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Functional identification of oleate 12-desaturase and ω-3 fatty acid desaturase genes from Perilla frutescens var. frutescens

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Abstract

Key message

We described identification, expression, subcellular localization, and functions of genes that encode fatty acid desaturase enzymes in Perilla frutescens var. frutescens.

Abstract

Perilla (Perilla frutescens var. frutescens) seeds contain approximately 40 % of oil, of which α-linolenic acid (18:3) comprise more than 60 % in seed oil and 56 % of total fatty acids (FAs) in leaf, respectively. In perilla, endoplasmic reticulum (ER)-localized and chloroplast-localized ω-3 FA desaturase genes (PfrFAD3 and PfrFAD7, respectively) have already been reported, however, microsomal oleate 12-desaturase gene (PfrFAD2) has not yet. Here, four perilla FA desaturase genes, PfrFAD2-1, PfrFAD2-2, PfrFAD3-2 and PfrFAD7-2, were newly identified and characterized using random amplification of complementary DNA ends and sequence data from RNAseq analysis, respectively. According to the data of transcriptome and gene cloning, perilla expresses two PfrFAD2 and PfrFAD3 genes, respectively, coding for proteins that possess three histidine boxes, transmembrane domains, and an ER retrieval motif at its C-terminal, and two chloroplast-localized ω-3 FA desaturase genes, PfrFAD7-1 and PfrFAD7-2. Arabidopsis protoplasts transformed with perilla genes fused to green fluorescence protein gene demonstrated that PfrFAD2-1 and PfrFAD3-2 were localized in the ER, and PfrFAD7-1 and PfrFAD7-2 were localized in the chloroplasts. PfrFAD2 and perilla ω-3 FA desaturases were functional in budding yeast (Saccharomyces cerevisiae) indicated by the presence of 18:2 and 16:2 in yeast harboring the PfrFAD2 gene. 18:2 supplementation of yeast harboring ω-3 FA desaturase gene led to the production of 18:3. Therefore, perilla expresses two functional FAD2 and FAD3 genes, and two chloroplast-localized ω-3 FA desaturase genes, which support an evidence that P. frutescens cultivar is allotetraploid plant.

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Abbreviations

PUFA:

Polyunsaturated fatty acid

FA:

Fatty acid

ER:

Endoplasmic reticulum

PC:

Phosphatidylcholine

UTR:

Untranslated region

FAME:

Fatty acid methyl ester

Nt:

Nucleotide

Bp:

Base pair

Aa:

Amino acid

PG:

Phosphatidylglycerol

DAG:

Diacylglycerol

PA:

Phosphatidic acid

TMDs:

Transmembrane domains

References

  • Abdel-Reheem M, Hildebrand D (2013) Activity of Brassica napus and Perilla frutescens microsomal ω-3 desaturases expressed in yeast (Saccharomyces cerevisiae). Turk J Biol 37:591–605

    Article  CAS  Google Scholar 

  • Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11:R106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Andreu V, Collados R, Testillano PS, del C Risueño M, Picorel R, Alfonso M (2007) In situ molecular identification of the plastid ω3 fatty acid desaturase FAD7 from soybean: evidence of thylakoid membrane localization. Plant Physiol 145:1336–1344

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Arondel V, Lemleux B, Hwang I, Gibson S, Goodman HM, Somerville CR (1992) Map-based cloning of a gene controlling omega-3 fatty acid desaturation in Arabidopsis. Science 258:1353–1355

    Article  CAS  PubMed  Google Scholar 

  • Asif M (2011) Health effects of omega-3,6,9 fatty acids: Perilla frutescens is a good example of plant oils. Orient Pharm Exp Med 11:51–59

    Article  PubMed  PubMed Central  Google Scholar 

  • Banik M, Duguid S, Cloutier S (2011) Transcript profiling and gene characterization of three fatty acid desaturase genes in high, moderate, and low linolenic acid genotypes of flax (Linum usitatissimum L.) and their role in linolenic acid accumulation. Genome 54:471–483

    Article  CAS  PubMed  Google Scholar 

  • Browse J, McConn M, James D Jr, Miquel M (1993) Mutants of Arabidopsis deficient in the synthesis of α-linolenate. Biochemical and genetic characterization of the endoplasmic reticulum linoleoyl desaturase. J Biol Chem 268(22):16345–16351

    CAS  PubMed  Google Scholar 

  • Chung CH, Kim JL, Lee YC, Choi YL (1999) Cloning and characterization of a seed-specific ω-3 fatty acid desaturase cDNA from Perilla frutescens. Plant Cell Physiol 40:114–118

    Article  CAS  PubMed  Google Scholar 

  • Ciftci ON, Przybylski R, Rudzińska M (2012) Lipid components of flax, perilla, and chia seeds. Eur J Lipid Sci Technol 114:794–800

    Article  CAS  Google Scholar 

  • Cox MP, Peterson DA, Biggs PJ (2010) SolexaQA: at-a-glance quality assessment of Illumina second-generation sequencing data. BMC Bioinform 11:485

    Article  Google Scholar 

  • Dyer JM, Mullen RT (2001) Immunocytological localization of two plant fatty acid desaturases in the endoplasmic reticulum. FEBS Lett 494:44–47

    Article  CAS  PubMed  Google Scholar 

  • Dyer JM, Chapital DC, Kuan JCW, Mullen RT, Turner C, McKeon TA, Pepperman AB (2002) Molecular analysis of a bifunctional fatty acid conjugase/desaturase from tung. Implications for the evolution of plant fatty acid diversity. Plant Physiol 130:2027–2038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dyer JM, Chapital DC, Kuan JCW, Shepherd HS, Tang F, Pepperman AB (2004) Production of linolenic acid in yeast cells expressing an omega-3 desaturase from tung (Aleurites fordii). J Am Oil Chem Soc 81:647–651

    Article  CAS  Google Scholar 

  • Emanuelsson O, Nielsen H, von Heijne G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8:978–984

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ferro M, Salvi D, Brugière S, Miras S, Kowalski S, Louwagie M, Garin J, Joyard J, Rolland N (2003) Proteomics of the chloroplast envelope membranes from Arabidopsis thaliana. Mol Cell Proteom 2:325–345

    CAS  Google Scholar 

  • Flores T, Karpova O, Su X, Zeng P, Bilyeu K, Sleper DA, Nguyen HT, Zhang ZJ (2008) Silencing of GmFAD3 gene by siRNA leads to low α-linolenic acids (18:3) of fad3-mutant phenotype in soybean [Glycine max (Merr.)]. Transgenic Res 17:839–850

    Article  CAS  PubMed  Google Scholar 

  • Gibson S, Arondel V, Iba K, Somerville C (1994) Cloning of a temperature-regulated gene encoding a chloroplast ω-3 desaturase from Arabidopsis thaliana. Plant Physiol 106:1615–1621

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Heppard EP, Kinney AJ, Stecca KL, Miao GH (1996) Developmental and growth temperature regulation of two different microsomal ω-6 desaturases in soybean. Plant Physiol 110:311–319

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hernandez ML, Mancha M, Martinez-Rivas JM (2005) Molecular cloning and characterization of genes encoding two microsomal oleate desaturases (FAD2) from olive. Phytochemistry 66:1417–1426

    Article  CAS  PubMed  Google Scholar 

  • Honda G, Yuba A, Kojima T, Tabata M (1994) Chemotaxonomic and cytogenetic studies on Perilla frutescens var. citriodora (“Lemon Egoma”). Nat Med 48:185–190

    CAS  Google Scholar 

  • Hongtrakul V, Slabaugh MB, Knapp SJ (1998) A seed specific Δ-12 oleate desaturase is duplicated, rearranged, and weakly expressed in high oleic acid sunflower lines. Crop Sci 38:1245–1249

    Article  CAS  Google Scholar 

  • Iba K, Gibson S, Nishiuch T, Fuse T, Nishimura M, Arondel V, Hugly S, Somerville C (1993) A gene encoding a chloroplast ω-3 fatty acid desaturase complements alterations in fatty acid desaturation and chloroplast copy number of the fad7 mutant of Arabidopsis thaliana. J Biol Chem 268:24099–24105

    CAS  PubMed  Google Scholar 

  • Jin JB, Kim YA, Kim SJ, Lee SH, Kim DH, Cheong GW, Hwang I (2001) A new dynamin-like protein, ADL6, is involved in trafficking from the trans-Golgi network to the central vacuole in Arabidopsis. Plant Cell 13:1511–1526

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Joyard J, Block MA, Malherbe A, Maréchal E, Douce R (1993) Origin and synthesis of galactolipid and sulfolipid head groups. In: Moore TS Jr (ed) Lipid metabolism in plants. CRC Press, Boca Raton, pp 231–258

    Google Scholar 

  • Joyard J, Ferro M, Masselon C, Seigneurin-Berny D, Salvi D, Garin J, Rolland N (2010) Chloroplast proteomics highlights the subcellular compartmentation of lipid metabolism. Prog Lipid Res 49:128–158

    Article  CAS  PubMed  Google Scholar 

  • Jung JH, Kim H, Go YS, Lee SB, Hur C-G, Kim HU, Suh MC (2011) Identification of functional BrFAD2-1 gene encoding microsomal delta-12 fatty acid desaturase from Brassica rapa and development of Brassica napus containing high oleic acid contents. Plant Cell Rep 30:1881–1892

    Article  CAS  PubMed  Google Scholar 

  • Kang J, Snapp AR, Lu C (2011) Identification of three genes encoding microsomal oleate desaturase (FAD2) from the oilseed crop Camelina sativa. Plant Physiol Biochem 49:223–229

    Article  CAS  PubMed  Google Scholar 

  • Kim HU, Lee K-R, Shim D, Lee JH, Chen GQ, Hwang S (2016) Transcriptome analysis and identification of genes associated with ω-3 fatty acid biosynthesis in Perilla frutescens (L.) var. frutescens. BMC Genom 17:474

    Article  Google Scholar 

  • Kurdrid P, Subudhi S, Hongsthong A, Ruengjitchatchawalya M, Tanticharoen M (2005) Functional expression of Spirulina-Δ6 desaturase gene in yeast, Saccharomyces cerevisiae. Mol Biol Rep 32:215–226

    Article  CAS  PubMed  Google Scholar 

  • Lee S-K, Kim K-H, Kwon M-S, Hwang Y-S (2001a) Molecular cloning and characterization of expression patterns of a plastid ω-3 fatty acid desaturase cDNA from Perilla frutescens. Agric Chem Biotechnol 44:6–11

    CAS  Google Scholar 

  • Lee YJ, Kim DH, Kim Y-W, Hwang I (2001b) Identification of a signal that distinguishes between the chloroplast outer envelope membrane and the endomembrane system in vivo. Plant Cell 13:2175–2190

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee K-R, Kim SH, Go Y-S, Jung SM, Roh KH, Kim J-B, Suh M-C, Lee S, Kim HU (2012) Molecular cloning and functional analysis of two FAD2 genes from American grape (Vitis labrusca L.). Gene 509:189–194

    Article  CAS  PubMed  Google Scholar 

  • Lee K-R, Sohn SI, Jung JH, Kim SH, Roh KH, Kim JB, Suh MC, Kim HU (2013) Functional analysis and tissue-differential expression of four FAD2 genes in amphidiploid Brassica napus derived from Brassica rapa and Brassica oleracea. Gene 531:253–262

    Article  CAS  PubMed  Google Scholar 

  • Li L, Wang X, Gai J, Yu D (2007) Molecular cloning and characterization of a novel microsomal oleate desaturase gene from soybean. J Plant Physiol 164:1516–1526

    Article  CAS  PubMed  Google Scholar 

  • Liu HL, Yin ZJ, Xiao L, Xu YN, le Qu Q (2012) Identification and evaluation of ω-3 fatty acid desaturase genes for hyperfortifying α-linolenic acid in transgenic rice seed. J Exp Bot 63:3279–3287

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Los DA, Murata N (1998) Structure and expression of fatty acid desaturases. Biochim Biophys Acta 1394:3–15

    Article  CAS  PubMed  Google Scholar 

  • Machettira AB, Groß LE, Tillmann B, Weis BL, Englich G, Sommer MS, Königer M, Schleiff E (2012) Protein-induced modulation of chloroplast membrane morphology. Front Plant Sci 2:118

    Article  PubMed  PubMed Central  Google Scholar 

  • McCartney AW, Dyer JM, Dhanos PK, Kim PK, Andrews DW, McNew JA, Mullen RT (2004) Membrane-bound fatty acid desaturases are inserted co-translationally into the ER and contain different ER retrieval motifs at their carboxy termini. Plant J 37:156–173

    Article  CAS  PubMed  Google Scholar 

  • McConn M, Hugly S, Browse J, Somerville C (1994) A mutation at the fad8 locus of Arabidopsis identifies a second chloroplast ω-3 desaturase. Plant Physiol 106:1609–1614

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Miquel M, James D, Dooner H, Browse J (1993) Arabidopsis requires polyunsaturated lipids for low-temperature survival. Proc Natl Aca Sci USA 90:6208–6212

    Article  CAS  Google Scholar 

  • Nitta M, Lee JK, Kang CW, Katsuta M, Yasumoto S, Liu D, Nagamine T, Ohnishi O (2005) The distribution of Perilla species. Genet Resour Crop Ev 52:797–804

    Article  Google Scholar 

  • Ohlrogge J, Browse J (1995) Lipid biosynthesis. Plant Cell 7:957–970

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Okuley J, Lightner J, Feldmann K, Yadav N, Lark E, Browse J (1994) Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis. Plant Cell 6:147–158

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • O’Quin JB, Bourassa L, Zhang D, Shockey JM, Gidda SK, Fosnot S, Chapman KD, Mullen RT, Dyer JM (2010) Temperature-sensitive post-translational regulation of plant omega-3 fatty-acid desaturases is mediated by the endoplasmic reticulum-associated degradation pathway. J Biol Chem 285:21781–21796

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Reed DW, Schäfer UA, Covello PS (2000) Characterization of the Brassica napus extraplastidial linoleate desaturase by expression in Saccharomyces cerevisiae. Plant Physiol 122:715–720

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Routaboul J-M, Fischer SF, Browse J (2000) Trienoic fatty acids are required to maintain chloroplast function at low temperatures. Plant Physiol 124:1697–1705

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schulz MH, Zerbino DR, Vingron M, Birney E (2012) Oases: robust de novo RNA-seq assembly across the dynamic range of expression levels. Bioinformatics 28:1086–1092

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shanklin J, Whittle E, Fox BG (1994) Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase. Biochemistry 33:12787–12794

    Article  CAS  PubMed  Google Scholar 

  • Shin HS, Kim SW (1994) Lipid composition of perilla seed. J Am Oil Chem Soc 71:619–622

    Article  CAS  Google Scholar 

  • Smith MA, Moon H, Chowrira G, Kunst L (2003) Heterologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis thaliana. Planta 217:507–516

    Article  CAS  PubMed  Google Scholar 

  • Somerville C, Browse J, Jaworski JG, Ohlrogge JB (2000) Lipids. In: Buchanan BB, Gruissem W, Jones RL (eds) Biochemistry and molecular biology of plants. American Society of Plant Physiologists, Rockville, pp 456–527

    Google Scholar 

  • Tang GQ, Novitzky WP, Griffin HC, Huber SC, Dewey RE (2005) Oleate desaturase enzymes of soybean: evidence of regulation through differential stability and phosphorylation. Plant J 44:433–446

    Article  CAS  PubMed  Google Scholar 

  • Torres-Franklin M-L, Repellin A, Huynh V-B, d’Arcy-Lameta A, Zuily-Fodil Y, Pham-Thi A-T (2009) Omega-3 fatty acid desaturase (FAD3, FAD7, FAD8) gene expression and linolenic acid content in cowpea leaves submitted to drought and after rehydration. Environ Exp Bot 65:162–169

    Article  CAS  Google Scholar 

  • von Heijne G, Steppuhn J, Herrmann RG (1989) Domain structure of mitochondrial and chloroplast targeting peptides. Eur J Biochem 180:535–545

    Article  Google Scholar 

  • Vrinten P, Hu Z, Munchinsky M-A, Rowland G, Qiu X (2005) Two FAD3 desaturase genes control the level of linolenic acid in flax seed. Plant Physiol 139:79–87

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yadav NS, Wierzbicki A, Aegerter M, Caster CS, Pérez-Grau L, Kinney AJ, Hitz WD, Booth JR Jr, Schweiger B, Stecca KL, Allen SM, Blackwell M, Reiter RS, Carlson TJ, Russell SH, Feldmann KA, Pierce J, Browse J (1993) Cloning of higher plant ω-3 fatty acid desaturases. Plant Physiol 103:467–476

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yang Q, Fan C, Guo Z, Qin J, Wu J, Li Q, Fu T, Zhou Y (2012) Identification of FAD2 and FAD3 genes in Brassica napus genome and development of allele-specific markers for high oleic and low linolenic acid contents. Theor Appl Genet 125:715–729

    Article  CAS  PubMed  Google Scholar 

  • Zerbino DR, Birney E (2008) Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821–829

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang D, Pirtle IL, Park SJ, Nampaisansuk M, Neogi P, Wanjie SW, Pirtle RM, Chapman KD (2009) Identification and expression of a new delta-12 fatty acid desaturase (FAD2-4) gene in upland cotton and its functional expression in yeast and Arabidopsis thaliana plants. Plant Physiol Biochem 47:462–471

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The perilla seeds used in this study were kindly provided by Dr. Myung-Hee Lee of the Dept. of the Southern Area Crop Science, National Institute of Crop Science, in Miryang, Republic of Korea. This study was conducted with the support of the Research Program for Agricultural Science & Technology Development (Project No. PJ01007504), the National Institute of Agricultural Science, Rural Development Administration, and the Next-Generation BioGreen 21 Program (SSAC, Grant No. PJ01108101), Republic of Korea as well as the faculty research fund (Project No. 20160163) of Sejong University.

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Authors and Affiliations

  1. Department of Agricultural Biotechnology, National Institute of Agricultural Science, Rural Development Administration, Jeonju, 54874, Republic of Korea

    Kyeong-Ryeol Lee, Eun-Ha Kim, Seul-Bee Lee, Kyung Hee Roh, Jong-Bum Kim & Han-Chul Kang

  2. Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, 37673, Republic of Korea

    Yongjik Lee

  3. Department of Bioindustry and Bioresource Engineering, Plant Engineering Research Institute, Sejong University, Seoul, 05006, Republic of Korea

    Hyun Uk Kim

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  1. Kyeong-Ryeol Lee
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Correspondence to Hyun Uk Kim.

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Communicated by J. Sheop Shin.

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299_2016_2053_MOESM1_ESM.pptx

Supplementary Fig. S1 Multiple sequence alignment of two FAD2 genes from perilla leaves and developing seeds tissues. Black box and dotted box indicate PfrFAD2-1 and PfrFAD2-2 specific sequence, respectively. Multiple sequence alignment was performed using DNASTAR® MegAlign (Ver. 8.1.4) (PPTX 887 kb)

299_2016_2053_MOESM2_ESM.pptx

Supplementary Fig. S2 Multiple sequence alignment of two FAD3 genes from diverse perilla cultivar. Black box and dotted box indicate PfrFAD3-1 and PfrFAD3-2 specific sequence, respectively. Exceptionally, there is a single nucleotide polymorphism at 300 bp position in F2 and F4. 13 sequences except PfrFAD3-1 and PfrFAD3-2 were from perilla cultivar as follows. A1,2, Dayudeulkkae; B2, K131012; C1,3, K135903; D4, K131017; E5,6, Anyu; F2,3,4, K135858; G2,3, K126202. These cultivars were obtained from National Agrobiodiversity Center, National Institute of Agricultural Sciences, Republic of Korea. Multiple sequence alignment was performed using DNASTAR® MegAlign (Ver. 8.1.4) (PPTX 588 kb)

299_2016_2053_MOESM3_ESM.pptx

Supplementary Fig. S3 Relative expression levels of PfrFAD2, PfrFAD3, PfrFAD7-1, and PfrFAD7-2 genes from leaves of perilla treated or untreated at low temperature (15˚C) for two weeks. Unattached any letter in front of gene name and LT-gene name indicate untreated and treated low temperature, respectively. Experiments were performed in triplicate and error bars indicate standard deviations (PPTX 48 kb)

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Lee, KR., Lee, Y., Kim, EH. et al. Functional identification of oleate 12-desaturase and ω-3 fatty acid desaturase genes from Perilla frutescens var. frutescens . Plant Cell Rep 35, 2523–2537 (2016). https://doi.org/10.1007/s00299-016-2053-4

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