Skip to main content
SpringerLink
Log in

Functional Analysis of Sesame Diacylglycerol Acyltransferase and Phospholipid: Diacylglycerol Acyltransferase Genes Using In Silico and In Vitro Approaches

  • Original Paper
  • Published:
Plant Molecular Biology Reporter Aims and scope Submit manuscript

Abstract

Sesamum indicum L. is one of the major oilseed crops of India. Sesame oil is stable with a good source of unsaturated fatty acids. To know the mechanism of oil accumulation in sesame, genes coding for the key enzymes involved in triacylglycerol (TAG) synthesis pathway, namely diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) and phospholipid:diacylglycerol acyltransferase (PDAT; EC 2.3.1.158), were retrieved from sesame full genome sequences by comparative analysis using Arabidopsis genes. All isoforms of SiDGAT and SiPDAT genes were analyzed in silico for their sequence and structural similarity with the existing members from other plants. Phylogenetic analysis delineated the sesame DGAT and PDAT genes into four separate clades. Most of the members had several transmembrane domains except SiDGAT1 A1, SiDGAT1 A2, SiDGAT1 B1, and SiPDAT2 which do not have any transmembrane domain suggesting a soluble function. In vitro gene isolation followed by mRNA expression analysis of SiDGAT1, SiDGAT2, SiPDAT1, and SiPDAT2 were carried out. Expression of these genes at the transcription level at the leaf, stem, root, flower, developing seeds, and mature seeds were examined by quantitative real-time PCR experiments. Functional analysis of these sesame genes was performed in a yeast quadruple mutant lacking TAG synthesis genes. Transcript abundance of SiDGAT1 and SiDGAT2 genes was highest in mature seeds, SiPDAT1 at flowering stage and developing seeds, and SiPDAT2 in stem and developing seeds. Heterologous expression of sesame genes in the yeast system indicated higher oil content in both SiDGAT and SiPDAT gene–transformed mutants. In addition, SiPDAT1-expressing mutants had higher polyunsaturated (C18:1; C18:2) fatty acid content. The present study indicates both sesame DGAT and PDAT genes are good candidates for not only oil yield increase but also for higher PUFA content.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Arslan C, Uzun B, Ülger S, İlhan CM (2007) Determination of oil content and fatty acid composition of sesame mutants suited for intensive management conditions. J Am Oil Chem Soc 84:917–920

    Article  CAS  Google Scholar 

  • Bates PD, Browse J (2012) The significance of different diacylgycerol synthesis pathways on plant oil composition and bioengineering. Front Plant Sci 3:147

    Article  PubMed  PubMed Central  Google Scholar 

  • Bouvier-Nave P, Benveniste P, Oelkers P, Sturley SL, Schaller H (2000) Expression in yeast and tobacco of plant cDNAs encoding acyl CoA: diacylglycerol acyltransferase. Eur J Biochem 267:85–96

    Article  CAS  PubMed  Google Scholar 

  • Browse J, McCourt PJ, Somerville CR (1986) Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal Biochem 152:141–145

    Article  CAS  PubMed  Google Scholar 

  • Burgal J, Shockey J, Lu C, Dyer J, Larson T, Graham I, Browse J (2008) Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil. Plant Biotechnol J 6(8):819–831

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cao H, Shockey JM, Klasson KT, Chapital DC, Mason CB (2013) Developmental regulation of diacylglycerol acyltransferase family gene expression in tung tree tissues. PLoS One 8:e76946

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chi X, Hu R, Zhang X, Chen M, Chen N, Pan L, Wang T, Wang M, Yang Z, Wang Q, Yu S (2014) Cloning and functional analysis of three diacylglycerol acyltransferase genes from peanut (Arachis hypogaea L.). PLoS One 9:e105834

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne H (2000) Phospholipid: diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. PNAS 97:6487–6492

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Durrett TP, McClosky DD, Tumaney AW, Elzinga DA, Ohlrogge J (2008) A distinct DGAT with sn-3 acetyltransferase activity that synthesizes unusual, reduced viscosity oils in euonymus and transgenic seeds. PNAS 107:9464–9469

    Article  Google Scholar 

  • Jako C, Kumar A, Wei Y, Zou J, Barton DL, Giblin EM, Covello PS, Taylor DC (2001) Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight. Plant Physiol 126(2):861–874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jilian F, Yan C, Shanklin J, Xu C (2013) Dual role for phospholipid: diacylglycerol acyltransferase: enhancing fatty acid synthesis and diverting fatty acids from membrane lipids to triacylglycerol in Arabidopsis leaves. Plant Cell 25:3506–3518

    Article  CAS  Google Scholar 

  • Jin Y, Yuan Y, Gao L, Sun R, Chen L, Li D, Zheng Y (2017) Characterization and functional analysis of a type 2 diacylglycerol acyltransferase (dgat2) gene from oil palm (Elaeis guineensis jacq.) mesocarp in Saccharomyces cerevisiae and transgenic Arabidopsis thaliana. Front Plant Sci 17:1791

    Article  Google Scholar 

  • Kalscheuer R, Steinbüchel A (2003) A novel bifunctional wax ester synthase/acyl-CoA: diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J Biol Chem 278:8075–8082

    Article  CAS  PubMed  Google Scholar 

  • Ke T, Dong C, Mao H, Zhao Y, Chen H, Liu H, Liu S (2011) Analysis of expression sequence tags from a full-length-enriched cDNA library of developing sesame seeds (Sesamum indicum). BMC Plant Biol 11:180

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kelly W (2013) Experimental characterization of next-generation expanded-bed adsorbents for capture of a recombinant protein expressed in high-cell-density yeast fermentation. Biotechnol Appl Biochem 60:510–520

    Article  CAS  PubMed  Google Scholar 

  • Kim HU, Lee KR, Go YS, Jung JH, Suh MC, Kim JB (2011) Endoplasmic reticulum-located PDAT1-2 from castor bean enhances hydroxy fatty acid accumulation in transgenic plants. Plant Cell Physiol 52:983–993

    Article  CAS  PubMed  Google Scholar 

  • Knittelfelder OL, Kohlwein SD (2017) Lipid extraction from yeast cells. Cold Spring Harb Protoc 2017(5):pdb-prot085449

    Article  Google Scholar 

  • Li Q, Hu L, Guo J, Yang T, Chen L (2016) Molecular characterization of two type I acyl-CoA: diacylglycerol acyltransferase genes in maize. Biotechnol Biotechnol Equip 30:453–461

    Article  CAS  Google Scholar 

  • Liu XY, Ouyang LL, Zhou ZG (2016) Phospholipid: diacylglycerol acyltransferase contributes to the conversion of membrane lipids into triacylglycerol in Myrmecia incisa during the nitrogen starvation stress. Sci Rep 6:26610

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lu Y (2011) Making yeast competent cells and yeast cell transformation

  • Lung SC, Weselake RJ (2006) Diacylglycerol acyltransferase: a key mediator of plant triacylglycerol synthesis. Lipids 41:1073–1088

    Article  CAS  PubMed  Google Scholar 

  • Pan X, Siloto RM, Wickramarathna AD, Mietkiewska E, Weselake RJ (2013) Identification of a pair of phospholipid: diacylglycerol acyltransferases from developing flax (Linum usitatissimum L.) seed catalyzing the selective production of trilinolenin. J Biol Chem 288:24173–24188

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Peng D, Zhang L, Tan X, Yuan D, Liu X, Zhou B (2016) Increasing seed oil content and altering oil quality of Brassica napus L. by over-expression of diacylglycerol acyltransferase 1 (SsDGAT1) from Sapium sebiferum (L.) Roxb. Mol Breed 36:136

    Article  CAS  Google Scholar 

  • Saha S, Enugutti B, Rajakumari S, Rajasekharan R (2006) Cytosolic triacylglycerol biosynthetic pathway in oilseeds. Molecular cloning and expression of peanut cytosolic diacylglycerol acyltransferase. Plant Physiol 141:1533–1543

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Salanoubat M, Lemcke K, Rieger M, Ansorge W, Unseld M, Fartmann B, Valle G, Blöcker H, Perez-Alonso M, Obermaier B, Delseny M, Boutry M, Grivell LA, Mache R, Puigdomènech P, de Simone V, Choisne N, Artiguenave F, Robert C, Brottier P, Wincker P, Cattolico L, Weissenbach J, Saurin W, Quétier F, Schäfer M, Müller-Auer S, Gabel C, Fuchs M, Benes V, Wurmbach E, Drzonek H, Erfle H, Jordan N, Bangert S, Wiedelmann R, Kranz H, Voss H, Holland R, Brandt P, Nyakatura G, Vezzi A, D’Angelo M, Pallavicini A, Toppo S, Simionati B, Conrad A, Hornischer K, Kauer G, Löhnert TH, Nordsiek G, Reichelt J, Scharfe M, Schön O, Bargues M, Terol J, Climent J, Navarro P, Collado C, Perez-Perez A, Ottenwälder B, Duchemin D, Cooke R, Laudie M, Berger-Llauro C, Purnelle B, Masuy D, de Haan M, Maarse AC, Alcaraz JP, Cottet A, Casacuberta E, Monfort A, Argiriou A, flores M, Liguori R, Vitale D, Mannhaupt G, Haase D, Schoof H, Rudd S, Zaccaria P, Mewes HW, Mayer KF, Kaul S, Town CD, Koo HL, Tallon LJ, Jenkins J, Rooney T, Rizzo M, Walts A, Utterback T, Fujii CY, Shea TP, Creasy TH, Haas B, Maiti R, Wu D, Peterson J, van Aken S, Pai G, Militscher J, Sellers P, Gill JE, Feldblyum TV, Preuss D, Lin X, Nierman WC, Salzberg SL, White O, Venter JC, Fraser CM, Kaneko T, Nakamura Y, Sato S, Kato T, Asamizu E, Sasamoto S, Kimura T, Idesawa K, Kawashima K, Kishida Y, Kiyokawa C, Kohara M, Matsumoto M, Matsuno A, Muraki A, Nakayama S, Nakazaki N, Shinpo S, Takeuchi C, Wada T, Watanabe A, Yamada M, Yasuda M, Tabata S, European Union Chromosome 3 Arabidopsis Sequencing Consortium, Institute for Genomic Research, Kazusa DNA Research Institute (2000) Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana. Nature 408:820–822

    Article  CAS  PubMed  Google Scholar 

  • Sandager L, Gustavsson MH, Ståhl U, Dahlqvist A, Wiberg E, Banas A, Stymne S (2002) Storage lipid synthesis is non-essential in yeast. J Biol Chem 277:6478–6482

    Article  CAS  PubMed  Google Scholar 

  • Schmutz J, Cannon SB, Schlueter J, ma J, Mitros T, Nelson W, Xu D (2010) genome sequence of the palaeopolyploid soybean. Nature 463:178

    Article  CAS  PubMed  Google Scholar 

  • Settlage SB, Kwanyuen P, Wilson RF (1998) Relation between diacylglycerol acyltransferase activity and oil concentration in soybean. J Am Oil Chem Soc 75:775–781

    Article  CAS  Google Scholar 

  • Shyu YS, Hwang LS (2002) Antioxidative activity of the crude extract of lignan glycosides from unroasted Burma black sesame meal. Food Res Int 35:357–365

    Article  CAS  Google Scholar 

  • Ståhl U, Carlsson AS, Lenman M, Dahlqvist A, Huang B, Banaś W, Stymne S (2004) Cloning and functional characterization of a phospholipid: diacylglycerol acyltransferase from Arabidopsis. Plant Physiol 135:1324–1335

    Article  PubMed  PubMed Central  Google Scholar 

  • Taylor DC, Zhang Y, Kumar A, Francis T, Giblin EM, Barton DL, Ferrie JR, Laroche A, Shah S, Zhu W, Snyder CL (2009) Molecular modification of triacylglycerol accumulation by over-expression of DGAT1 to produce canola with increased seed oil content under field conditions. Botany. 87(6):533–543

    Article  CAS  Google Scholar 

  • Turchetto-Zolet AC, Maraschin FS, de Morais GL, Cagliari A, Andrade CM, Margis-Pinheiro M, Margis R (2011) Evolutionary view of acyl-CoA diacylglycerol acyltransferase (DGAT), a key enzyme in neutral lipid biosynthesis. BMC Evol Biol 11:263

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Van Erp H, Bates PD, Burgal J, Shockey J (2011) Castor phospholipid: diacylglycerol acyltransferase facilitates efficient metabolism of hydroxy fatty acids in transgenic Arabidopsis. Plant Physiol 155:683–693

    Article  CAS  PubMed  Google Scholar 

  • Wagner M, Hoppe K, Czabany T, Heilmann M, Daum G, Feussner I, Fulda M (2010) Identification and characterization of an acyl-CoA: diacylglycerol acyltransferase 2 (DGAT2) gene from the microalga O. tauri. Plant Physiol Biochem 48:407–416

    Article  CAS  PubMed  Google Scholar 

  • Wang Z, Huang W, Chang J, Sebastian A, Li Y, Li H, Li W (2014) Overexpression of SiDGAT1, a gene encoding acyl-CoA: diacylglycerol acyltransferase from Sesamum indicum L. increases oil content in transgenic Arabidopsis and soybean. Plant Cell Tissue Organ Cult 119:399–410

    Article  CAS  Google Scholar 

  • Wei X, Zhu X, Yu J, Wang L, Zhang Y, Li D, Zhang X (2016) Identification of sesame genomic variations from genome comparison of landrace and variety. Front Plant Sci 7:1169–1129

    PubMed  PubMed Central  Google Scholar 

  • Xue JA, Mao X, Yang ZR, Wu YM, Jia XY, Zhang L, Li RZ (2013) Expression of yeast acyl-CoA-∆ 9 desaturase leads to accumulation of unusual monounsaturated fatty acids in soybean seeds. Biotechnol Lett 35:951–959

    Article  CAS  PubMed  Google Scholar 

  • Yoon K, Han D, Li Y, Sommerfeld M, Hu Q (2012) Phospholipid: diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii. Plant Cell 24:3708–3724

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yuan L, Mao X, Zhao K, Ji X, Ji C, Xue J, Li R (2017) Characterisation of phospholipid: diacylglycerol acyltransferases (PDATs) from Camelina sativa and their roles in stress responses. Biol Open 6:1024–1034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zheng L, Shockey J, Bian F, Chen G, Shan L, Li X, Wan S, Peng Z (2017) Variant amino acid residues alter the enzyme activity of peanut type 2 diacylglycerol acyltransferases. Front Plant Sci 8

Download references

Acknowledgements

We thank Dr. Sten Stymne and Dr. Jenny Lindberg Yilmaz for providing the H1246 mutant strain.

Funding

The authors thank the Department of Biotechnology, PSG College of Technology, and Department of Science and Technology, Government of India, for providing the funding and infrastructure.

Author information

Authors and Affiliations

  1. Department of Biotechnology, PSG College of Technology, Coimbatore, Tamil Nadu, 641004, India

    Muthulakshmi Chellamuthu, Kanimozhi Kumaresan, Selvi Subramanian & Hemashree Muthumanickam

Authors
  1. Muthulakshmi Chellamuthu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kanimozhi Kumaresan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Selvi Subramanian
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hemashree Muthumanickam
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Selvi Subramanian.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflicts of interest.

Research Involving Human Participants and/or Animals

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic Supplementary Material

Supplementary Table 1

(DOCX 20 kb)

ESM 1

(DOCX 262 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chellamuthu, M., Kumaresan, K., Subramanian, S. et al. Functional Analysis of Sesame Diacylglycerol Acyltransferase and Phospholipid: Diacylglycerol Acyltransferase Genes Using In Silico and In Vitro Approaches. Plant Mol Biol Rep 37, 146–156 (2019). https://doi.org/10.1007/s11105-019-01144-7

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11105-019-01144-7

Keywords

Search

Navigation