The mRNA abundance of pepc2 gene is negatively correlated with oil content in Chlamydomonas reinhardtii

b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 1 8 1 1 e1 8 1 7

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The mRNA abundance of pepc2 gene is negatively correlated with oil content in Chlamydomonas reinhardtii Xiaodong Deng a, Yajun Li a, Xiaowen Fei a,b,* a

Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, Haikou 571101, China b Department of Biochemistry, Hainan Medical College, Haikou 571101, China

article info

abstract

Article history:

Many microalgae accumulate oils, especially under stress conditions, thus have attracted

Received 20 October 2010

more and more attention as a potential feedstock for biodiesel production. However, little

Received in revised form

information is available for molecular mechanisms that control oil accumulation in micro-

23 December 2010

algae. In this study, we investigated the accumulation of oils during nitrogen (N) deprivation

Accepted 5 January 2011

in the model green algae Chlamydomonas reinhardtii. Our results indicated that oil content

Available online 4 February 2011

significantly increased after N deficiency in C. reinhardtii, which was accompanied by a drastic

Keywords:

enzyme that converts phosphoenolpyruvate (PEP) to oxaloacetate (OAA), which conjugates

Phosphoenolpyruvate carboxylase

with acetyl-CoA to generate citric acid. Furthermore, down regulation of pepc2 gene by RNAi

gene

silencing in C. reinhardtii led to 14e28% increase in oil content. These results revealed

Oil accumulation

a negative correlation between pepc2 mRNA abundance and oil content in C. reinhardtii.

decrease (63e91%) in the transcript level of phosphoenolpyruvate carboxylase 2 (PEPC2), an

ª 2011 Elsevier Ltd. All rights reserved.

RNAi Chlamydomonas reinhardtii Nitrogen deprivation

1.

Introduction

In recent years, the quick depletion of fossil fuels and the ever increasing concern about negative environmental impact resulting from the consumption these conventional energy sources have made renewable energy such as biodiesel fuel a very attractive alternative to power a sustainable economic development. Microalgae, a large and diverse group of simple organisms of unicellular and multicellular forms, are of growing importance as potential sources for biodiesel production. Compared to traditional feedstocks such as soybean and rapeseed, microalgae have higher oil content and higher growth rates, usually capable of doubling their biomass within one day. These organisms often contain 20e50% oils in their dry biomass [1]. Moreover, microalgae assimilate carbon

dioxide (CO2) as the carbon source for growth, thus can contribute significantly to the mitigation of atmospheric CO2. Finally, the production of microalgae does not need arable land, thus will not compete natural resources with food production. Because of these advantages, microalgae biodiesel has been considered by many scientists as the only feasible source to significantly or even completely replace fossil fuels in the foreseeable future [1e3]. Microalgae tend to accumulate higher oil content under unfavorable growth conditions like nutrient deficiency. For example, numerous reports have showed that nitrogen deprivation induces oils accumulation in many microalgae species [4e13]. However, very little is known about the molecular mechanisms underlying such induction. Phosphoenolpyruvate carboxylase (PEPC) is a ubiquitous cytoplasmic enzyme present in a wide spectrum of organisms,

* Corresponding author. Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, Haikou 571101, China. Tel.: þ86 898 66960173; fax: þ86 898 66890978. E-mail address: [email protected] (X. Fei). 0961-9534/$ e see front matter ª 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biombioe.2011.01.005

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including archaeal, (cyano)bacterial, unicellular green algal and vascular plants [14e16]. In C4 and crassulacean acid metabolism (CAM) plants, PEPC plays an important role in CO2 fixation. However, in C3 plants and non-photosynthetic tissue, this enzyme catalyzes the irreversible conversion of phosphoenolpyruvate (PEP) to oxaloacetate (OAA), and thus is involved in the citric acid cycle for the production of a series of intermediate metabolites used as carbon bones in basic metabolisms [17,18]. A few studies have showed that high activity of PEPC negatively affects oil accumulation in higher plants. For example, Chen et al. [19] reported that oil content increased by 6.4e18% in the transgenic rapeseed lines in which the expression of pepc was blocked by antisense RNA. However, whether similar results can be obtained in microalgae is unknown. Green algae Chlamydomonas reinhardtii harbor two isoforms of pepc, pepc1 and pepc2 [20e23]. PEPC1 is a homotetramer of w102 kDa with full catalytic activity. On the other hand, PEPC2 is a multi-subunit complex composed of a heteromeric complex that includes a w102 kDa subunit and several immunologically unrelated polypeptides of w65 kDa, w73 kDa and w130 kDa in Selenastrum minutum and of w50e70 kDa and w130 kDa in C. reinhardtii. In C. reinhardtii, the abundant PEPC2 has higher catalytic efficiency and higher affinity for the substrate PEP than PEPC1. In this study, we investigated the expression profiles of pepc2 during the C. reinhardtii cells maintained in nitrogen (N)-normal and N-deficient media by using real-time RT-PCR analysis. Our results indicated that in C. reinhardtii, the levels of pepc2 transcript are significantly lower under the N deprivation condition (P < 0.01). Interestingly, lower pepc2 transcript was accompanied by higher oil content in this organism. To further investigate the correlation between pepc2 transcription and oil accumulation in C. reinhardtii, we built an RNAi construct against pepc2 to reveal that oil content in C. reinhardtii CC124 can be significantly increased by down regulating pepc2. Thus, pepc2 mRNA abundance negatively correlates with oil content in C. reinhardtii.

2.

Materials and methods

2.1. assay

Algal strain, cultivation conditions and biomass

C. reinhardtii CC124 (mt-) was purchased from the Chlamydomonas Genetic Center of Duke University. Cells grown on Trisacetate-phosphate (TAP) [24] agar plate were inoculated into 100 mL Erlenmeyer flasks containing 50 mL of HSM and N-deficient HSM (HSM-N) media, respectively. HSM medium is composed of NH4Cl 0.500 g L1, MgSO4$7H2O 0.020 g L1, CaCl2$2H2O 0.010 g L1, K2HPO4 1.440 g L1, KH2PO4 0.720 g L1, NaAc 2.000 g L1, H3BO3 0.001 g L1, MnCl2$4H2O 0.005 g L1, ZnSO4$7H2O 0.022 g L1, FeSO4$7H2O 0.005 g L1, CoCl2$6H2O 0.002 g L1, Na2Mo7O24$4H2O 0.002 g L1, Na2$EDTA 0.050 g L1and the HSM-N medium contains the same components in which NaCl was replaced with NH4Cl. Other media and the corresponding N-restriction media used in this work are showed in Table 1. All cultures were maintained in an incubator shaker with 230 rpm at 25  C, and exposed to a continuous illumination at a light intensity of 150 mmol m2 s1.

Table 1 e Other media and their N-restriction media used in this work.

NaNO3 K2HPO4$3H20 MgSO4$7H2O CaCl2$2H2O Citric acid FeC6H5O7 EDTA NaCO3 A5 þ Co solution

NaNO3 K2HPO4$3H20 MgSO4$7H2O CaCl2$2H2O KH2PO4 NaCl FeCl3$6H20 Fe-EDTA A5 solution

Tris-Base Glacial acetic acid K2HPO4 KH2PO4 NH4Cl MgSO4$7H2O CaCl2$2H2O Trace

BG11 (mg L1)

BG11-N (mg L1)

250 40 75 36 6 6 1 20 1 ml

NaCl/172 40 75 36 6 6 1 20 1 ml

SE (mg L1)

SE-N (mg L1)

250 75 75 25 175 25 5 10 1 ml

NaCl/172 75 75 25 175 25 5 10 1 ml

TAP (mg L1)

TAP-N (mg L1)

2420 1 ml/L 119 61 400 100 50 1 ml

2420 1 ml/L 119 61 NaCl/437 100 50 Trace-N

Biomass concentration (g/L) was determined by measuring the optical density of samples at 490 nm (OD490). To produce the standard curve, a series of C. reinhardtii CC124 samples of different biomass concentrations were collected. Their OD490 value and cell dry weight were determined gravimetrically after drying the algal cells to plot the standard curve of OD490 versus biomass concentration (g/L). Samples were diluted by appropriate ratios to ensure that the measured OD490 values were in the range of 0.15e0.75 if applicable. Biomass concentration was then calculated by following formula: Cell dry weight (g/L) ¼ 0.5484  OD4900.0411.

2.2.

Oil content analysis

The Nile Red fluorescence method was used to determine neutral lipids levels [25]. The algal cells were directly stained with 0.1 mg/mL (final concentration) Nile Red for 10 min, and then fluorescence was measured on a GloMax-Multi Detection System (Promega, USA), with excitation and emission wavelengths of 470 nm and 570 nm, respectively. The fluorescence value was calculated by the equation: FD (470/ 570) ¼ (A2A1), where A2 is the fluorescence value of algal cells after staining with Nile Red, A1 is that of the algal cells before staining. For establishing the relationship of the fluorescence value of the samples and their neutral lipid content, a standard curve graph had been drew by preparing different concentration of Triolein (Sigma, USA) and detecting their fluorescence value after stained with Nile Red. The lipid content of the algal

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cell was then calculated using the following formula: Lipid content (g/g) ¼ [0.0004  FD (470/570)  0.0038]  0.05/cell dry weight. For microscopic assay, after staining the cells with Nile Red (10 mg/mL final concentration), images were acquired using a Nikon 80i Fluorescence Microscopes. Nile Red signals were captured using an excitation wavelength of 480 nm, and emission was collected between 560 nm and 600 nm [26,27].

2.3.

RNA extraction

Total RNA was prepared using the Trizol Reagent (Shanghai Sangon, China). After a series of phenol-chloroform extraction, nucleic acids were precipitated with 2 volumes of absolute ethanol, and then washed with 75% ethanol. Finally, the air-dried pellet was dissolved in 40 mL of RNase free water. Concentration of the RNA was determined by spectrophotometry and the integrity was examined by agarose gel electrophoresis.

amplified by PCR using primers Pepc2F 50 -CGGGTAAGGCAAGTGGT-30 and Pepc2R 50 -TCGGAGCTTTATGTGAGAAT-30 . PCR reactions were performed in a final volume of 25 ml containing 1 PCR reaction buffer, 2 mM MgCl2, 0.4 mmol of each primer, 0.25 mM dNTPs, 1 mL DMSO, 0.5 M Betain and 0. 5 U Taq DNA polymerase (Promega, USA) using the following program: 4 min at 95  C; 35 cycles of denaturation for 40 s at 95  C, annealing for 40 s at 58  C, and elongation for 20 s at 72  C; 10 min at 72  C. After purification using the EZ Spin Column DNA Gel Extraction Kit (BBI, Canada), the DNA was inserted into vector pMD18-T following the manufacturer’s instructions (TaKaRa, Japan). The resulting plasmid was designated as pMD18T-Pepc2. DNA sequence analyzed was performed by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China).

2.5. 2.4.

Construction of a RNAi vector against the pepc2 gene

Cloning of a fragment of the pepc2 gene

lipid content (g/g).

4

Cell dry weight (g/L)..

6

HSM

0.4

HSM-N

0.1 0

SE

0.4

4

6

0.3 0.2 0.1 0

0 0.4

2

4

BG11

6 BG11-N

0.1 0

4

A

0

6

8

2

4

6

HSM

0.5 0.4 0.3

8

HSM-N

0.2 0.1 0

0

2 SE

0.15

4

6

8

SE-N

0.1 0.05 0

0

0.2

2

0.2

8

0.3

0

0.4

8

SE-N

TAP-N

0.6

0

0.2

2

TAP

0.8

8

0.3

0

lipid content (g/g).

2

Cell dry weight (g/L)..

lipid content (g/g).

0

TAP-N

Cell dry weight (g/L)..

TAP

0.25 0.2 0.15 0.1 0.05 0

To construct the RNAi vector, a fragment of C. reinhardtii 18S gene was amplified with primers 50 -CGAACTTCTGCGAAAGCAT-30 and 50 - TCAGCCTTGCGACCATACT-30 and inserted into pMD18-T to give pMD18Te18S. The fragment of pepc2 and its

Cell dry weight (g/L)..

lipid content (g/g).

The first strand of cDNA was synthesized by SuperScript Ⅲ Reverse Transcriptase (Invitrogen, USA) according to manufacturer’s instructions. A fragment of the pepc2 gene was

2

4

BG11

0.15

6

8

BG11-N

0.1 0.05 0

0

2

4

6

8

B

Fig. 1 e The lipid content (A) and growth curve (B) of C. reinharadtii CC124 in TAP, TAP-N, HSM, HSM-N, SE, SE-N, BG11, and BG11-N medium.

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0.3

N+

selective media. pMaa7IR/XIR and pMaa7IR/Pepc2IR transformants were selected on TAP medium containing 1.5 mM Ltryptophan, 5 mg/mL paromomycin, and 5 mM 5-FI. Plates were incubated under dim light (approximately 50 mmol m2 s1 photosynthetically active radiation). Isolated transgenic strains were kept under constant selective pressure to prevent the loss of integrated IR transgenes.

N-

lipid content(g/g)

0.25 0.2 0.15 0.1 0.05

2.7.

0 24h

mRNA abundance

30

+N

48h

72h

-N

25 20 15 10 5 0 24 h

48 h

72 h

96 h

PEPC2

Fig. 2 e The mRNA abundance of pepc2 gene in HSM or HSM-N media and corresponding lipid content of the cells in HSM or HSM-N media.

reverse complementary sequences were lifted from pMD18TPepc2 with KpnI/BamHI and HindIII/SalI digestion, respectively, and were inserted into the corresponding cloning sites of pMD18Te18S to form vector pMD18-pepcF-18S-pepcR, which contained inverted repeat sequence of pepc2 ( pepc2 IR). pMD18-pepcF-18S-pepcR was double digested with KpnI and HindIII to obtain the pepc2 IR. Finally, the pepc2 IR was inserted into as a blunt-end fragment into EcoRI digested pMaa7/XIR to give pMaa7IR/Pepc2IR.

2.6.

Quantitative real-time PCR

96h

Transformation

Transformation of C. reinhardtii strain CC124 was performed as described by Kindle [28]. C. reinhardtii cells were grown in TAP medium to a cell density of 1e2  106 cells/mL. Cells were collected by centrifugation, washed twice and resuspended in TAP medium to a cell density of approximately 1  108 cells/mL. Plasmid DNA was introduced into the cells by the glass beads procedure [28]. In each case, 2 mg of plasmid DNA included in a mixture containing 400 mL cells, 100 mL 20% polyethylene glycol and 300 mg sterile glass beads. The reaction was mixed for 15 s on a bench-top vortex. To permit induction of RNAi, cells were allowed to recover for 1 day before plating onto

Cells for real-time PCR analysis were cultured in HSM or HSM-N liquid medium. RNA was extracted by using the TRIzol Reagent (Shanghai Sangon Biological Engineering Technology & Service Co.). Single strand cDNA was synthesized by Invitrogen SuperScript Ⅲ cDNA synthesis kit using 100 ng RNA and random primers performed at as 65  C 5 min, 25  C 5 min, 42  C 50 min. The real-time PCR was performed on a BioRad iCycler iQ Real-Time PCR Detection System using SYBR Green as the fluorescent dye. Each reaction was performed in a final volume of 25 mL with the following components: 0.2 pmoles of each primer, 1 mL of cDNA, 12.5 ml of SYBR Green Mix (Invitrogen SYBR Greener QPCR), and water was used to adjust the volume to 25 mL. The iCycler protocol was: denaturing at 95  C, 5 min; 40 cycles of (denaturing at 95  C, 30 s; annealing at 54  C, 30 s; amplification at 72  C, 15 s). The specificity of the PCR amplification was examined by a melting curve program (55e100  C with a heating rate of 0.5  C/s). The 18S rRNA was used as controls with the primers, 18SrRNAF (50 -TCAACTTTCGATGGTAGGATAGTG-30 ) and 18SrRNAR (50 -CCGTGTCAGGATTGGGTAATTT-30 ). Expression of this target gene was measured and found to be constant under all the conditions used in this work. Primers, Pepc2-F (50 -AGCTTACCGGCCCATTACTC-30 ) and Pepc2-R (50 -TCGGATGCGTTCAATCTTCT-30 ) were used to evaluate the quantity of pepc2 cDNA. The amplification rate of each transcript (Ct) was calculated by the PCR Base Line Subtracted method performed in the iCycler software at a constant fluorescence level. Cts were determined over three repeats. Relative fold differences were calculated based on the relative quantification analytical method (2DDCT) using 18s rRNA amplification as internal standard [29].

3.

Results

3.1. N deprivation induced oil accumulation in C. reinhardtii To analyze the effect of N deficiency in oil accumulation in C. reinhardtii, we grew this organism in several media with and

Fig. 3 e The schematic diagram of pMaa7IR/Pepc2IR (a) The plasmid of pMaa7IR/XIR; (b) Construct pMD18-pepcF-18S-pepcR with the structure of Pepc2 inverted repeat sequence. B, BamHI; E, EcoRI; H, HindIII; K, KpnI; S, SalI; Xb, XbaI; Xh, XhoI.

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0.3

Lipid content(g/g)

0.25 0.2 0.15 0.1 0.05 0 CC124

Maa7-RNAi8 Pepc2-RNAi6

Pepc2RNAi27

Pepc2RNAi33

Pepc2RNAi78

Pepc2RNAi113

Fig. 4 e The lipid content of transgenic alga and non transgenic wild type after 12-days cultivation in HSM medium. CC124, non transgenic wild type C. reinhaditti CC124; Maa7-RNAi8, pMaa7IR/XIR transgenic strain; Pepc2-RNAi6 e Pepc2-RNAi113, pMaa7IR/Pepc2IR transgenic strains.

without normal nitrogen supplement. As expected, compared to N-normal media, C. reinhardtii displayed lower growth rates in TAP, HSM, SE and BG11 without nitrogen (Fig. 1B). However, cells grown in N-deficient media accumulated significantly higher oil, with those grown in N-deficient HSM, high carbon (HSM-N) condition contained the highest oil level, reaching more than 3 times. Thus, HSM medium was used to culture C. reinhardtii for the subsequent experiments (Fig. 1A).

3.2. pepc2 mRNA abundance negatively correlated with oil accumulation in C. reinhardtii To determine the relationship between oil accumulation and pepc2 gene expression in C. reinhardtii, cells grown in HSM and HSM-N media were sampled at 24, 48, 72 and 96 h after subculture. Collected cells were assayed for biomass, oil content and pepc2-specific mRNA analysis. In cells grown in HSM medium, oil content was 5%, 7%, 8%, 8% of weight of dry biomass for samples cultured for 1, 2, 3 and 4 days, respectively (Fig. 2). On the other hand, in cells grown in N-deficient HSM, oil content drastically increased to 19.1% after 24 h and reached up to 25.4% after 96 h of incubation (Fig. 2), indicating that N starvation induced oil accumulation in C. reinhardtii. Interestingly, the transcript levels of pepc2, were significantly

lower in cells grown in N-deficient condition, decreasing by 84e92% in the 24e96 h time frame (P < 0.01) (Fig. 2). Thus, in C. reinhardtii, the increase in oil content was accompanied by the decrease in pepc2 mRNA abundance.

3.3. RNAi-mediated silencing of pepc2 led to increase in oil content in C. reinhardtii To determine whether the observed relationship between oil content and pepc2 mRNA abundance is specific to N deficiency, we examined the effect of artificial silencing of pepc2 on oil content in C. reinhardtii. Using the pepc2 gene (Protein ID: 182821) sequence retrieved from the JGI C. reinhardtii v4.0 database, we designed a pair of primers: PEPC2F: 50 CGGGTAAGGCAAGTGGT-30 , PEPC2R: 50 -TCGGAGCTTTATGTGAGAAT-30 to amply a 500 base pairs fragment from the coding region of pepc2. This DNA fragment was subcloned and then used to generate the pepc2 gene RNAi construct pMaa7IR/ Pepc2IR (Fig. 3). After transforming pMaa7/Pepc2IR into C. reinhardtii CC124, we isolated 135 positive transformants and cultivated them in HSM medium for 12 days before measuring oil content by the Nile Red fluorescence method. Wild type and C. reinhardtii similarly transformed with pMaa7/XIR were used as controls. Analysis of these transgenic lines of

Fig. 5 e Microscopic observation of transgenic algae Pepc2-RNAi78 (400X Nikio 80i) (a) C. reinhardtii CC124; (b) Transgenic algae Pep2-RNAi78.

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increase in oil content of these transgenic strains, we concluded that pepc2 negatively affected oil accumulation in C. reinhardtii.

Pepc2 mRNA Level

1.2 1 0.8 0.6

4.

0.4 0.2 0 CC124

Maa7RNAi8

Pepc2RNAi6

Pepc2RNAi27

Pepc2RNAi33

Pepc2Pepc2RNAi78 RNAi113

Fig. 6 e Comparison of the mRNA abundance of pepc2 gene in transgenic alga and non transgenic wild type C. reinhaditti CC124. CC124, non transgenic wild type C. reinhaditti CC124; Maa7-RNAi8, pMaa7IR/XIR transgenic strain; Pepc2-RNAi6 e Pepc2-RNAi113, pMaa7IR/Pepc2IR transgenic strains.

C. reinhardtii harboring the pepc2 silencing constructs, our results showed that oil content significantly increased by 14e28% in C. reinhardtii transformants compared to the control (Fig. 4). Transgenic strain Pepc-RNAi6 and PepcRNAi78 could accumulate 0.26169 g/g and 0.2734 g/g oil, compared to that of 0.2137 g/g in wild type C. reinhardtii CC124. For microscopy analysis (Fig. 5), the number of lipid droplets was calculated in the transgenic strains and controls, respectively. Our results showed that the difference between the transgenic strains and controls was that small lipid droplets in the former were more than that of the latter. For instance, transgenic strain Pepc2-RNAi78 had 17  4 small lipid droplets per cell, whereas controls had only 4  1 per cell (Table 2). In contrast, the number of large lipid droplets was similar between the transformants and controls.

3.4. pepc2 mRNA abundance decreased in the transgenic C. reinhardtii strains To evaluate the effectiveness of pepc2 gene silencing caused by our constructs, similarly grown C. reinhardtii strains were analyzed for the abundance of pepc2-specific mRNA by Realtime PCR using the SYBR green PCR master mix. As shown in Fig. 6, the pepc2 mRNA abundance decreased by 79e90% in transgenic C. reinhardtii strains, suggesting the high effectiveness of pepc2 silencing. With the results of a significant

Table 2 e The numbers of lipid droplets in algae cell. Strains C. reinhardtii CC-124 Maa7-RNAi8 Pepc2-RNAi6 Pepc2-RNAi27 Pepc2-RNAi33 Pepc2-RNAi78 Pepc2-RNAi113

Numbers of large lipid droplets

Numbers of small lipid droplets

21 21 21 21 21 21 21

41 41 12  3 83 92 17  4 84

Discussion

It appears that carbon allocation plays a critical role in the development of rapeseed [30,31]. A portion of carbon source is diverted to the Kennedy Pathway to synthesize oils with a key reaction catalyzed by Acetyl-CoA carboxylase (ACCase) whereas others are transported to the citric acid cycle to provide intermediates and energy for proteins synthesis and other primary metabolic pathways via the PEPC-catalyzed reaction. Thus, in rapeseed, ACCase and PEPC have been hypothesized to compete for the common substrate, PEP and the relative activity of these two enzymes determine carbon allocation to protein or oil biosynthesis. However, little is known about the molecular mechanisms of oil accumulation in microalgae. In this study, we investigate the expression pattern of pepc2 in the model green algae C. reinhardtii. Our results showed that the abundance of pepc2 mRNA exhibited a remarkable 63e91% decrease in response to nitrogen deficiency in C. reinhardtii. Consistently, the transcript levels of citrate synthase, another important enzyme involved in the citric acid cycle were also drastically decreased (data not shown). These results show us that the citric acid cycle was probably in inactivity status under N deficiency conditions. On the other hand, Oil content significantly increased in N-deficient cells of C. reinhardtii (Fig. 2). Therefore, we hypothesized that, similar to those in rapeseed, carbon source was mainly utilized to synthesize oils in C. reinhardtii grown in N-deficient medium. Our observation that silencing of pepc2 caused similar effects indicated that artificial blockage of protein biosynthesis pathways led to oil accumulation in C. reinhardtii. The shunt of the metabolic intermediates to oil biosynthesis may result in the observed growth inhibition. Although it is not experimentally established, accumulation of oil in the absence of nitrogen may help the cell to survive in the unfavorable condition. Further studies are required to elucidate the mechanisms underlying the exact signals triggering such switches. The tight link between oil accumulation and cell growth arrest presents a paradox in achieving the goal; our findings suggest that it was feasible to increase oil by suppressing the expression of pepc2 in microalgae after biomass accumulation has been ideally achieved. Furthermore, the success of inducing oil accumulation by silencing pepc2 validates the usefulness and effectiveness of biotechnology methods in manipulating microalgae to promote oil production.

Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (30860028, 30960032) and from National Nonprofit Institute Research Grants (CATASITBBZX0841) and from the National Natural Science Foundation of Hainan province (061012).

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Appendix. Supplementary material The supplementary data associated with this article can be found in the on-line version at doi:10.1016/j.biombioe.2011.01. 005.

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