Metabolic and gene expression changes triggered by nitrogen deprivation
in the photoautotrophically grown microalgae Chlamydomonas reinhardtii
and Coccomyxa sp. C-169
Joseph Msannea,d, Di Xub,1, Anji Reddy Kondac,d, J. Armando Casas-Mollanob,d, Tala Awadaa,
Edgar B. Cahoonc,d,⇑, Heriberto Ceruttib,d,⇑
aSchool of Natural Resources, University of Nebraska-Lincoln, Lincoln, NE 68588, USA
bSchool of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68588, USA
cDepartment of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, USA
dCenter for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, NE 68588, USA
a r t i c l e i n f o
Received 1 July 2011
Received in revised form 27 October 2011
Available online 5 January 2012
a b s t r a c t
Microalgae are emerging as suitable feedstocks for renewable biofuel production. Characterizing the met-
abolic pathways involved in the biosynthesis of energy-rich compounds, such as lipids and carbohy-
drates, and the environmental factors influencing their accumulation is necessary to realize the full
potential of these organisms as energy resources. The model green alga Chlamydomonas reinhardtii accu-
mulates significant amounts of triacylglycerols (TAGs) under nitrogen starvation or salt stress in medium
containing acetate. However, since cultivation of microalgae for biofuel production may need to rely on
sunlight as the main source of energy for biomass synthesis, metabolic and gene expression changes
occurring in Chlamydomonas and Coccomyxa subjected to nitrogen deprivation were examined under
strictly photoautotrophic conditions. Interestingly, nutrient depletion triggered a similar pattern of early
synthesis of starch followed by substantial TAG accumulation in both of these fairly divergent green mic-
roalgae. A marked decrease in chlorophyll and protein contents was also observed, including reduction in
ribosomal polypeptides and some key enzymes for CO2assimilation like ribulose-1,5-bisphosphate car-
boxylase/oxygenase. These results suggest that turnover of nitrogen-rich compounds such as proteins
may provide carbon/energy for TAG biosynthesis in the nutrient deprived cells. In Chlamydomonas, sev-
eral genes coding for diacylglycerol:acyl-CoA acyltransferases, catalyzing the acylation of diacylglycerol
to TAG, displayed increased transcript abundance under nitrogen depletion but, counterintuitively, genes
encoding enzymes for de novo fatty acid synthesis, such as 3-ketoacyl-ACP synthase I, were down-regu-
lated. Understanding the interdependence of these anabolic and catabolic processes and their regulation
may allow the engineering of algal strains with improved capacity to convert their biomass into useful
? 2011 Elsevier Ltd. All rights reserved.
Algae are a diverse group of eukaryotic organisms with impor-
tant roles in marine, freshwater and even terrestrial ecosystems.
For instance, 30–50% of the planetary net photosynthetic produc-
tivity (the difference between autotrophic gross photosynthesis
and respiration) is of marine origin and dependent on phytoplank-
ton biomass (Field et al., 1998; Boyce et al., 2010). Recently, the
great potential of algal species as feedstocks for renewable biofuel
production has also gained recognition (Hu et al., 2008; Scott et al.,
2010; Wijffels and Barbosa, 2010). Unicellular microalgae are capa-
ble of harnessing sunlight and CO2to produce energy-rich chemi-
cal compounds, such as lipids and carbohydrates, which can be
converted into fuels (Hu et al., 2008; Rodolfi et al., 2009; Wijffels
and Barbosa, 2010). However, the commercial production of algal
biofuels is currently hindered by limitations in the biological
productivity of strains, culture systems and harvesting/extraction
processes (Sheehan et al., 1998; Hu et al., 2008; Scott et al.,
2010; Wijffels and Barbosa, 2010). As recently proposed, a multi-
disciplinary approach, including advances in basic biology and
metabolic engineering of algal strains as well as in culture systems,
0031-9422/$ - see front matter ? 2011 Elsevier Ltd. All rights reserved.
⇑Corresponding authors. Address: Center for Plant Science Innovation, University
of Nebraska-Lincoln, Lincoln, NE 68588, USA. Tel.: +1 402 472 5611 (E.B. Cahoon),
+1 402 472 0247 (H. Cerutti).
E-mail addresses: firstname.lastname@example.org (E.B. Cahoon), email@example.com (H.
1Present address: Key Laboratory of Marine Genetics and Breeding of the Ministry
of Education, Ocean University of China, Qingdao 266003, Shandong, People’s
Republic of China.
Phytochemistry 75 (2012) 50–59
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/phytochem
bioprocessing and integrated biorefinery design, may be required
to realize the full potential of microalgae as sustainable biofuel
sources (Hu et al., 2008; Scott et al., 2010; Wijffels and Barbosa,
Desirable strain characteristics include rapid growth rate, high
product content, tolerance to variable environmental conditions,
resistance to predators and viruses and ease of harvest and extrac-
tion (Griffiths and Harrison, 2009; Rodolfi et al., 2009; Radakovits
et al., 2010). An additional algal feature for biodiesel production is
the suitability of the lipid composition, with triacylglycerols (TAGs)
being the preferred substrate (Schenk et al., 2008; Radakovits et al.,
2010). Algae synthesize, under optimal growth conditions, fatty
acids predominantly for esterification into glycerol-based mem-
brane lipids (Guschina and Harwood, 2006; Hu et al., 2008;
Khozin-Goldberg and Cohen, 2011). However, factors such as
temperature, irradiance and nutrient availability affect both lipid
composition and lipid content in many algal species (Guschina and
Harwood, 2006; Hu et al., 2008; Khozin-Goldberg and Cohen,
2011; Siaut et al., 2011). Upon certain environmental stresses
(particularly nutrient shortage), various algae accumulate energy-
rich storage compounds such as starch and TAGs (Guschina and
Harwood, 2006; Hu et al., 2008; Rodolfi et al., 2009; Wang et al.,
2009; Dean et al., 2010; Li et al., 2010; Moellering and Benning,
2010; Work et al., 2010; Fan et al., 2011; Siaut et al., 2011). Fatty
acids are the common precursors for the formation of both mem-
brane lipids (required for growth) and TAGs (involved in energy
storage and fatty acid homeostasis), but it remains to be elucidated
how algal cells coordinate the distribution of these precursors to
distinct destinationsin response
(Sheehan et al., 1998; Guschina and Harwood, 2006; Hu et al.,
2008; Radakovits et al., 2010). Interestingly, biomass productivity
and lipid content appear to be inversely correlated in many algae
(Sheehan et al., 1998; Hu et al., 2008; Rodolfi et al., 2009) and
nutrient limitation stimulatesTAG accumulation butat the expense
of growth (Rodolfi et al., 2009; Li et al., 2011).
Given the outlined constraints, improving algal strain perfor-
mance will require a greater understanding of carbon allocation
between biosynthetic pathways and of the regulatory mechanisms
controlling this distribution, particularly in response to environ-
mental stresses. This need is also emphasized by the limited
success in increasing total oil content in higher plants and algae
by the direct engineering of single lipid biosynthesis components
(Dunahay et al., 1996; Durrett et al., 2008; Li et al., 2010;
Radakovits et al., 2010; Work et al., 2010). The unicellular green
alga Chlamydomonas reinhardtii has emerged as a model system
for studying algal physiology, photosynthesis, metabolism and
flagellar structure and function (Harris, 2001; Merchant et al.,
2007). Its nuclear, plastid and mitochondrial genomes have been
sequenced and a set of genomic, molecular and genetic tools is also
available for this organism (Harris, 2001; Grossman et al., 2007;
Merchant et al., 2007). Chlamydomonas has been shown to
accumulate significant amounts of TAGs under nitrogen starvation
or salt stress (Wang et al., 2009; Dean et al., 2010; Li et al., 2010;
Moellering and Benning, 2010; Work et al., 2010; Fan et al.,
2011; James et al., 2011; Siaut et al., 2011). However, most exper-
iments characterizing storage lipid synthesis in C. reinhardtii have
been carried out under photoheterotrophic conditions, in acetate-
containing medium (Wang et al., 2009; Li et al., 2010; Moellering
and Benning, 2010; Work et al., 2010; Fan et al., 2011; James
et al., 2011; Siaut et al., 2011).
Large-scale cultivation of microalgae for biofuel production may
need to be based on sunlight, captured by photosynthesis, as the
main source of energy (Hu et al., 2008; Griffiths and Harrison,
2009; Rodolfi et al., 2009; Scott et al., 2010; Wijffels and Barbosa,
2010). Thus, metabolic and gene expression changes occurring in
Chlamydomonas subject to nitrogen deprivation under strictly
photoautotrophic conditions were examined. TAG and starch syn-
theses were also analyzed, under analogous environmental condi-
tions, in another alga with sequenced nuclear, plastid and
mitochondrial genomes, Coccomyxa sp. C-169 (Smith et al., 2011).
This microalga belongs to the class Trebouxiophyceae, within the
division Chlorophyta (Palmqvist et al., 1997; Zoller and Lutzoni,
2003), and it is very divergent in phylogeny and habitat from C.
reinhardtii (see below). Interestingly, nitrogen depletion triggered
a similar pattern of early synthesis of starch followed by significant
TAG accumulation in these fairly different green microalgae. The
results also suggest that turnover of nitrogen-rich compounds in
the nutrient starved cells may provide carbon/energy for TAG bio-
synthesis. Understanding the interdependence of these anabolic
and catabolic pathways and their regulation may be key for the
metabolic engineering of algal strains with improved biofuel
2.1. Cell growth and triacylglycerol accumulation in C. reinhardtii
subject to nitrogen deprivation
To analyze the effect of nitrogen (N) depletion on Chlamydo-
monas grown under photoautotrophic conditions, the wild-type
CC-125 strain was pre-cultured to middle logarithmic phase in
high salt (HS) minimal medium (Harris, 1989). Cells were then
Fig. 1. Growth and nonpolar lipid accumulation of Chlamydomonas reinhardtii CC-
125 subject to nitrogen deprivation. Cells were cultured photoautotrophically, for
the indicated times, in high salt medium (HS + N) or in the same medium lacking
nitrogen (HS ? N). (A) Growth curves displaying changes in cell density over time.
Each data point represents the average of three independent experiments (±SE). (B)
Fluorescence microscopy detection of nonpolar lipid accumulation by Nile Red
staining. The images shown are representative of typical cells at the different time
points. In each medium series, the upper panels correspond to merged transmitted
light and fluorescent images whereas the lower panels correspond to fluorescent
images. Scale bars equal 10 lm.
J. Msanne et al./Phytochemistry 75 (2012) 50–59
centrifuged, resuspended in either HS or HS ? N medium at a den-
sity of 0.5 ? 106cells mL?1and grown under photoautotrophic
conditions for 6 days. Cultures were sampled daily for specific
analyses. In nitrogen replete medium, cell density increased ?8-
fold during the examined period whereas in medium lacking nitro-
gen cells approximately doubled in number within the first 48 h
followed by an arrest in cell division (Fig. 1A). As reported for Chla-
mydomonas grown in medium containing acetate (Work et al.,
2010), it was also noticed in this study that the average cell size in-
creased during acclimation to nitrogen starvation (Fig. 1B), pre-
sumably as a consequence of the greater accumulation of carbon
storage compounds (see below).
To assess lipid accumulation during the growth period, cells
were examined by fluorescence microscopy after staining with
the nonpolar lipid fluorophore Nile Red (Greenspan et al., 1985;
Chen et al., 2009). Lipid body formation, as revealed by Nile Red
fluorescence, increased significantly in nitrogen-stressed cells rela-
tive to those cultured in nutrient-replete medium (Fig. 1B). The
levels of lipid-derived fatty acid methyl esters (FAMEs) were also
analyzed, by gas chromatography–flame ionization detection
(GC–FID), to evaluate if the detected increase in nonpolar lipids
during nitrogen deprivation corresponded to TAGs. Total cellular
lipids were extracted and either derivatized and quantified by
GC–FID or separated by thin layer chromatography (TLC). The
TAG fraction, identified by co-migration with a purified TAG stan-
dard, was recovered from the TLC plate and its fatty acid content
and composition also analyzed by GC–FID. Total fatty acid (FA)
content per cell remained relatively constant, and similar to that
of cells grown in nutrient replete medium, during the first 2 days
of nitrogen starvation (Fig. 2A). However, FA levels showed an in-
crease after 72 h in nutrient-depleted medium and doubled after
Fig. 2. Total fatty acid and TAG accumulation in Chlamydomonas CC-125 subject to
nitrogen deprivation under photoautotrophic conditions. Cells were incubated for
the indicated times in HS medium, either nutrient replete (+N) or nitrogen depleted
(?N). Values indicate the mean of three independent experiments (±SE). (A) Total
fatty acid content expressed as nanograms per 1000 cells. (B) Fatty acids in TAGs
expressed as nanograms per 1000 cells. (C) Fatty acids in the TAG fraction expressed
as percentage of the total FAs in a cell.
Fatty acid composition (wt.% of total FAs ± SD, n = 3) of the total lipid extract (TLE) or purified triacylglycerols (TAG) from C. reinhardtii cells cultured in HS ? N medium for the
16:016:1 16:3 16:4
17.0 ± 0.23.4 ± 0.23.1 ± 0.1 20.2 ± 0.3 2.2 ± 0.12.7 ± 0.1 3.8 ± 0.2 5.3 ± 0.75.5 ± 0.2 31.9 ± 0.43.7 ± 0.31.1
20.4 ± 1.32.9 ± 0.4 2.9 ± 0.1 17.3 ± 0.5 2.1 ± 0.16.5 ± 0.9 3.7 ± 0.26.6 ± 0.55.8 ± 0.7 27.3 ± 0.83.3 ± 0.4 1.2
21.9 ± 3.42.7 ± 0.53.1 ± 0.2 14.8 ± 3.32.3 ± 0.28.1 ± 2.2 3.9 ± 0.47.9 ± 1.15.9 ± 0.1 24.9 ± 3.03.0 ± 0.31.5
27.5 ± 0.31.8 ± 0.2 2.9 ± 0.1 9.7 ± 0.82.6 ± 0.1 10.6 ± 1.34.5 ± 0.3 9.7 ± 0.4 6.8 ± 0.4 19.9 ± 1.12.4 ± 0.21.5
30.6 ± 0.6 1.7 ± 0.12.7 ± 0.16.9 ± 0.22.9 ± 0.112.1 ± 0.54.4 ± 0.312.3 ± 0.26.6 ± 0.216.1 ± 0.62.1 ± 0.21.6
31.6 ± 0.51.9 ± 0.12.5 ± 0.15.6 ± 0.22.7 ± 0.115.6 ± 0.64.3 ± 0.313.3 ± 0.26.0 ± 0.212.9 ± 0.51.9 ± 0.21.8
Fig. 3. Starch, chlorophyll and protein contents in Chlamydomonas CC-125 subject
to nitrogen deprivation under photoautotrophic conditions. Cells were cultured for
the indicated times in HS medium, either nutrient replete (+N) or nitrogen depleted
(?N). Values indicate the mean of three independent experiments (±SE). (A) Starch
content expressed as nanograms per 1000 cells. (B) Chlorophyll amount expressed
as nanograms per 1000 cells. (C) Total protein content expressed as nanograms per
1000 cells. The hatched bars indicate soluble protein amounts.
J. Msanne et al./Phytochemistry 75 (2012) 50–59
6 day of nitrogen deprivation. The proportion of FAs in TAGs also
increased significantly in nitrogen-stressed cells (Fig. 2B), repre-
senting ?70% of the total fatty acids in the cells by the end of
the examined period (Fig. 2C).
The fatty acid composition of C. reinhardtii subject to nitrogen
starvation for 6 days was similar to that of the predominant TAG
fraction (Table 1 and Supplementary Table 1). Cells grown in nutri-
ent replete medium were rich in polyunsaturated FA species char-
acteristic of membrane lipids, in particular 16:4D4,7,10,13 and
18:3D9,12,15 (a-linolenic acid) (Table 1). In contrast, nitrogen
starved cells showed an increased abundance of saturated fatty
acids and of those with lower degree of unsaturation such as
16:0 (palmitic acid), 18:1D9 (oleic acid) and 18:2D9,12 (linoleic
acid) (Table 1). This compositional bias, similar to that of many
plant oils (Cahoon and Schmid, 2008), was even more pronounced
in the TAG fraction of cells starved for nitrogen during 6 days (Ta-
ble 1 and Supplementary Table 1).
2.2. Changes in starch, chlorophyll and protein content in C. reinhardtii
subject to nitrogen deprivation
To characterize further the metabolic changes triggered by
nitrogen shortage under photoautotrophic conditions, starch, chlo-
rophyll and protein levels in nutrient-stressed cells were also ana-
lyzed. Starch accumulation occurred very rapidly upon incubation
in nitrogen depleted medium, increasing by ?14-fold after 2 days
of nutrient deprivation and reaching maximal level 1 day latter,
at ?18-fold the amount of starch measured in cells cultured in reg-
ular HS medium (Fig. 3A). Interestingly, the majority of starch syn-
thesis appears to take place prior to the most significant
accumulation of TAGs (cf. Figs. 3A and 2B).
Nitrogen-starved cells had a yellowish appearance compared to
those grown under nutrient replete conditions, resulting from a
marked decrease in chlorophyll content with time of exposure to
nitrogen depleted medium (Fig. 3B). Indeed, cells incubated in
nitrogen free medium for 6 days had ?20% the chlorophyll amount
measured in cells cultured in standard HS medium. Similarly, the
content of total and soluble proteins also decreased substantially
in nutrient deprived cells (Fig. 3C). Chlamydomonas cells subjected
to nitrogen deprivation for 6 days had ?26% of the total protein
amount in cells grown under nutrient replete conditions. Immuno-
blot assays also indicated a pronounced reduction, triggered by
nitrogen shortage, in specific proteins such as the chloroplast lo-
cated large subunit of ribulose-1,5-bisphosphate carboxylase/oxy-
genase (Rubisco) (involved in CO2 fixation) and tryptophan
synthase b subunit (involved in tryptophan biosynthesis) as well
as in subunits of the cytosolic ribosome, like ribosomal protein
S16 (Fig. 4). These three polypeptides decreased to between 25%
and 30% of their normal levels after 6 days of nutrient depletion.
In contrast, nitrogen starvation only caused a minor reduction in
the steady-state amount of other proteins, such as histone H3
(Fig. 4). These observations, taken together, are consistent with
progressive loss of certain plastidial functions, such as photosyn-
thesis and amino acid biosynthesis, and of cytosolic protein trans-
lation capabilities triggered by nitrogen depletion. However,
histone proteins, that may be critical for preserving chromatin
organization, appear to be much less affected by nutrient
2.3. Triacylglycerol and starch accumulation in Coccomyxa sp. C-169
subject to nitrogen deprivation
To begin assessing the representativeness of Chlamydomonas as
a model system for studying metabolic processes triggered by
nutrient starvation in green microalgae, the lipid and starch con-
tents in cells of Coccomyxa sp. C-169 experiencing nitrogen deple-
tion were also investigated. Coccomyxa belongs to the class
Trebouxiophyceae (together with species of the genus Chlorella)
within the green algae and it is quite divergent phylogenetically
from Chlamydomonas which groups with members of the class
Chlorophyceae (Palmqvist et al., 1997; Zoller and Lutzoni, 2003;
Smith et al., 2011). In addition, strain C-169 originated from Mar-
ble Point Antarctica (Holm-Hasen, 1964) and is representative of
algae adapted to an extreme environment as opposed to Chlamydo-
monas CC-125 which was reportedly collected from a potato field
in Amherst, MA (Harris, 1989). Thus, it was reasoned that if similar
metabolic responses to nitrogen deprivation are observed in
Coccomyxa and Chlamydomonas, they might be indicative of
processes conserved in a wide spectrum of green microalgae.
However, Coccomyxa cells are smaller and grow slower, photo-
autotrophically under ambient levels of CO2, than those of
Chlamydomonas (data not shown). This limitation, which among
other species-specific differences may be related to the lack of a
carbon concentrating mechanism in Coccomyxa (Palmqvist et al.,
1997), required longer incubation times in nitrogen free medium
to assess metabolic changes triggered by nutrient depletion.
As observed in Chlamydomonas, Coccomyxa cells examined by
fluorescence microscopy, after staining with the nonpolar lipid
fluorophore Nile Red, also showed a significant increase in lipid
body formation when subjected to nitrogen deprivation (Fig. 5A).
The analysis of FAMEs by GC–FID established that total fatty acid
content per cell remained relatively constant during the first
4 days of nitrogen starvation but did rise markedly after that,
attaining an ?80% increase by the end of the examined period
(Fig. 5B). The proportion of FAs in TAGs also augmented consider-
ably in nitrogen-stressed cells (Fig. 5C), representing ?70% of the
total fatty acids in the cells after 11 days of nutrient deprivation
Coccomyxa cells grown in nutrient replete medium were rich in
polyunsaturated FA species, in particular 18:2 and 18:3 (Table 2
and Supplementary Table 2). In contrast, nitrogen starved cells
showed a marked increase in the abundance of the monounsatu-
rated fatty acid 18:1 (Table 2 and Supplementary Table 2). These
changes in FA composition were similar in trend to those occurring
in Chlamydomonas under nitrogen deprivation, that is an increase
in fatty acids with lower degree of unsaturation (cf. Tables 1 and
2). However, there were also species-specific differences. In
Chlamydomonas, palmitic acid became the predominant FA in cells
Fig. 4. Immunoblot analysis of specific polypeptides in C. reinhardtii CC-125 subject
to nitrogen deprivation under photoautotrophic conditions. Cells were grown for
the indicated times in HS medium with (+) or without (?) nitrogen. Whole cell
protein extracts were separated by SDS–PAGE, transferred to nitrocellulose and
probed with antibodies raised against ribosomal protein S16 (RPS16), tryptophan
synthase b subunit (TSb), the large subunit of ribulose-1,5-bisphosphate carbox-
ylase/oxygenase (Lg Sub Rubisco) or histone H3 (H3). The panels show represen-
tative images from one out of three independent experiments. Numbers below the
blots indicate the relative abundance of the examined proteins.
J. Msanne et al./Phytochemistry 75 (2012) 50–59
subject to nitrogen starvation whereas in Coccomyxa oleic acid
showed the greatest abundance.
Starch and chlorophyll contents in nitrogen-stressed Coccomyxa
cells were also analyzed. Starch synthesis occurred much rapidly
than TAG accumulation, reaching a peak after 4 days of nitrogen
deprivation (Fig. 6A). However, in relative terms, the amount of
synthesized starch was much lower in Coccomyxa than in Chla-
mydomonas cells (cf. Figs. 3A and 6A). Nutrient shortage also re-
sulted in a significant decrease in chlorophyll content in
Coccomyxa. Cells incubated in nitrogen free medium for 11 days
had ?25% of the chlorophyll amount measured in cells cultured
in standard medium (Fig. 6B). One caveat with the Coccomyxa
experiments is that the small cell size and solid cell wall made it
difficult to break the cells and extract metabolites, reducing the
accuracy of starch measurements and making the analysis of pro-
tein content unreliable (data not shown). Nonetheless, the obser-
vations made herein suggest that TAG and starch accumulation,
triggered by nitrogen depletion, followed similar overall patterns
in Chlamydomonas and Coccomyxa grown photoautotrophically.
2.4. Expression of lipid biosynthesis genes in C. reinhardtii subject to
Homologs of many proteins involved in eukaryotic lipid meta-
bolic pathways are encoded in the C. reinhardtii genome (Riekhof
et al., 2005; Merchant et al., 2007; Khozin-Goldberg and Cohen,
understood, asis the caseinmostmicroalgae(Hu et al., 2008;Miller
et al., 2010; Khozin-Goldberg and Cohen, 2011). Thus, steady-state
transcript levels for a few lipid biosynthesis genes were examined
to assess whether their expression was consistent with the rela-
tively late pattern of TAG accumulation under nitrogen stress
Fig. 5. Lipid accumulation in Coccomyxa sp. C-169 subject to nitrogen deprivation
under photoautotrophic conditions. Cells were incubated for the indicated times in
BBM medium, either nutrient replete (+N) or nitrogen depleted (?N). (A) Fluores-
cence microscopy detection of nonpolar lipid accumulation by Nile Red staining.
The images shown are representative of typical cells at the different time points. In
each medium series, the upper panels correspond to merged transmitted light and
fluorescent images whereas the lower panels correspond to fluorescent images.
Scale bars equal 2.5 lm. (B) Total fatty acid content expressed as nanograms per
1000 cells. (C) Fatty acids in TAGs expressed as nanograms per 1000 cells. (D) Fatty
acids in the TAG fraction expressed as percentage of the total FAs in a cell. Values
indicate the mean of three independent experiments (±SE).
Fatty acid composition (wt.% of total FAs ± SD, n = 3) of the total lipid extract (TLE) or purified triacylglycerols (TAG) from Coccomyxa sp. cells cultured in BBM ? N medium for the
16:016:116:2 16:3 18:118:218:3Other
0 day (TLE)
4 day (TLE)
6 day (TLE)
11 day (TLE)
11 day (TAG)
17.5 ± 0.7
18.1 ± 0.7
17.0 ± 0.1
16.1 ± 0.4
11.4 ± 0.3
8.7 ± 1.0
1.1 ± 0.5
0.8 ± 0.1
1.3 ± 0.2
0.4 ± 0.0
7.8 ± 0.5
2.9 ± 0.1
1.7 ± 0.3
1.5 ± 0.0
0.7 ± 0.1
10.8 ± 0.8
5.6 ± 0.1
4.6 ± 0.3
3.9 ± 0.1
2.5 ± 0.1
7.5 ± 0.1
30.4 ± 0.2
40.1 ± 2.6
44.3 ± 1.7
57.7 ± 0.3
20.8 ± 0.3
20.2 ± 0.3
16.6 ± 1.1
15.1 ± 0.2
13.7 ± 0.2
23.6 ± 2.4
17.4 ± 0.5
14.8 ± 0.9
13.1 ± 0.1
10.3 ± 0.3
Fig. 6. Starch and chlorophyll contents in Coccomyxa sp. C-169 subject to nitrogen
deprivation under photoautotrophic conditions. Cells were grown for the indicated
times in BBM medium, either nutrient replete (+N) or nitrogen depleted (?N). (A)
Starch content expressed as nanograms per 1000 cells. Values correspond to the
average of two independent experiments. (B) Chlorophyll amount expressed as
nanograms per 1000 cells. Values indicate the mean of three independent
J. Msanne et al./Phytochemistry 75 (2012) 50–59
(Fig. 2B). The Chlamydomonas genome contains six genes coding for
diacylglycerol:acyl-CoA acyltransferases (DGATs), which catalyze
the last step in the Kennedy pathway of TAG biosynthesis, the acyl-
ation of diacylglycerol to TAG (Weiss and Kennedy, 1956; Coleman
and Lee, 2004; Courchesne et al., 2009). Based on primary sequence
homology to functionally characterized eukaryotic enzymes, five
genes encode DGAT2-like enzymes whereas the remaining one
codes for a DGAT1 type isoform (Merchant et al., 2007; Khozin-
Goldberg and Cohen, 2011; Turchetto-Zolet et al., 2011). However,
no transcripts were detected, by reverse transcription-polymerase
chain reaction (RT-PCR) assays, for one of the DGAT2 genes (DGTT5;
ProtID: 536379)(datanot shown).The remainingDGAT2like genes,
DGTT1 (Prot ID: 536226), DGTT2 (Prot ID: 519435), DGTT3 (Prot ID:
523869) and DGTT4 (Prot ID: 190539), as well as the unique DGAT1
gene (Prot ID: 536378) were tested for expression during the nitro-
gen deprivation period by semi-quantitative RT-PCR (Fig. 7).
While this manuscript was being prepared, Miller et al. (2010)
reported a transcriptome study of C. reinhardtii subject to nitrogen
deprivation under photoheterotrophic conditions. Their analysis
focused predominantly on changes in transcript abundance occur-
ring at 48 h after nitrogen depletion in medium containing acetate.
Under these conditions, DGTT1 showed a large increase in steady-
state transcript levels whereas the other DGAT genes displayed
only minor or no change in expression (Miller et al., 2010). In the
current study, it was also observed, under strictly photoautotro-
phic conditions, an early and substantial up-regulation of DGTT1
in cells subject to nitrogen starvation (Fig. 7). Additionally, DGTT3
and DGTT4 were also expressed at significantly higher levels, in
particular after 6 days of nitrogen deprivation (Fig. 7). These obser-
vations suggest that DGTT1 may contribute to TAG synthesis early
during nitrogen starvation but several other DGAT genes may also
play a role during the period of maximal TAG accumulation under
photoautotrophic conditions. Intriguingly, DGAT1-like enzymes
are primarily involved in TAG biosynthesis in seeds of Arabidopsis
thaliana (Zhang et al., 2009), but the only gene encoding this type
of isoform in Chlamydomonas was expressed at relatively low levels
and not affected by nitrogen depletion (Fig. 7).
The transcript abundance for two genes involved in de novo
fatty acid synthesis was also examined. KASI, 3-ketoacyl-ACP Syn-
thase I, is a component of the multimeric fatty acid synthase II
(FASII) and catalyzes the acyl–acyl carrier protein (acyl-ACP)
dependent elongation steps from C4 to C16 in higher plants (Baud
and Lepiniec, 2010; Miller et al., 2010). The only gene in the Chla-
mydomonas genome coding for KASI (KASI; Prot ID: 205887) was
found to decrease in expression, under photoautotrophic condi-
tions, in cells subject to nitrogen depletion (Fig. 7). KAR, 3-ketoa-
cyl-ACP reductase, is also part of the FASII complex and reduces
the carbon 3 ketone to a hydroxyl group during FA synthesis (Baud
and Lepiniec, 2010). This enzyme is also encoded by a single gene
in the Chlamydomonas genome (KAR; Prot ID: 335991) and its tran-
script abundance decreased slightly in nitrogen deprived cells
(Fig. 7). These results were somewhat surprising since doubling
of the FA content in cells starved for nitrogen (Fig. 2A) is strongly
suggestive of de novo fatty acid synthesis. Moreover, experiments
with cerulenin, a specific inhibitor of the 3-ketoacyl-ACP synthase
of FASII, have also implicated de novo FA synthesis in TAG accumu-
lation in nitrogen-deprived Chlamydomonas, albeit under photo-
heterotrophic conditions (Fan et al., 2011). Interestingly, in the
transcriptome analysis of Miller et al. (2010) the KASI mRNA in-
creased by ?2-fold after 48 h of nitrogen deprivation in acetate
containing medium. As discussed below, FASII enzymatic activity
may not be limiting for TAG accumulation under our experimental
conditions but differences in substrate availability under photoau-
totrophic or photoheterotrophic conditions may cause variations in
the response of algal metabolic pathways to nitrogen depletion.
C. reinhardtii accumulates both starch and TAGs when subject to
a number of stresses such as nitrogen deprivation, high salinity,
sulfur depletion or when exposed to high light (Klein, 1987; Ball
et al., 1990; Matthew et al., 2009; Wang et al., 2009; Dean et al.,
2010; Doebbe et al., 2010; Li et al., 2010; Moellering and Benning,
2010; Work et al., 2010; Fan et al., 2011; Kropat et al., 2011; James
et al., 2011; Siaut et al., 2011). Several groups have begun charac-
terizing the metabolic pathways involved in TAG synthesis in
nutrient stressed cells of this organism (Wang et al., 2009; Dean
et al., 2010; Li et al., 2010; Miller et al., 2010; Moellering and
Benning, 2010; Work et al., 2010; Fan et al., 2011; Siaut et al.,
2011). However, most studies have been performed under photo-
heterotrophic conditions (Wang et al., 2009; Li et al., 2010; Miller
et al., 2010; Moellering and Benning, 2010; Work et al., 2010; Fan
et al., 2011; James et al., 2011; Siaut et al., 2011). In this case,
acetate in the medium appears to contribute substantially to the
accumulated TAGs (Fan et al., 2011 and data not shown) and, pos-
sibly, to the synthesized starch (Ball et al., 1990; Work et al., 2010).
Coccomyxa species that participate, as lichen photobionts, in sym-
biotic associations with fungi are also capable of accumulating
TAGs but the effect of environmental factors on lipid metabolism
is poorly understood in these algae (Guschina et al., 2003).
Microalgae are considered a promising source of renewable
biofuels. Yet, for the production of algal biofuels to become
economically feasible, various studies have underlined the neces-
sity of low-cost culture systems, using sunlight as the sole or main
Fig. 7. Expression of lipid biosynthesis genes in Chlamydomonas reinhardtii CC-125
subject to nitrogen deprivation under photoautotrophic conditions. Cells were
grown for the indicated times in HS medium with (+N) or without (?N) nitrogen.
Transcript abundance corresponding to specific genes involved in lipid synthesis
was analyzed by semi-quantitative RT-PCR. Reactions were performed as described
under experimental procedures in the presence (+RT) or absence (?RT) of reverse
transcriptase. The panels show representative images of agarose resolved RT-PCR
products stained with ethidium bromide. Examined genes included those encoding
diacylglycerol:acyl-CoA acyltransferases type 2 (DGTT1, DGTT2, DGTT3 and DGTT4)
and type 1 (DGAT1), 3-ketoacyl-ACP synthase I (KASI) and 3-ketoacyl-ACP reductase
(KAR). Amplification of the mRNAs corresponding to ACT1 (encoding actin), COX4
(encoding mitochondrial cytochrome c oxidase subunit Vb) and COX12 (encoding
mitochondrial cytochrome c oxidase subunit VIb) were used as controls for equal
amounts of input RNA and for the efficiency of the RT-PCRs.
J. Msanne et al./Phytochemistry 75 (2012) 50–59
energy source for biomass synthesis (Hu et al., 2008; Griffiths and
Harrison, 2009; Rodolfi et al., 2009; Scott et al., 2010; Wijffels and
Barbosa, 2010). Thus, metabolite accumulation in Chlamydomonas
and Coccomyxa cells subject to nitrogen deprivation was examined
under strictly photoautotrophic conditions, so that the information
gained may be useful for understanding the biology of production
strains (Morowvat et al., 2010). Nutrient depletion led to an arrest
in cell division and an increase in starch and TAG content. In the
absence of the nitrogen necessary for protein synthesis and cell
growth, excess carbon from photosynthesis appears to be chan-
neled into storage molecules, such as starch and TAGs. However,
a detailed time course analysis of the metabolic changes triggered
by nitrogen depletion suggests that turnover of non-lipid cellular
components may also play a role in TAG accumulation. The
involvement of two processes, photosynthesis and recycling of pre-
viously assimilated carbon, in the synthesis of lipids has also been
proposed for the diatom Cyclotella cryptica subjected to silicon
deficiency (Roessler, 1988).
Upon nitrogen deprivation, Chlamydomonas cells initially accu-
mulate starch, which increases its content ?14-fold during the first
2 days of stress (Fig. 3A). Starch biosynthesis likely involves newly
fixed carbon through photosynthesis since chlorophyll and protein
contents remain relatively high at the beginning of the nutrient
deprivation (Figs. 3B, C and 4). In contrast, there is virtually no
change in total fatty acid levels on a per cell basis during this period
first 48 h of stress (Fig. 1A), maintaining a steady-state amount of
fatty acids per cell implies the occurrence of de novo synthesis. The
majority of the FAs are likely devoted to preserving membrane
homeostasis but a small fraction may also be used for incipient
TAG biosynthesis (Fig. 2B). Although, our data cannot discriminate
whether FAs employed in early TAG production are synthesized de
novo and/or recycled from pre-existing lipids such as those of the
plastid membranes (Wang et al., 2009; Siaut et al., 2011).
At latter timepoints, Chlamydomonas cells show clear accumula-
tent (Fig. 2B and C). Conversely, starch levels display a slight decline
afterprolonged incubationinnitrogendeprived medium,which be-
comes more obvious after 10 days of nutrient stress (data not
shown). These observations suggest considerable de novo fatty acid
synthesis in cells depleted of nitrogen for several days, but the syn-
synthetically) assimilated carbon since chlorophyll and enzymes
like Rubisco, that are essential for CO2fixation, are greatly dimin-
in photosynthesis and overall anabolic processes has been reported
phic and photoheterotrophic conditions (Martin and Goodenough,
1975; Bulte and Wollman, 1992; Li et al., 2010; Miller et al., 2010;
Work et al., 2010). Thus, in C. reinhardtii cultured under photoauto-
trophic conditions in nitrogen depleted medium, fatty acids for TAG
biosynthesis may be partly obtained at the expense of the carbon/
energy assimilated in other cellular components, in particular pro-
teins which decrease substantially in content (Figs. 3C and 4), ribo-
somal RNAs recycled as a consequence of ribosome degradation
and, to some degree, starch and chlorophyll (Fig. 3A and B). Conver-
lar lipids,undernitrogenlimited conditions, hasalso beenproposed
Coccomyxa cells cultured in nitrogen deprived medium accumu-
lated less starch, in relative terms, than Chlamydomonas but a sim-
ilar proportion of TAGs, representing ?70% of the total fatty acids
in the cells after 11 days of nutrient deprivation (Figs. 5 and 6).
The predominant fatty acid in Coccomyxa triacylglycerols was oleic
acid, whereas palmitic acid showed the greatest abundance in
Chlamydomonas. Interestingly, despite these species-specific differ-
ences, both Coccomyxa sp. C-169 and C. reinhardtii CC-125 dis-
played similar trends in the accumulation of starch and TAGs and
in the reduction of chlorophyll content triggered by nitrogen depri-
vation. Given the substantial divergence in habitat and phylogeny
between these algal species (see Section 2.3), these observations
suggest that certain metabolic responses to N shortage may be
shared by a broad range of green microalgae.
TAG biosynthesis can occur by several enzymaticmechanisms in
eukaryotes (Hu et al., 2008; Baud and Lepiniec, 2010; Khozin-
Goldberg and Cohen, 2011). In the Kennedy pathway, DGATs cata-
lyze the acylation of diacylglycerol to TAGs (Weiss and Kennedy,
1956; Coleman and Lee, 2004; Courchesne et al., 2009). Although
the specific mechanisms of TAG synthesis in microalgae are poorly
characterized, increased abundance of the transcripts for several
DGAT homologs (this work and Miller et al., 2010), particularly at
latter time points during nitrogen starvation, suggests that these
enzymes may play a role in Chlamydomonas. In contrast, transcripts
for KASI and KAR, subunits of the fatty acid synthase II complex
(Baud and Lepiniec, 2010), appear to decrease as cells are incubated
photoautotrophically in nitrogen depleted medium. These results
are counterintuitive since doubling of the FA content in nitrogen
starved Chlamydomonas cells is strongly suggestive of de novo fatty
of KASI, have implicated de novo FA synthesis in TAG accumulation
in medium containing acetate (Fan et al., 2011). Additionally, KASI
tion in the presence of acetate (Miller et al., 2010). With the caveat
matic activities, we hypothesize that expression of (some) genes
In this context, normal FASII activity may not be limiting for TAG
accumulation under photoautotrophic conditions, as precursors
for fatty acid synthesis may be scarce, likely derived at least in part
from the recycling of previously assimilated carbon in proteins,
ribosomal RNAs, chlorophyll and possibly starch. However, KASI
up-regulation may be necessary when exogenous acetate provides
an abundant precursor for lipid synthesis (Fan et al., 2011). This
interpretation is consistent with the ?35% greater accumulation of
TAGs in cells subject to nitrogen deprivation for 6 days in acetate
containing medium in comparison with those cultured in minimal
medium (data not shown).
The primary product of CO2fixation, 3-phosphoglycerate, feeds
directly into the starch biosynthesis pathway but it can also be
used as a precursor of acetyl-CoA for FA synthesis and of the glyc-
erol backbone of TAGs (Hu et al., 2008; Radakovits et al., 2010).
This prompted several groups to investigate whether biosynthesis
of starch and TAGs compete with each other and whether Chla-
mydomonas mutants defective in starch synthesis would accumu-
late higher levels of lipids under nitrogen starvation (Wang et al.,
2009; Li et al., 2010; Work et al., 2010; Siaut et al., 2011). Despite
some promising results (Wang et al., 2009; Li et al., 2010; Work
et al., 2010) the relationship between these two metabolic path-
ways appears to be more complex than mere competition (Work
et al., 2010; Li et al., 2011; Siaut et al., 2011), emphasizing our
incomplete understanding of the interdependence and regulation
of metabolic processes in microalgae. Additionally, the results pre-
sented here suggest that recycling of other non-lipid cellular com-
ponents may also contribute carbon/energy for TAG synthesis
under nitrogen depletion.
4. Concluding remarks
Nutrient deprivation is one of the common stresses encoun-
tered by microorganisms in nature (Elser et al., 2007). Under
J. Msanne et al./Phytochemistry 75 (2012) 50–59
nutrient replete conditions, growth is promoted via increased tran-
scription and translation, processes that require large amounts of
anabolic structural components such as ribosomes (Warner,
1999). Indeed, proteins are the dominant fraction in the biomass
of fast-growing photosynthetic microorganisms (Langner et al.,
2009; Huo et al., 2011). In contrast, under nutrient depletion,
growth is inhibited and most of the anabolic machinery becomes
superfluous (Acquisti et al., 2009). In this context, anabolic struc-
tural components, such as ribosomes and chlorophyll, may be tar-
geted for degradation and nutrient recycling (Kraft et al., 2008).
When exogenous nitrogen is depleted, selective autophagic pro-
cesses may function to make available endogenous nitrogen for
limited de novo protein synthesis so that cells can adapt and
change their fate, including in the case of Chlamydomonas differen-
tiation into sexual gametes and acquisition of the ability to survive
a prolonged nutrient stress (Martin and Goodenough, 1975; Ball
et al., 1990; Wang et al., 2009). The recycled excess carbon likely
provides substrates/energy for the synthesis of nitrogen-poor stor-
age compounds such as TAGs. A greater understanding of these
metabolic processes may allow the genetic engineering of algal
strains with increased capacity to convert their biomass into useful
5.1. Strains and culture conditions
C. reinhardtii CC-125 and Coccomyxa sp. C-169 were used in all
the reported experiments. Unless stated otherwise, cultures were
incubated under continuous illumination (180 lmol m?2s?1pho-
tosynthetically active radiation) on an orbital shaker (190 rpm) at
25 ?C and ambient levels of CO2. Cells were initially grown photo-
autotrophically to the middle of the logarithmic phase in nitrogen
replete high salt (HS) medium (Sueoka, 1960), in the case of
Chlamydomonas, or Bold’s basal medium (BBM) (Bold, 1949), in
the case of Coccomyxa. These pre-cultured cells were collected by
centrifugation and resuspended at a density of 0.5–1.0 ? 106
cells mL?1in regular HS or BBM media or in the same media lack-
ing nitrogen (HS ? N or BBM ? N). Samples for analysis were taken
immediately after resuspension (0 day) and at the times indicated
in the figures and tables. Culture growth was monitored by count-
ing cells with a hemocytometer (Harris, 1989).
5.2. Fluorescence microscopy
To assess the effect of nitrogen deprivation on nonpolar lipid
accumulation, cells were stained with the lipophilic fluorophore
Nile Red (Greenspan et al., 1985) as previously described (Chen
et al., 2009). Images were acquired with a laser scanning confocal
microscope (Olympus Fluoview 500), using an 100? oil immersion
lens, and analyzed with the Fluoview (v4.3) software. Laser excita-
tion was at an emission wavelength of 443 nm and Nile Red fluo-
rescence was detected between 560 and 590 nm using band-pass
filtering. In the presence of nonpolar lipids, Nile Red emits a yel-
low-gold fluorescence (kmax= 580 nm) (Greenspan et al., 1985),
which is shown as red pseudo coloring in Figs. 1B and 5A.
5.3. Lipid analyses
Total lipids were extracted with a Bligh and Dyer (1959) proce-
dure, transmethylated and quantified as fatty acid methyl esters
(FAMEs) by gas chromatography with flame ionization detection
(GC–FID) as described previously (Cahoon et al., 2006). Briefly,
?1.5 ? 108
13 ? 100 mm glass test tube with Teflon-lined screw cap. After
cellswere collectedby centrifugation ina
(300 lg) (Nu-Chek Prep) was added as an internal standard from
a stock solution at a concentration of 10 mg mL?1in toluene. Lipids
were then extracted from the cell pellet using a modification of the
method described by Bligh and Dyer (1959). MeOH:CHCl3(3 mL,
2:1, v/v) containing 0.01% (w/v) butylated hydroxytoluene were
added to the cell pellet and incubated at 25 ?C for 30 min. After
addition of CHCl3(1 mL) and H2O (1.8 mL), the tube was shaken
vigorously and the content partitioned into two phases by centrifu-
gation at 1000g. The upper phase was discarded and the lower or-
ganic phase, containing the extracted lipids, was transferred to a
new glass tube. Extracted lipids were dried under a stream of N2
and resuspended in CHCl3:MeOH (0.5 mL, 6:1, v/v). One fourth of
this extract was used for measuring total fatty acid content and
the remainder was used for measuring TAGs (see below). For anal-
ysis of the content and composition of fatty acids an aliquot of the
lipid extract (125 lL) was transferred to another glass screw cap
test tube, dried under N2, and resuspended in toluene (250 lL)
and H2SO4in MeOH (1 mL, 2.5%, v/v). The tube was capped under
N2and heated at 90 ?C for 30 min. Upon cooling, H2O (0.5 mL) and
heptane (0.7 mL) were added. The tube was shaken vigorously, and
the contents were separated into two phases by centrifugation. The
upper heptane layer containing FAMEs was analyzed using an Agi-
lent 7890 gas chromatograph (Agilent Technologies) fitted with an
Agilent INNOWax column (0.25 mm inner diameter ? 30 cm
length). The oven temperature was programmed from 185 ?C
(1 min hold) to 235 ?C (2.5 min hold) at a rate of 7 ?C/min with
hydrogen as the carrier gas. FAMEs levels were quantified relative
to the methyl heptadecanoate from the internal standard. Different
fatty acid methyl esters were identified by mobility relative to
standards as well as by gas chromatography–mass spectrometry
using an Agilent 7890A gas chromatograph interfaced with an Agi-
lent 5975C mass selective detector. Chromatography conditions in
the latter case were the same as described above, except for the use
of helium as the carrier gas.
For analysis of TAGs, the remainder of the total lipid extract was
dried under N2and resuspended in CHCl3:MeOH (100 lL, 6:1, v/v).
This extract was applied to a silica 60 thin layer chromatography
plate (Sigma–Aldrich) and neutral lipids were resolved using a sol-
vent system of 70:30:1 (v/v/v) heptane:Et2O:AcOH. The TAG band
was identified by co-migration with a TAG standard, stained lightly
with iodine vapors, in an adjacent lane. The TAG fraction was then
recovered from the plate and lipids resuspended in 0.4 mL of
toluene and H2SO4in MeOH (1.5 mL, 2.5%, v/v). Fatty acid methyl
esters were prepared, extracted, and analyzed by gas chromatogra-
phy as described above.
Double bond positions of unsaturated fatty acids were con-
firmed by GC–MS of derivatives generated from fatty acid methyl
esters. Pyrrolidine derivatives were prepared and analyzed for dou-
ble bond positions of polyunsaturated fatty acids (Andersson et al.,
1975), and dimethyl disulfide derivatives were prepared and ana-
lyzed for double bond positions of monounsaturated fatty acids
(Cahoon et al., 1994). Analyses of fatty acid derivatives was con-
ducted with an Agilent 7890A gas chromatograph interfaced with
an Agilent 5975C mass selective detector fitted with a DB-1MS col-
umn (Agilent; 30 m length ? 0.25 mm inner diameter ? 0.25 lM
film thickness). The GC oven was programmed from 185 ?C
(1 min hold) at 10 ?C/min to 320 ?C (10 min hold).
of culture mediaby aspiration, triheptadecanoin
5.4. Starch assays
Starch measurements were performed using an EtOH-washed
chlorophyll-free cell pellet (Ball et al., 1990). In brief, ?2.0 ? 107
cells were harvested by centrifugation, resuspended in an ethanol
solution for chlorophyll extraction and then centrifuged again.
Pellets were resuspended in distilled H2O and boiled for 10 min
J. Msanne et al./Phytochemistry 75 (2012) 50–59
to solubilize the starch. In the case of Coccomyxa, resuspended pel-
lets were autoclaved for 15 min at 120 ?C for starch solubilization.
Total starch was quantified using a commercial kit (Starch Assay
Kit SA-20; Sigma–Aldrich), based on amyloglucosidase digestion
to convert starch to glucose, according to the manufacturer’s
5.5. Chlorophyll measurements
Chlorophyll content was determined using EtOH extraction
(Arnon, 1949). An aliquot (1 mL) of culture, at a concentration of
1.0 ? 107cells mL?1, was centrifuged and the pellet resuspended
in 96% ethanol and vortexed to extract pigments. Cellular debris
was pelleted by centrifugation and chlorophyll a and b levels were
determined spectrophotometrically, in the supernatant, by mea-
suring optical absorbance at 645 and 663 nm. Calculations of total
chlorophyll (lg mL?1) were performed as previously described
(Arnon, 1949; Harris, 1989).
5.6. Protein determination
The concentration of solubilized proteins extracted from
?5.0 ? 106Chlamydomonas cells was determined using the Bio-
Rad Protein Assay, by measuring absorbance at 595 nm with a
microplate reader. Briefly, cells were sonicated for 20 s (3?) in lysis
buffer (250 lL, 50 mM Tris–HCl, pH 8.0, 20% guanidine hydrochlo-
ride, 10 mM EDTA, 10 mM DTT, 0.4 lM PMSF) and, after addition of
0.05% Triton X-100, centrifuged at 12,000g for 10 min. The super-
natant was used for protein determination by the Bradford assay
(Bradford, 1976) following the manufacturer’s instructions (Bio-
Rad). Dilutions of bovine serum albumin were used to prepare a
series of protein standards. Total protein was measured by Lowry
analysis with a commercially available kit (Sigma–Aldrich).
5.7. Immunoblot analyses
The steady-state level of selected proteins in Chlamydomonas
was examined by Western blotting as previously described (van
Dijk et al., 2005). Histone H3 and Ribosomal Protein S16 were de-
tected with commercially available antibodies (ab1791 and
ab26159, respectively; Abcam). The antibody against the large sub-
unit of Rubisco was a generous gift of Robert Spreitzer (University
of Nebraska-Lincoln) whereas the antibody against tryptophan
synthase b subunit was kindly provided by Thomas McKnight
(Texas A&M University).
5.8. Semi-quantitative RT-PCR assays
Total RNA was isolated from Chlamydomonas cells with TRI re-
agent (Molecular Research Center), in accordance with the manu-
facturer’s instructions, and treated with DNase I (Ambion) to
remove contaminating DNA. Reverse transcription reactions were
carried out as previously described (Carninci et al., 1998) and the
synthesized cDNAs were then used as template in standard PCR
reactions (Sambrook and Russell, 2001). The numbers of cycles
showing a linear relationship between input RNA and the final
product were determined in preliminary experiments. Most prim-
ers were designed to match exonic sequences flanking one or more
introns to distinguish contaminating PCR products possibly gener-
ated by amplification of any remaining DNA. Controls also included
the use as template of reactions without reverse transcriptase and
verification of PCR products by hybridization with specific probes
(data not shown). The PCR conditions for amplification of most
templates were 30 cycles at 94 ?C for 30 s, at 56 ?C for 30 s and
at 72 ?C for 20 s. Aliquots (5 ll) of each RT-PCR were resolved on
1.5% agarose gels and visualized by ethidium bromide staining.
The primer sequences were as follows: for DGAT1 511566-RT-F1,
50-ACTGGTGGAATGCGGCTAC-30and 511566-RT-R1 50-TAGCAGCT
CGTGGAACACAG-30; for DGTT1 285889-RT-F1 50-GAAGCAGGTG
TCT-30; for DGTT2 184281-RT-F1 50-GCGCCGCAACATTTACATGG-30
and 184281-RT-R1 50-CAGCCGTACTCGGTCTTGTG-30; for DGTT3
188937-RT-F1 50-GTCAGAGCCAAGTGCTGGAC-30and 188937-RT-
R1 50-TCCACCTCCTTGTCGAACTC-30; for DGTT4 190539-RT-F1 50-
GCATGTTTGGGCAGTACGGC-30and 190539-RT-R1 50-GCCTTGTGC
TTGTCGTACAG-30; for DGTT5 141301-RT-F1 50-AGTCACTGCAGCA
GCTGTCG-30and 141301-RT-R1 50-GCCCACACACATCATGAGCG-30;
for KASI 205887-RT-F1 50-CAGTGTGCTGCGGAATGC-30and 205887-
RT-R1 50-GGTCACACAAACACACATTTGA-30; for KAR 335991-RT-F1
50-GTCATCGGCCTGACCAAG-30and 335991-RT-R1 50-ATGCCCTT-
GACCTCTAC-30and ACT-Cod-R 50-GATCCACATTTGCTGGAAGGT-30;
for COX4 187882-RT-F1 50-GGATAAGTTCGGCACTGAGG-30
195712-RT-F1 50-CACTGCTTTGTGCGATTCAA-30and 195712-RT-
and for COX12
This research was funded in part by the NSF EPSCoR Research
Infrastructure Improvement Grant Track 1: Nanohybrid Materials
and Algal Biology (EPS-1004094 to E.B.C. and H.C.) and by the
Center for Advanced Biofuel Systems (CABS), an Energy Frontier
Research Center funded by the U.S. Department of Energy, Office
of Science, Office of Basic Energy Sciences under Award Number
DE-SC0001295 to E.B.C. The authors also acknowledge the support
of the U.S. Department of Energy Research for Developing Renew-
able Biofuels from Algae (DE-FG36-08GO88055 to H.C.). J.M. was
supported by a graduate scholarship from the School of Natural
Resources, University of Nebraska-Lincoln.
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