Cloning, characterization and functional analysis of two type 1 diacylglycerol acyltransferases (DGAT1s) from Tetraena mongolica.

Two cDNAs encoding putative type 1 acyl-CoA: diacylglycerol acyltransferases (DGAT1, EC 2.3.1.20), were cloned from Tetraena mongolica, an extreme xerophyte with high oil content in the stems. The 1 488-bp and 1 485-bp of the open reading frame (ORF) of the two cDNAs, designated as TmDGAT1a and TmDGAT1b, were both predicted to encode proteins of 495 and 494 amino acids, respectively. Southern blot analysis revealed that TmDGAT1a and TmDGAT1b both had low copy numbers in the T. mongolica genome. In addition to ubiquitous expression with different intensity in different tissues, including stems, leaves and roots, TmDGAT1a and TmDGAT1b, were found to be strongly induced by high salinity, drought and osmotic stress, resulting in a remarkable increase of triacylglycerol (TAG) accumulation in T. mongolica plantlets. TmDGAT1a and TmDGAT1b activities were confirmed in the yeast H1246 quadruple mutant (DGA1, LRO1, ARE1, ARE2) by restoring DGAT activity of the mutant host to produce TAG. Overexpression of TmDGAT1a and TmDGAT1b in soybean hairy roots as well as in T. mongolica calli both resulted in an increase in oil content (ranging from 37% to 108%), accompanied by altered fatty acid profiles.

[1]  J. Ohlrogge,et al.  Acyl-Lipid Metabolism , 2013, The arabidopsis book.

[2]  I. Graham,et al.  A Cytosolic Acyltransferase Contributes to Triacylglycerol Synthesis in Sucrose-Rescued Arabidopsis Seed Oil Catabolism Mutants1[W][OA] , 2012, Plant Physiology.

[3]  S. Park,et al.  Overexpression of Cinnamate 4-Hydroxylase Gene Enhances Biosynthesis of Decursinol Angelate in Angelica gigas Hairy Roots , 2012, Molecular Biotechnology.

[4]  J. Ohlrogge,et al.  Compartmentation of Triacylglycerol Accumulation in Plants* , 2011, The Journal of Biological Chemistry.

[5]  Y. Hao,et al.  MdVHP1 encodes an apple vacuolar H(+)-PPase and enhances stress tolerance in transgenic apple callus and tomato. , 2011, Journal of plant physiology.

[6]  G. Di Sansebastiano,et al.  Localization of seed oil body proteins in tobacco protoplasts reveals specific mechanisms of protein targeting to leaf lipid droplets. , 2011, Journal of integrative plant biology.

[7]  V. Lefebvre,et al.  Agrobacterium rhizogenes-dependent production of transformed roots from foliar explants of pepper (Capsicum annuum): a new and efficient tool for functional analysis of genes , 2011, Plant Cell Reports.

[8]  A. Zarka,et al.  Cloning and molecular characterization of a novel acyl‐CoA:diacylglycerol acyltransferase 1‐like gene (PtDGAT1) from the diatom Phaeodactylum tricornutum , 2011, The FEBS journal.

[9]  M. Margis-Pinheiro,et al.  Evolutionary view of acyl-CoA diacylglycerol acyltransferase (DGAT), a key enzyme in neutral lipid biosynthesis , 2011, BMC Evolutionary Biology.

[10]  D. Taylor,et al.  Brassica carinata – a new molecular farming platform for delivering bio‐industrial oil feedstocks: case studies of genetic modifications to improve very long‐chain fatty acid and oil content in seeds , 2010 .

[11]  J. Ohlrogge,et al.  A distinct DGAT with sn-3 acetyltransferase activity that synthesizes unusual, reduced-viscosity oils in Euonymus and transgenic seeds , 2010, Proceedings of the National Academy of Sciences.

[12]  H. Koprowski,et al.  Tobacco as a production platform for biofuel: overexpression of Arabidopsis DGAT and LEC2 genes increases accumulation and shifts the composition of lipids in green biomass. , 2010, Plant biotechnology journal.

[13]  Runzhi Li,et al.  DGAT1, DGAT2 and PDAT Expression in Seeds and Other Tissues of Epoxy and Hydroxy Fatty Acid Accumulating Plants , 2010, Lipids.

[14]  Jingmei Liu,et al.  Enhanced flavonoid production in hairy root cultures of Glycyrrhiza uralensis Fisch by combining the over-expression of chalcone isomerase gene with the elicitation treatment , 2009, Plant Cell Reports.

[15]  E. Belarbi,et al.  Cloning and Molecular Characterization of the Acyl-CoA:Diacylglycerol Acyltransferase 1 (DGAT1) Gene from Echium , 2009, Lipids.

[16]  D. Taylor,et al.  Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content. , 2008, Plant biotechnology journal.

[17]  Chaofu Lu,et al.  Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil. , 2008, Plant biotechnology journal.

[18]  D. Oliver,et al.  Role of triacylglycerols in leaves , 2008 .

[19]  J. Harwood,et al.  Metabolic control analysis is helpful for informed genetic manipulation of oilseed rape (Brassica napus) to increase seed oil content , 2008, Journal of experimental botany.

[20]  Shirong Zhang,et al.  A phenylalanine in DGAT is a key determinant of oil content and composition in maize , 2008, Nature Genetics.

[21]  Geliang Wang,et al.  Tetraena mongolica Maxim can accumulate large amounts of triacylglycerol in phloem cells and xylem parenchyma of stems. , 2007, Phytochemistry.

[22]  G. Du,et al.  3′ End cDNA amplification using classic RACE , 2006, Nature Protocols.

[23]  J. Kroon,et al.  Identification and functional expression of a type 2 acyl-CoA:diacylglycerol acyltransferase (DGAT2) in developing castor bean seeds which has high homology to the major triglyceride biosynthetic enzyme of fungi and animals. , 2006, Phytochemistry.

[24]  G. Du,et al.  5′ end cDNA amplification using classic RACE , 2006, Nature Protocols.

[25]  Randall J. Weselake,et al.  Diacylglycerol acyltransferase: A key mediator of plant triacylglycerol synthesis , 2006, Lipids.

[26]  J. Shockey,et al.  Tung Tree DGAT1 and DGAT2 Have Nonredundant Functions in Triacylglycerol Biosynthesis and Are Localized to Different Subdomains of the Endoplasmic Reticulum[W] , 2006, The Plant Cell Online.

[27]  R. Rajasekharan,et al.  Cytosolic Triacylglycerol Biosynthetic Pathway in Oilseeds. Molecular Cloning and Expression of Peanut Cytosolic Diacylglycerol Acyltransferase1[W] , 2006, Plant Physiology.

[28]  P. Gantet,et al.  Hairy root research: recent scenario and exciting prospects. , 2006, Current opinion in plant biology.

[29]  Grace Q Chen,et al.  Cloning and characterization of a cDNA encoding diacylglycerol acyltransferase from castor bean , 2004, Lipids.

[30]  M. Hills,et al.  Heterogeneity of the endoplasmic reticulum with respect to lipid synthesis in developing seeds of Brassica napus L. , 1996, Planta.

[31]  G. Jiang,et al.  Effect of various temperatures on phosphatidylglycerol biosynthesis in thylakoid membranes. , 2003, Physiologia plantarum.

[32]  Li Bo,et al.  Interpopulation difference in growth and reproduction of endemic species Tetraena mongolica in Ordos Plateau , 2003 .

[33]  Chaofu Lu,et al.  Arabidopsis Mutants Deficient in Diacylglycerol Acyltransferase Display Increased Sensitivity to Abscisic Acid, Sugars, and Osmotic Stress during Germination and Seedling Development1 , 2002, Plant Physiology.

[34]  X. Deng,et al.  Agrobacterium-mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene , 2002, Plant Cell Reports.

[35]  R. Weselake,et al.  Characterization of cDNAs encoding diacylglycerol acyltransferase from cultures of Brassica napus and sucrose-mediated induction of enzyme biosynthesis. , 2002, Biochimica et biophysica acta.

[36]  S. Stymne,et al.  Storage Lipid Synthesis Is Non-essential in Yeast* , 2002, The Journal of Biological Chemistry.

[37]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[38]  T. Voelker,et al.  DGAT2 Is a New Diacylglycerol Acyltransferase Gene Family , 2001, The Journal of Biological Chemistry.

[39]  P. Covello,et al.  Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight. , 2001, Plant physiology.

[40]  M. Davis,et al.  ACAT1 and ACAT2 membrane topology segregates a serine residue essential for activity to opposite sides of the endoplasmic reticulum membrane. , 2000, Molecular biology of the cell.

[41]  S. Stymne,et al.  Phospholipid:diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[42]  J. Widholm,et al.  High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode , 2000, Planta.

[43]  J. Harwood,et al.  Changes in Kennedy pathway intermediates associated with increased triacylglycerol synthesis in oil-seed rape , 1999 .

[44]  H. Sul,et al.  Acyltransferases of de novo glycerophospholipid biosynthesis. , 1999, Progress in lipid research.

[45]  A. Kumar,et al.  The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene. , 1999, The Plant journal : for cell and molecular biology.

[46]  S. B. Settlage,et al.  Relation between diacylglycerol acyltransferase activity and oil concentration in soybean , 1998 .

[47]  S. B. Settlage,et al.  Localization of diacylglycerol acyltransferase to oil body associated endoplasmic reticulum , 1995 .

[48]  E. Firoozabady,et al.  Agrobacterium-Mediated Transformation , 1995 .

[49]  R. Elble.  A simple and efficient procedure for transformation of yeasts. , 1992, BioTechniques.

[50]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[51]  W. F. Thompson,et al.  Rapid isolation of high molecular weight plant DNA. , 1980, Nucleic acids research.

[52]  F. Skoog,et al.  A revised medium for rapid growth and bio assays with tobacco tissue cultures , 1962 .

[53]  Kennedy Ep.  Biosynthesis of complex lipids. , 1961 .

[54]  W. J. Dyer,et al.  A rapid method of total lipid extraction and purification. , 1959, Canadian journal of biochemistry and physiology.