Identification of two genes encoding microsomal oleate desaturases (FAD2) from the biodiesel plant Pongamia pinnata L.

Published Date

August 2016, Volume 30, Issue 4, pp 1351-1360

First online: 09 March 2016

Title 

Identification of two genes encoding microsomal oleate desaturases (FAD2) from the biodiesel plant Pongamia pinnata L.

• Author 

• Ramesh Aadi Moolam
• , Anuma Singh
• , Rahul G. Shelke
• , Paul T. Scott
• , Peter M. Gresshoff
• , Latha Rangan

Abstract

Key message

The current study dissect microsomal oleate desaturase genes having differential expression pattern with respect to temperature from the seeds ofPongamia pinnata, and are grouped with other legumes and biofuel plants.

Abstract

Biofuel often is available as plant oil or products derived thereafter, such as biodiesel. In view of the anticipated fossil fuel shortage, the biochemical and genetic basis of vegetable oil biosynthesis is vital. We are focusing on the versatile oil-yielding legume tree Pongamia pinnata. Microsomal oleate desaturase (FAD2) is the key enzyme responsible for the production of linoleic acid in non-photosynthetic tissues. This work reports on the isolation of two full length cDNA clones, tissue expression and copy number detection of Pongamia FAD2 genes, and also on the transcriptional regulation at low and high temperatures. The deduced amino acid sequences of both PpFAD2 proteins share 83 % identity and display three typical histidine boxes that are characteristics of all membrane-bound microsomal oleate desaturases. Both sequences possess aromatic amino acid containing sequence motifs at the C-terminal end necessary for maintaining endoplasmic reticulum (ER) localization. Southern blot analysis is consistent with the presence of at least two copies of FAD2 in the pongamia genome. Quantitative real-time PCR analysis showed that PpFAD2-1 is expressed strongly in developing seeds and, showing very low levels of expression in vegetative tissues, whereas PpFAD2-2 is constitutively expressed in both vegetative tissues and the developing seeds, with higher transcript levels in roots, stems and leaves. In response to low temperature stress PpFAD2-1 and PpFAD2-2 were differentially expressed in various tissues. The PpFAD2-2 transcript dramatically decreased in roots, stems and leaves under low temperatures, whereas PpFAD2-1 showed a significant increase in these tissues.

References

1. Aadi MR, Kesari V, Latha R (2014) Characterization of a stearoyl-acyl carrier protein desaturase gene from potential biofuel plant, Pongamia pinnata. Gene 542:113–121CrossRef
2. Altschul SF (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nuc Acids Res 25:3389–3402CrossRef
3. Biswas B, Kazakoff SH, Jiang Q, Samuel S, Gresshoff PM, Scott PT (2013) Genetic and genomic analysis of the tree legume Pongamia pinnata as a feedstock for biofuel. Plant Genome. doi:10.​3835/​plantgenome2013.​05.​0015
4. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159CrossRefPubMed
5. Dereeper A (2008) Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nuc Acids Res 3:465–469CrossRef
6. Durrett TP, Benning C, Ohlrogge J (2008) Plant triacyglycerols as feed stocks for the production of biofuels. Plant J 54:593–607CrossRefPubMed
7. Falcone DL, Gibson SG, Lemieux B, Somerville C (1994) Identification of a gene complements and Arabidopsismutant deficient in chloroplast ω-6desaturase activity. Plant Physiol 106:1453–1459CrossRefPubMedPubMedCentral
8. Heppard EP, Kinney AJ, Stecca KL, Miao GH (1996) Developmental and growth temperature regulation of two different microsomal ω-6 desaturase genes in soybean. Plant Physiol 110:311–319CrossRefPubMedPubMedCentral
9. Hernandez ML, Mancha M, Martinez Rivas JM (2005) Molecular cloning and characterization of genes encoding two microsomal oleate desaturases (FAD2) from olive. Phytochem 66:1417–1426CrossRef
10. Hernandez ML, Padilla MN, Sicardo MD, Mancha M, Marinez-Rivas JM (2011) Effect of different environmental stresses on the expression of oleate desaturase genes and fatty acid composition in olive fruit. Phytochem 72(2–3):178–187CrossRef
11. Hirokawa T, Boon-Chieng S, Mitaku S (1998) SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinfo 14:378–379CrossRef
12. Holdsworth M, Kurup S, McKibbin R (1999) Molecular and genetic mechanisms regulating the transition from embryo development to germination. Trends Plant Sci 4:275–280CrossRef
13. Hu XY, Mandy SG, Gupta M, Steven A (2006) Mapping of the loci controlling oleic and linoleic acid contents and development of fad2 and fad3 allele-specific markers in canola (Brassica napus L.). Theor Appl Genet 113:497–507CrossRefPubMed
14. Kargiotidou A, Deli D, Galanopoulou D, Tsaftaris A, Farmaki T (2008) Low temperature and light regulate delta 12 fatty acid desaturases (FAD2) at a transcriptional level in cotton (Gossypium hirsutum). J Exp Bot 59:2043–2056CrossRefPubMedPubMedCentral
15. Kazakoff S, Gresshoff PM, Scott PT (2011) Pongamia pinnata, a sustainable feedstock for biodiesel production. In: Energy Crops (eds). Nigel Halford and Angela Karp. Royal Society Chem pp: 233–254
16. Kesari V, Sudarshan M, Das A, Rangan L (2009) PCR amplification of the genomic DNA from the seeds of Ceylon Ironwood, Jatropha and Pongamia. Biomass Bioenergy 33:1724–1728CrossRef
17. Kesari V, Das A, Rangan L (2010) Physico-chemical characterization and antimicrobial activity from seed oil of Pongamia pinnata, a potential biofuel crop. Biomass Bioenergy 34:108–115CrossRef
18. Kesari V, Ramesh AM, Rangan L (2012) High frequency direct organogenesis and evaluation of genetic stability for in vitro regenerated Pongamia pinnata, a valuable biodiesel plant. Biomass Bioenergy 44:23–32CrossRef
19. Kumar V, Muthuraj M, Palabhanvi B, Ghoshal AK, Das D (2014) Evaluation and optimization of two stage sequential in situ transesterification process for fatty acid methyl ester quantification from microalgae. Renew Energy 68:560–569CrossRef
20. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 57:105–132CrossRef
21. Li LY, Wang XL, Gai JY, Yu DY (2007) Molecular cloning and characterization of a novel microsomal oleate desaturase gene from soybean. J Plant Physiol 164:1516–1526CrossRefPubMed
22. Ling- Liang G, Yuan BW, Hao S, Kai H, Ying WX, Wei W (2012) Molecular cloning and expression analysis of genes encoding two microsomal oleate desaturases (FAD2) from safflower (Carthamus tinctorius L.). Plant Mol Biol Rep 30:139–148CrossRef
23. Lingyong L, Xiaolin W, Junyi G, Deyue Y (2007) Molecular cloning and characterization of a novel microsomal oleate desaturase gene from soybean. J Plant Physiol 164:1516–1526CrossRef
24. López Y, Nadaf HL, Smith OD, Connell JP, Reddy AS, Fritz AK (2000) Isolation and characterization of the delta 12-fatty acid desaturase in peanut (Arachis hypogaea L.) and search for polymorphisms for the high oleate trait in Spanish market -type lines. Theor Appl Genet 101:1131–1138CrossRef
25. Los DA, Murata N (1998) Structure and expression of fatty acid desaturase. Biochemica Biophysica Acta 1394:3–15CrossRef
26. Mansfield SG, Briarty LG (1991) Cotyledon cell development in Arabidopsis thaliana during reserve deposition. Can J Bot 70:151–164CrossRef
27. Martinez-Rivas JM, Sperling P, Luhs W, Heinz E (2001) Spatial and temporal regulation of three different microsomal oleate desaturase genes (FAD2) from normal type and high-oleic varieties of sunflower (Helianthus annuus L.). Mol Breed 8:159–168CrossRef
28. McCartney AW, Dyer JM, Dhanoa PK, Kim PK, Andrews DW, McNew JA (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 37:156–173CrossRefPubMed
29. Mekhedov S, De Ilarduya OM, Ohlrogge J (2000) Toward a functional catalog of the genome. A survey of genes for lipid biosynthesis. Plant Physiol 122:389–401CrossRefPubMedPubMedCentral
30. Murphy DJ, Piffanelli P (1998) Fatty acid desaturases: structure, mechanism and regulation. In: Plant Lipid Biosynthesis: fundamentals and agricultural applications. Harwood JL (ed), Cambridge University Pres: pp 95–130
31. Neidleman SL (1987) Effects of temperature on lipid unsaturation. Biotech Genet Eng Rev 5:245–268CrossRef
32. Ohlrogge J (1997) Regulation of fatty acid synthesis. Ann Rev Plant Physiol Plant Mol Biol 48:109–136CrossRef
33. Ohlrogge J, Browse J (1995) Lipid biosynthesis. Plant Cell 7:957–970CrossRefPubMedPubMedCentral
34. Okuley J, Lighatner J, Feldmann K, Yadav N, Lark E, Browse J (1994) Arabidopsis FAD2 gene encode enzyme that is essential for polyunsaturated lipid synthesis. Plant Cell 6:147–158CrossRefPubMedPubMedCentral
35. Pavithra HR, Balakrishna G, Rajesh KK, Prasanna KT, Shivanna MB (2012) Oil, fatty acid profile and karanjin content in developing Pongamia pinnata (L.) Pierre seeds. J Am Oil Chem 89:2237–2244CrossRef
36. Rahamatalla AB, Babiker EE, Krishna AG, El Tinay AH (2001) Changes in fatty acids composition during seed growth and physicochemical characteristics of oil extracted from four safflower cultivars. Plant Foods Human Nutr 56:385–395CrossRef
37. Rangan L, Rout A, Sudarshan M, Gregorio G (2009) Molecular cloning, expression and mapping of the translational initiation factor elf1 gene in Oryza sativa. Functional Plant Biol 36:442–452CrossRef
38. Schlueter JA, Vasylenko-Sanders IF, Deshpande S, Yi J, Siegfried M, Roe BA, Schlueter SD, Scheffler BE, Shoemaker RC (2007) The FAD2 gene family of soybean: insights into the structural and functional divergence of a paleopolyploid genome. Crop Sci 47(1):14–26
39. Scott PT, Pregelj L, Chen N, Hadler JS, Djordjevic MA, Gresshoff PM (2008) Pongamia pinnata: an untapped resource for the biofuels industry of the future. BioEnergy Res 1:2–11CrossRef
40. Seffens WS, Almoguera D, Wilde HD, Vonder HRA, Thomas TL (1990) Molecular analysis of a phylogenetically conserved carrot gene: developmental and environmental regulation. Dev Genet 11:65–76CrossRefPubMed
41. Shanklin J, Cahoon EB (1998) Desaturation and related modifications of fatty acids. Ann Rev Plant Physiol Plant Mol Biol 49:611–641CrossRef
42. 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 33:12787–12794CrossRefPubMed
43. Shijiang C, Xue RZ, Craig CW, Allan GG, Surinder PS, Lixia L, Qing L (2013) A large and functionally diverse family of Fad2 genes in safflower (Carthamus tinctorius L.). BioMed Central 13(5):1–18
44. Sorensen BM, Furukawa-Stoffer TL, Marshall KS, Page EK, Mir Z, Forster RJ, Weselake RJ (2005) Storage lipid accumulation and acyltransferase action in developing flaxseed. Lipids 40:1042–1049CrossRef
45. Stoutjesdijk PA, Singh SP, Liu Q, Hurlstone CJ, Waterhouse PA, Green AG (2002) hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efficient and stable silencing. Plant Physiol 129:1723–1731CrossRefPubMedPubMedCentral
46. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599CrossRefPubMed
47. Tang GQ, Novitzky WP, Carol Griffin H, Huber SC, Dewey RE (2005) Oleate desaturase enzymes of soybean: evidence of regulation through different stability and phosphorylation. Plant J 44:433–446CrossRefPubMed
48. Teixeira MC, Coelho N, Olsson ME, Brodelius PE, Carvalho IS, Brodelius M (2009) Molecular cloning and expression analysis of three omega-6 desaturase genes from purlane (Portulaca oleracea L.). Biotech Lett 31:1089–1101CrossRef
49. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nuc Acids Res 25:4876–4882CrossRef
50. Voelker T, Kinney AJ (2001) Variations in the biosynthesis of seed storage lipids. Annu Rev Plant Physiol Plant Mol Biol 52:335–361CrossRefPubMed
51. Weber H (2002) Fatty acid-derived signals in plants. Trends in Plant Sci 7:217–224CrossRef
52. Wiberg E, Banas A, Stymne S (1997) Fatty acid distribution and lipid metabolism in developing seeds of laurate producing rape (Brassica napus L.). Planta 203:341–348CrossRefPubMed
53. Wu P, Sheng Z, Lin Z, Yaping C, Meiru L, Huawu J, Wu G (2013) Functional characterization of two microsomal fatty acid desaturases from Jatropha curcas L. J Plant Physiol 170:1360–1366CrossRefPubMed
54. Xiao G, Zhang ZQ, Liu RY, Yin CF, Wu XM, Tan TL, Guan CY (2013) Molecular cloning and characterization of a novel gene involved in fatty acid synthesis in Brassica napus L. J Int Agr 12:962–970CrossRef

For further details log on website :
http://link.springer.com/article/10.1007/s00468-016-1371-z