
with previous studies which suggest that LHCSR proteins protect the cell from photodamage induced by low-CO2 and highlight conditions (10, 39). The massive induction of LHCBM9
against other LHCBM isoforms is more surprising. They share
a striking number of nucleotide and polypeptide similarities,
but their individual functions are not known. Confirmed by the
biochemical analyses of the PSII-LHC, our data suggest that
LHCBM9 becomes the dominant LHCBM isoform during Lhc
remodeling under S starvation. This could be explained by the
fact that LHCBM9 displays the smallest S contents (only four
atoms) compared to all other LHCBM proteins that contain
either six or seven S atoms each.
Remodeling of light-harvesting complexes, for example via
the state transition process, is a common adaption of photosynthetic organisms in response to environmental changes
(36), in particular to optimize light energy capture and distribution to PSI and PSII and dissipation of excess energy to
minimize photooxidative damage to the PSII reaction centers.
For example, under high-light conditions, LHCII proteins can
be phosphorylated, causing them to dissociate from PSII and
to migrate to PSI where they are functionally integrated. By
1978
NGUYEN ET AL.
using pulse amplitude modulation to obtain fluorescence yield
measurements, Melis (28) and Wykoff et al. (47) showed that
the maximal fluorescence (Fmax) of S-starved cells was only
76% that of unstarved cells, indicating a larger amount of light
energy absorbed by LHCII was dissipated as heat in starved
cells. However, it is unlikely that LHCM9 replaces other
LHCBM polypeptides to increase the energy dissipation capacity,
as this can probably be better achieved by reducing the antenna
size. Moreover, LHCBM9 was found to be downregulated under CO2-limiting conditions (31), suggesting it might actively
contribute to photosynthesis. As LHCBM9 has the least number of sulfur-containing amino acids of all the major lightharvesting proteins of PSII and the relative abundance of
LHCBM9 transcript under S starvation was about one to two
times the abundance of all other LHCBM-encoding transcripts
combined under normal conditions, one could suggest that under
S-depleted conditions, other major light-harvesting complex proteins are replaced by LHCBM9 to compensate for the sulfur
shortage. According to Turkina et al. (45), LHCBM9 contains
two phosphorylation sites, which both become phosphorylated
under high-light conditions. From our additional study of the
regulation of LHCBM9 transcription by sulfur alone or anaerobiosis, which shows that further activation of LHCBM9 occurred under anaerobic conditions, one could also suggest that
LHCBM9 plays a role in state transition and/or in the cyclic
electron flow that takes place under anaerobic conditions including light. However, despite of the overall reduction in
photosynthesis due to S starvation, part of this system remains
functional or even slightly upregulated during the H2 production phase. As indicated by the qRT-PCR and the microarray
results, a number of photosynthesis genes were actually upregulated during the course of PBHP (PRK, LHCBM9,
LHCSR1, and LHCSR3). Such a selective maintenance indicates that not only the light-harvesting system but the whole
photosynthesis system are remodeled in response to S starvation and later to hydrogen production. Photosystems do not
simply decline but undergo significant transformations to prepare for hydrogen production. Further studies are required to
characterize the unknown target genes and to compare the
expression of identified genes at the protein level.
ACKNOWLEDGMENTS
We thank Elizabeth Harris (Duke University) for providing the C.
reinhardtii strain CC-137 and Arthor Grossman (Standford University)
for providing the microarrays. We are also grateful to Bernie Degnan
for the usage of the GenePix scanner and Rosanne Casu (University of
Queensland, Australia) for the help with microarray data analysis and
Alun Jones (University of Queensland, Australia) for providing us with
advice and material for MS-MS analysis.
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