The Roles of acyl-CoA: Diacylglycerol ... - Molecular Plant

Molecular Plant • Pages Volume 1–3, 5 • 2012 Number 4 • Pages 945–947 • July 2012
LETTER TO THE EDITOR
LETTER TO THE EDITOR
The Roles of acyl-CoA: Diacylglycerol
Acyltransferase 2 Genes in the Biosynthesis of
Triacylglycerols by the Green Algae
Chlamydomonas reinhardtii
Dear Editor,
Triacylglycerols (triglycerides) (TAGs), as the major storage
forms of energy, mainly are stored in adipocytes, myocytes,
enterocytes, hepatocytes, and mammary epithelial cells in
mammals, oilseeds in plants, and lipid droplets in microorganisms
(Yen et al., 2008). Aside from energy storage, TAGs have
essential functions in multiple physiological processes. In
plants, TAGs are crucial for seed oil accumulation, germination,
and seedling development (Zhang et al., 2005, 2009).
Notably, TAGs derived from plants and microorganisms could
serve as the feedstock for biofuels production (Deng et al., 2009).
Therefore, understanding of the molecular basis of TAGs biosynthesis
and storage is of considerable economic importance.
The terminal step of TAGs biosynthesis is catalyzed by the
acyl-CoA:diacylglycerol acyltransferase (DGAT) enzyme. This
reaction allows covalent linkage of diacylglycerol (DAG) to
long-chain fatty acyl-CoAs. At least two DGAT families have
been identified—DGAT1 and DGAT2, which share no sequence
similarity. Genes coding for DGAT1 family members are homologous
to acyl-CoA:cholesterol acyltransferase (ACAT) (Cases
et al., 1998), whereas genes belong to DGAT2 family show similarity
to acyl-CoA:monoacylglycerol acyltransferase and wax
monoester synthase (Cheng et al., 2003). Using Arabidopsis
dgat2 genes as search entries in the Blastp and Blastn
programs, we obtained five of DGAT2 homologous genes
in Chlamydomonas from the JGI Chlamydomonas database
(E-value , 1 E-10), namely crDGAT2-1, CrDGAT2-2, CrDGAT2-
3, CrDGAT2-4, and CrDGAT2-5. These genes were amplified
by PCR and cloned into vector pMD18T for sequence determination.
The amino acid sequence alignment of the CrDGAT2-1
to CrDGAT2-5 (Supplemental Figure 1) revealed that these proteins
differ significantly at the amino acid level but all contain
the diacylglycerol acyltransferase domain shared by members
of the superfamily (http://pfam.sanger.ac.uk/search). Clustering
analysis shows that CrDGAT2-4 and Arabidopsis AtDGAT2
belong to one group; CrDGAT2-5 and yeast ScDGA1 (Oelkers
et al., 2002) are in one group whereas CrDGAT2-1, CrDGAT2-2,
and CrDGAT2-3 are in a different group (Supplemental Figure
2). The CrDGAT1, AtDGAT1, ScARE1, ScARE2, and AhDGAT3 are
in an independent group (Lu and Hills 2002; Saha et al., 2006).
Analysis of the CrDGAT2s by TMHMM Server v 2.0 indicates
that CrDGAT2-1 contains three transmembrane domains,
whereas CrDGAT2-2, CrDGAT2-3, and CrDGAT2-4 harbor
two and CrDGAT2-5 has no predicted transmembrane region.
CrDGAT2s are predicted (by Euk-mPLoc 2.0) to localize in the
endoplasmic reticulum. Transcriptional analysis revealed that
CrDGATs exhibits different expression patterns under nitrogensufficient
and -deficient conditions. Among these, CrDGAT2-1
and CrDGAT2-5 are highly expressed in cells grown in N-sufficient
medium, with mRNA levels more abundant than those of
CrDGAT1, CrDGAT2-2, CrDGAT2-3, and CrDGAT2-4. Under
N-deficient conditions, the CrDGAT1 mRNA increased in cells,
while all transcripts of the CrDGAT2 genes decreased. On the
other hand, CrDGAT2-1 and CrDGAT2-5 are transcribed at higher
levels. These results suggest that CrDGAT2-1 and CrDGAT2-5
play dominant roles in TAG biosynthesis in C. reinhardtii under
N-sufficient conditions; CrDGAT1, CrDGAT2-1, and CrDGAT2-5
appear to contribute to TAG accumulation under N-deficient
conditions (Supplemental Figure 3).
To further determine the roles of CrDGAT2-1 to CrDGAT2-5
in lipid biosynthesis, we examined the effects of artificial silencing
of the five homologous genes on lipid content in C.
reinhardtii. The results revealed that, in the CrDGAT2-1 or
CrDGAT2-5 transgenic silencing strains, the lipid content decreased
by 16%–24% or 28%–37%, respectively. On the other
hand, transformants carrying the siRNA against CrDGAT2-2 or
CrDGAT2-3 showed no detectable change in lipid content.
Interestingly, transformants harboring CrDGAT2-4 exhibited
24%–34% increase in lipid content (Figure 1A). To evaluate
the effectiveness of our RNAi constructs, we analyzed the
abundance of target gene-specific mRNA by real-time PCR
ª The Author 2012. Published by the Molecular Plant Shanghai Editorial
Office in association with Oxford University Press on behalf of CSPB and
IPPE, SIBS, CAS.
doi: 10.1093/mp/sss040
Received 15 January 2012; accepted 5 March 2012
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2 | Letter to the Editor
946 Letter to the Editor
Figure 1. CrDGAT2-1 and CrDGAT2-5 Play Major Roles in Triglycerides Synthesis in C. reinhardtii.
(A) Silencing of CrDGAT2-1 to CrDGAT2-5 by RNAi led to decrease in lipid content. Maa7-RNAi, pMaa7IR/XIR transgenic algae (control) were
analyzed for lipid content as described in the text; DGAT2-1RNAi to DGAT2-5RNAi indicate CrDGAT2-1 to CrDGAT2-5 RNAi transgenic algae
lines, respectively.
(B) The lipid content of CrDGAT2-1 and CrDGAT2-5 transgenic algae in HSM (N-sufficient) medium.
(C) The lipid content of CrDGAT2-1 and CrDGAT2-5 transgenic algae differed significantly in HSM-N (N-limited) medium.
(D) Lipid content in transgenic alga lines detected by Nile red staining. After 12 d of cultivation in HSM medium, the oil droplets of CrDGAT2-1
and CrDGAT2-5 transgenic algae were stained Nile red and detected by microscopic analysis. Strains: C.r CC425, wild-type; pCAMBIA1302,
C. reinhardtii CC425 transformed with pCAMBIA1302; CrDGAT2-1 and CrDGAT2-5, CrDGAT2-1 and CrDGAT2-5 transgenic algae. Statistical
analysis was performed using SPSS statistical software. Significance is indicated as * P , 0.05; ** P , 0.01. Bar = 5 lm.
in transgenic algae. The CrDGAT2-1 to CrDGAT2-5 mRNA abundance
decreased by 72.2%–82.21%, 82.71%–86.53%, 80%–
85.29%, 76.92%–85.71%, and 77.57%–83%, respectively
(Supplemental Figure 4), indicating high-efficiency silencing
by these constructs. Similar results were obtained in the Nile
red staining analysis, in which little oil droplets with yellow florescence
were found in CrDGAT2-1 RNAi transgenic algae, and
almost no yellow florescence was detected in transgenic algae
harboring RNAi against CrDGAT2-5 (Supplemental Figure 5).
The observation that RNAi silencing of CrDGAT2-1 and
CrDGAT2-5 caused a decrease in lipid content suggested that
CrDGAT2-1 and CrDGAT2-5 genes are critical for biosynthesis
of triglycerides in C. reinhardtii. We thus set to determine
whether overexpression of CrDGAT2-1 or CrDGAT2-5 could
lead to increase in lipid content in C. reinhardtii. Indeed,
overexpression of these genes led to significant increases in lipid
content. For examples, after 6 d of growth in HSM medium, the
lipid content of algae transformed with pCAMBIA1302,
CrDGAT2-1, and CrDGAT2-5 was 7.01%, 10.6%, and 12.35%, respectively,
whereas the lipid content of the C. reinhardtii CC425
was 8.33%, which corresponds to a 27.25 and 48.25% increase,
respectively (Figure 1B). Under the N-limited conditions, the
lipid content of CrDGAT2-1 and CrDGAT2-5 transgenic algae
also increased. In these samples, the lipid content of C. reinhardtii
CC425 and lines transformed with pCAMBIA1302, CrDGAT2-
1, and CrDGAT2-5 was 41.56, 41.43, 49.75, and 59.01%, respectively,
which corresponds to a 20.08 and 43.82% increase,
respectively (Figure 1C). A similar increase in lipid content
was observed by the Nile red dye staining method (Figure 1D).
In order to validate the gene functions of CrDGAT2-1 and
CrDGAT2-5, we took advantage of yeast mutants by producing
derivatives of the yeast dga1D mutant expressing CrDGAT2-1 or
CrDGAT2-5. Vectors pRS426TDGAT2-1 and pRS426TDGAT2-5
that allow overexpression of these genes were transformed
the mutant dga1D (YOR245C) (Sorger and Daum, 2002). Five independent
transformants growing on uracil-deficient medium
were selected for lipid content analysis. Compared to the dga1D
mutant and its transformants harboring the vector pRS426T, the
lipid content of strains expressing CrDGAT2-1 and CrDGAT2-5 increased
by 12%;17% (Supplemental Figure 6), implying that
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Letter to the Editor | 3
Letter to the Editor 947
expression of CrDGAT2-1 and CrDGAT2-5 from Clamydomonas
could induce lipid content in yeast.
In the present study, five CrDGAT2 homologous genes
were analyzed in C. reinhardtii. Interestingly, RNAi silencing of
these five genes caused different patterns of lipid accumulation.
Silencing of CrDGAT2-1 or CrDGAT2-5 resulted in a significant
decrease in oil content, whereas no significant changes in lipid
were observed in content when CrDGAT2-2 or CrDGAT2-3 was
silenced. On the contrary, oil content was slightly increased in
transgenic strains in which expression of CrDGAT2-4 was interfered
with. Together, these results suggest that CrDGAT2-1
and CrDGAT2-5 play main roles in triglycerides synthesis in
C. reinhardtii. These results are consistent with the observation
that overexpression of CrDGAT2-1 or CrDGAT2-5 leads to a significant
increase in lipid content in C. reinhardtii (Figure 1B and 1C).
Similarly, lipid content was enhanced in S. cerevisiae DGA1
mutant dga1D (YOR245C) by overexpression of CrDGAT2-1
or CrDGAT2-5. Although CrDGAT homologous genes involved
in fatty acid biosynthesis are present in C. reinhardtii, their
roles in triglycerides biosynthesis vary greatly. The identification
of genes that dominate this process may allow future manipulation
of their expression to maximize oil production by the
fast-growing algae species.
SUPPLEMENTARY DATA
Supplementary Data are available at Molecular Plant Online.
ACKNOWLEDGMENTS
This work was supported by grants from the National Natural Science
Foundation of China (30860028, 30960032, 31000117) and from
National Nonprofit Institute Research Grants (CATAS-ITBBZX0841).
No conflict of interest declared.
Xiao-Dong Deng a , Bo Gu a , Ya-Jun Li a , Xin-Wen Hu c ,
Jian-Chun Guo a and Xiao-Wen Fei a,b,1
a Key Laboratory of Biology and Genetic Resources of Tropical Crops,
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;
c College of Agronomy, Hainan University, Haikou, 571101, China;
1 To whom correspondence should be addressed. E-mail Feixw2000@
hotmail.com, tel. +86-898-66960173, fax +86-898–66890978.
REFERENCES
Cases, S., et al. (1998). Identification of a gene encoding an
acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol
synthesis. Proc. Natl Acad. Sci. U S A. 95, 13018–
13023.
Cheng, D., et al. (2003). Identification of acyl coenzyme A:monoacylglycerol
acyltransferase 3, an intestinal specific enzyme
implicated in dietary fat absorption. J. Biol. Chem. 278,
13611–13614.
Deng, X.D., Li, Y.J., and Fei, X.W. (2009). Microalgae: a promising
feedstock for biodiesel. African Journal of Microbiology Research.
3, 1008–1014.
Lu, C., and Hills, M.J. (2002). Arabidopsis mutants deficient in diacylglycerol
acyltransferase display increased sensitivity to abscisic
acid, sugars, and osmotic stress during germination and seedling
development. Plant Physiol. 129, 1352–1358.
Oelkers, P., et al. (2002). The DGA1 gene determines a second
triglyceride synthetic pathway in yeast. J. Biol. Chem. 277,
8877–8881.
Saha, S., Enugutti, B., Rajakumari, S., and Rajasekharan, R.
(2006). Cytosolic triacylglycerol biosynthetic pathway in
oilseeds: molecular cloning and expression of peanut cytosolic
diacylglycerol acyltransferase. Plant Physiol. 141, 1533–
1543.
Sorger, D., and Daum, G. (2002). Synthesis of triacylglycerols by
the acyl-coenzyme A: diacyl-glycerol acyltransferase Dga1p in
lipid particles of the yeast Saccharomyces cerevisiae. J. Bacterial.
184, 519–524.
Yen, C.E., et al. (2008). DGAT enzymes and triacylglycerol biosynthesis.
J. Lipid Res. 49, 2283–2301.
Zhang, F.Y., Yang, M.F., and Xu, Y.N. (2005). Silencing of DGAT1
in tobacco causes a reduction in seed oil content. Plant Sci.
169, 689–694.
Zhang, M., Fan, J., Taylor, D.C., and Ohlrogge, J.B. (2009).
DGAT1 and PDAT1 acyltransferases have overlapping functions
in Arabidopsis triacylglycerol biosynthesis and are essential
for normal pollen and seed development. Plant Cell. 21,
3885–3901.
Downloaded from http://mplant.oxfordjournals.org/ by guest on April 23, 2014