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The Roles of acyl-CoA: Diacylglycerol Acyltransferase 2 Genes in the Biosynthesis of Triacylglycerols by the Green Algae Chlamydomonas reinhardtii
Molecular Plant Advance Access published April 17, 2012 Molecular Plant
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Pages 1–3, 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 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
Downloaded from http://mplant.oxfordjournals.org/ at University of Mississippi on January 20, 2015
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, CrDGAT23, 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
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Letter to the Editor
(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, CrDGAT21, 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
Downloaded from http://mplant.oxfordjournals.org/ at University of Mississippi on January 20, 2015
Figure 1. CrDGAT2-1 and CrDGAT2-5 Play Major Roles in Triglycerides Synthesis in C. reinhardtii.
Letter to the Editor
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.
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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.
Xiao-Dong Denga, Bo Gua, Ya-Jun Lia, Xin-Wen Huc, Jian-Chun Guoa and Xiao-Wen Feia,b,1
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.
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;
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/ at University of Mississippi on January 20, 2015
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.
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Pages 1–3, 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 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
Downloaded from http://mplant.oxfordjournals.org/ at University of Mississippi on January 20, 2015
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, CrDGAT23, 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
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Letter to the Editor
(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, CrDGAT21, 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
Downloaded from http://mplant.oxfordjournals.org/ at University of Mississippi on January 20, 2015
Figure 1. CrDGAT2-1 and CrDGAT2-5 Play Major Roles in Triglycerides Synthesis in C. reinhardtii.
Letter to the Editor
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.
3
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.
Xiao-Dong Denga, Bo Gua, Ya-Jun Lia, Xin-Wen Huc, Jian-Chun Guoa and Xiao-Wen Feia,b,1
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.
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;
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/ at University of Mississippi on January 20, 2015
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.
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