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Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism

A Corrigendum to this article was published on 12 June 2017

Abstract

The amount of cholesterol made by many plants is not negligible. Whereas cholesterogenesis in animals was elucidated decades ago, the plant pathway has remained enigmatic. Among other roles, cholesterol is a key precursor for thousands of bioactive plant metabolites, including the well-known Solanum steroidal glycoalkaloids. Integrating tomato transcript and protein co-expression data revealed candidate genes putatively associated with cholesterol biosynthesis. A combination of functional assays including gene silencing, examination of recombinant enzyme activity and yeast mutant complementation suggests the cholesterol pathway comprises 12 enzymes acting in 10 steps. It appears that half of the cholesterogenesis-specific enzymes evolved through gene duplication and divergence from phytosterol biosynthetic enzymes, whereas others act reciprocally in both cholesterol and phytosterol metabolism. Our findings provide a unique example of nature's capacity to exploit existing protein folds and catalytic machineries from primary metabolism to assemble a new, multi-step metabolic pathway. Finally, the engineering of a ‘high-cholesterol’ model plant underscores the future value of our gene toolbox to produce high-value steroidal compounds via synthetic biology.

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Figure 1: The cholesterogenesis pathway in plants and its relationship to phytosterol metabolism and cholesterogenesis in humans.
Figure 2: A transcriptomics and proteomics-based co-expression approach reveals a set of putative cholesterol pathway genes.
Figure 3: Phylogenetic analysis suggests that some of the cholesterol biosynthesis enzymes evolved by duplication and divergence from the phytosterol biosynthesis enzymes.
Figure 4: Distinct pairs of SMOs are involved in cholesterol and phytosterol biosynthesis.
Figure 5: Common enzymes in the cholesterol and phytosterol biosynthetic pathways.
Figure 6: C5-SD2 and 7-DR2 catalyse the last two steps in plant cholesterogenesis.

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References

  1. Nes, W. D. Biosynthesis of cholesterol and other sterols. Chem. Rev. 111, 6423–6451 (2011).

    Article  CAS  Google Scholar 

  2. Benveniste, P. Biosynthesis and accumulation of sterols. Annu. Rev. Plant Biol. 55, 429–457 (2004).

    Article  CAS  Google Scholar 

  3. Piironen, V., Lindsay, D. G., Miettinen, T. A., Toivo, J. & Lampi, A. M. Plant sterols: biosynthesis, biological function and their importance to human nutrition. J. Sci. Food Agric. 80, 939–966 (2000).

    Article  CAS  Google Scholar 

  4. Schaller, H. The role of sterols in plant growth and development. Prog. Lipid Res. 42, 163–175 (2003).

    Article  CAS  Google Scholar 

  5. Behrman, E. J. & Gopalan, V. Concepts in biochemistry cholesterol and plants. J. Chem. Educ. 82, 1791–1793 (2005).

    Article  CAS  Google Scholar 

  6. Jäpelt, R. B. & Jakobsen, J. Vitamin D in plants: a review of occurrence, analysis, and biosynthesis. Front. Plant Sci. 4, 136 (2013).

    Article  Google Scholar 

  7. Milner, S. E. et al. Bioactivities of glycoalkaloids and their aglycones from Solanum species. J. Agric. Food Chem. 59, 3454–3484 (2011).

    Article  CAS  Google Scholar 

  8. Dinan, L. Phytoecdysteroids: biological aspects. Phytochemistry 57, 325–339 (2001).

    Article  CAS  Google Scholar 

  9. Cárdenas, P. D. et al. The bitter side of the nightshades: genomics drives discovery in Solanaceae steroidal alkaloid metabolism. Phytochemistry 113, 24–32 (2014).

    Article  Google Scholar 

  10. Bloch, K. The biological synthesis of cholesterol. Science 150, 19–28 (1965).

    Article  CAS  Google Scholar 

  11. Ohyama, K., Suzuki, M., Kikuchi, J., Saito, K. & Muranaka, T. Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis. Proc. Natl Acad. Sci. USA 106, 725–730 (2009).

    Article  CAS  Google Scholar 

  12. Diener, A. C. et al. STEROL METHYLTRANSFERASE 1 controls the level of cholesterol in plants. Plant Cell 12, 853–870 (2000).

    Article  CAS  Google Scholar 

  13. Arnqvist, L., Dutta, P. C., Jonsson, L. & Sitbon, F. Reduction of cholesterol and glycoalkaloid levels in transgenic potato plants by overexpression of a type 1 sterol methyltransferase cDNA. Plant Physiol. 131, 1792–1799 (2003).

    Article  CAS  Google Scholar 

  14. Sawai, S. et al. Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal glycoalkaloids in potato. Plant Cell 26, 3763–3774 (2014).

    Article  CAS  Google Scholar 

  15. Cárdenas, P. D. et al. GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway. Nat. Commun. 7, 10654 (2016).

    Article  Google Scholar 

  16. Itkin, M. et al. Biosynthesis of antinutritional alkaloids in Solanaceous crops is mediated by clustered genes. Science 341, 175–179 (2013).

    Article  CAS  Google Scholar 

  17. Itkin, M. et al. GLYCOALKALOID METABOLISM1 is required for steroidal alkaloid glycosylation and prevention of phytotoxicity in tomato. Plant Cell 23, 4507–4525 (2011).

    Article  CAS  Google Scholar 

  18. Rahier, A. Dissecting the sterol C-4 demethylation process in higher plants from structures and genes to catalytic mechanism. Steroids 76, 340–352 (2011).

    Article  CAS  Google Scholar 

  19. Rahier, A. & Karst, F. Plant cyclopropylsterol-cycloisomerase: key amino acids affecting activity and substrate specificity. Biochem. J. 459, 289–299 (2014).

    Article  CAS  Google Scholar 

  20. Kushiro, M. et al. Obtusifoliol 14-α-demethylase (CYP51) antisense Arabidopsis shows slow growth and long life. Biochem. Biophys. Res. Commun. 285, 98–104 (2001).

    Article  CAS  Google Scholar 

  21. Schrick, K. et al. FACKEL is a sterol C-14 reductase required for organized cell division and expansion in Arabidopsis embryogenesis. Genes Dev. 14, 1471–1484 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Souter, M. et al. Hydra mutants of Arabidopsis are defective in sterol profiles and auxin and ethylene signaling. Plant Cell 14, 1017–1031 (2002).

    Article  CAS  Google Scholar 

  23. Moses, T. et al. Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α-hydroxylase from Bupleurum falcatum. Proc. Natl Acad. Sci. USA 111, 1634–1639 (2014).

    Article  CAS  Google Scholar 

  24. Kushiro, T., Shibuya, M. & Ebizuka, Y. β-Amyrin synthase-cloning of oxidosqualene cyclase that catalyzes the formation of the most popular triterpene among higher plants. Eur. J. Biochem. 256, 238–244 (1998).

    Article  CAS  Google Scholar 

  25. Darnet, S. & Rahier, A. Plant sterol biosynthesis: identification of two distinct families of sterol 4α-methyl oxidases. Biochem. J. 378, 889–898 (2004).

    Article  CAS  Google Scholar 

  26. Suza, W. P. & Chappell, J. Spatial and temporal regulation of sterol biosynthesis in Nicotiana benthamiana. Physiol. Plant. 157, 120–134 (2016).

    Article  CAS  Google Scholar 

  27. Rahier, A., Darnet, S., Bouvier, F., Camara, B. & Bard, M. Molecular and enzymatic characterizations of novel bifunctional 3β-hydroxysteroid dehydrogenases/C-4 decarboxylases from Arabidopsis thaliana. J. Biol. Chem. 281, 27264–27277 (2006).

    Article  CAS  Google Scholar 

  28. Pascal, S., Taton, M. & Rahier, A. Plant sterol biosynthesis: identification of a NADPH dependant plant sterone reductase involved in the sterol-4-demethylation. Arch. Biochem. Biophys. 312, 260–271 (1994).

    Article  CAS  Google Scholar 

  29. Rahier, A., Pierre, S., Riveill, G. & Karst, F. Identification of essential amino acid residues in a sterol 8,7-isomerase from Zea mays reveals functional homology and diversity with the isomerases of animal and fungal origin. Biochem. J. 414, 247–259 (2008).

    Article  CAS  Google Scholar 

  30. Ashman, W. H., Barbuch, R. J., Ulbright, C. E., Jarrett, H. W. & Bard, M. Cloning and disruption of the yeast C-8 sterol isomerase gene. Lipids 26, 628–632 (1991).

    Article  CAS  Google Scholar 

  31. Palermo, L. M., Leak, F. W., Tove, S. & Parks, L. W. Assessment of the essentiality of ERG genes late in ergosterol biosynthesis in Saccharomyces cerevisiae. Curr. Genet. 32, 93–99 (1997).

    Article  CAS  Google Scholar 

  32. Husselstein, T., Schaller, H., Gachotte, D. & Benveniste, P. Δ7-sterol-C5-desaturase: molecular characterization and functional expression of wild-type and mutant alleles. Plant Mol. Biol. 39, 891–906 (1999).

    Article  CAS  Google Scholar 

  33. Choe, S. et al. The Arabidopsis dwf7/ste1 mutant is defective in the Δ7-sterol C-5 desaturation step leading to brassinosteroid biosynthesis. Plant Cell 11, 207–221 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Choe, S. et al. Lesions in the sterol delta reductase gene of Arabidopsis cause dwarfism due to a block in brassinosteroid biosynthesis. Plant J. 21, 431–443 (2000).

    Article  CAS  Google Scholar 

  35. Gaylor, J. L. Membrane-bound enzymes of cholesterol synthesis from lanosterol. Biochem. Biophys. Res. Commun. 1146, 1139–1146 (2008).

    Google Scholar 

  36. Caldas, H. & Herman, G. E. NSDHL, an enzyme involved in cholesterol biosynthesis, traffics through the Golgi and accumulates on ER membranes and on the surface of lipid droplets. Human Mol. Genetics 12, 2981–2991 (2003).

    Article  CAS  Google Scholar 

  37. Silvestro, D., Andersen, T. G., Schaller, H. & Jensen, P. E. Plant sterol metabolism. Δ7-Sterol-C5-desaturase (STE1/DWARF7), Δ5,7-sterol-Δ7-reductase (DWARF5) and Δ24-sterol-Δ24-reductase (DIMINUTO/DWARF1) show multiple subcellular localizations in Arabidopsis thaliana (Heynh) L. PLoS ONE 8, e56429 (2013).

    Article  CAS  Google Scholar 

  38. Ycas, M. On earlier states of biochemical systems. J. Theor. Biol. 44, 145–160 (1974).

    Article  CAS  Google Scholar 

  39. Jensen, R. A. Enzyme recruitment in evolution of new function. Annu. Rev. Microbiol. 30, 409–425 (1976).

    Article  CAS  Google Scholar 

  40. Maechler, M . et al. Cluster: Cluster Analysis Basics and Extensions. R package v.1.15.1 (CRAN, 2014); http://cran.r-project.org/web/packages/cluster/index.html

  41. Ihaka, R. & Gentleman, R. R: a language for data analysis and graphics. J. Comp. Graph. Stat. 5, 299–314 (1996).

    Google Scholar 

  42. Mi, H., Muruganujan, A. & Thomas, P. D. PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees. Nucleic Acids Res. 41, D377–D386 (2013).

    Article  CAS  Google Scholar 

  43. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

    Article  CAS  Google Scholar 

  44. Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729 (2013).

    Article  CAS  Google Scholar 

  45. Itkin, M. et al. TOMATO AGAMOUS-LIKE is a component of the fruit ripening regulatory network. Plant J. 60, 1081–1095 (2009).

    Article  CAS  Google Scholar 

  46. Senthil-Kumar, M. & Mysore, K. S. Tobacco rattle virus–based virus-induced gene silencing in Nicotiana benthamiana. Nat. Protoc. 9, 1549–1562 (2014).

    Article  CAS  Google Scholar 

  47. Wretensjö, I. & Karlberg, B. Characterization of sterols in borage oil by GC-MS. J. Am. Chem. Oil Soc. 79, 1069–1074 (2004).

    Article  Google Scholar 

  48. Zhang, X. et al. Separation of Δ5-and Δ7-phytosterols by adsorption chromatography and semipreparative reversed phase high-performance liquid chromatography for quantitative analysis of phytosterols in foods. J. Agric. Food Chem. 54, 1196–1202 (2006).

    Article  CAS  Google Scholar 

  49. Yang, B., Karlsson, R. M., Oksman, P. H. & Kallio, H. P. Phytosterols in sea buckthorn (Hippophae rhamnoides L.) berries: identification and effects of different origins and harvesting times. J. Agric. Food Chem. 49, 5620–5629 (2001).

    Article  CAS  Google Scholar 

  50. Kamal-Eldin, A., Appelqvist, L. A., Yousif, G. & Iskander, G. M. Seed lipids of Sesamum indicum and related wild species in Sudan. The sterols. J. Sci. Food Agric. 59, 327–334 (1992).

    Article  CAS  Google Scholar 

  51. Expósito-Rodríguez, M., Borges, A. A., Borges-Pérez, A. & Pérez, J. A. Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol. 8, 131–142 (2008).

    Article  Google Scholar 

  52. Rotenberg, D., Thompson, T. S., German, T. L. & Willis, D. K. Methods for effective real-time RT-PCR analysis of virus-induced gene silencing. J. Virol. Methods 138, 49–59 (2006).

    Article  CAS  Google Scholar 

  53. Karimi, M., Inzé, D. & Depicker, A. GATEWAYTM vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 7, 193–195 (2002).

    Article  CAS  Google Scholar 

  54. Taton, M., Husselstein, T., Benveniste, P. & Rahier, A. Role of highly conserved residues in the reaction catalyzed by recombinant Δ7-sterol-C5(6)-desaturase studied by site-directed mutagenesis. Biochemistry 39, 701–711 (2000).

    Article  CAS  Google Scholar 

  55. Zou, L., Li, L. & Porter, T. D. 7-dehydrocholesterol reductase activity is independent of cytochrome P450 reductase. J. Steroid Biochem. Mol. Biol. 127, 435–438 (2011).

    Article  CAS  Google Scholar 

  56. Sarrion-Perdigones, A. et al. Goldenbraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 162, 1618–1631 (2013).

    Article  CAS  Google Scholar 

  57. Clough, S. & Bent, A. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    Article  CAS  Google Scholar 

  58. Sparkes, I. A., Runions, J., Kearns, A. & Hawes, C. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat. Protoc. 1, 2019–2025 (2006).

    Article  CAS  Google Scholar 

  59. Nelson, B. K., Cai, X. & Nebenführ, A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 51, 1126–1136 (2007).

    Article  CAS  Google Scholar 

  60. DiCarlo, J. E. et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 4336–4343 (2013).

    Article  CAS  Google Scholar 

  61. Schiestl, R. H. & Gietz, R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr. Genet. 16, 339–346 (1989).

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to D. Twafik for useful suggestions in phylogenetic analysis. A.A. is the incumbent of the Peter J. Cohn Professorial Chair. We thank the Adelis Foundation, the Leona M. and Harry B. Helmsley Charitable Trust, the Jeanne and Joseph Nissim Foundation for Life Sciences, Tom and Sondra Rykoff Family Foundation Research and the Raymond Burton Plant Genome Research Fund for supporting the laboratory activity of A.A. The work was supported by the Israel Science Foundation (ISF Grant No. 1805/15) and the European Research Council (ERC; SAMIT-FP7) personal grants to A.A. P.D.S. is grateful to the Planning and Budgeting Committee of the Council for Higher Education, Israel for the VATAT fellowship. The research in the laboratory of A.G. was financially supported by the VIB International PhD Fellowship Program (fellowship to P.A.) and the Research Foundation Flanders (postdoctoral fellowships to J.P. and L.P.). A.K was supported by a short-term EMBO fellowship (EMBO-ASTF-146-2014). The research in the laboratories of A.A. and A.G. was supported by the European Union Seventh Framework Program FP7/2007–2013 under grant agreement no. 613692–TriForC.

Author information

Authors and Affiliations

Authors

Contributions

P.D.S. designed experiments, performed the research and wrote the paper. J.P., P.A., L.P. and A.G. designed part of the experiments and performed all yeast complementation assays and wrote the paper. S.P. assisted in the VIGS experiments. J.S. and E.S. assisted in the co-expression data analysis. H.M. performed the confocal imaging experiments for localization studies. I.R. and S. Meir assisted with metabolomics data analysis and operated the LCMS. S. Malitsky assisted with GC-S metabolomics data analysis and operated the GCMS. M.Y. and T.U. performed recombinant protein expression in insect cells and isolated microsomes fractions. P.D.C. assisted in wild tomato accessions RNA sequencing. A.M. assisted in sterol extractions and tissue culture work. A.K. and A.P.G. designed part of the research and wrote the paper. H.S. assisted in data analysis and manuscript preparation. A.A. designed the research and wrote the paper.

Corresponding author

Correspondence to Asaph Aharoni.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information. (PDF 32015 kb)

Supplementary Data 1

Detailed list of genes. (XLSX 18 kb)

Supplementary Data 2

Amino acid sequences. (PDF 286 kb)

Supplementary File 1

Chemical structure: 4α-methyl-5α-cholest-7-en-3βol. (CDX 3 kb)

Supplementary File 2

Chemical structure: 4α-methyl-ergostatrienol. (CDX 3 kb)

Supplementary File 3

Chemical structure: 4α-methyl-24,25-dihydrozymosterol. (CDX 3 kb)

Supplementary File 4

Chemical structure: 4α-methylcholesta-8,14-dien-3β-ol. (CDX 3 kb)

Supplementary File 5

Chemical structure: 4α-methyl-ergostadienol. (CDX 3 kb)

Supplementary File 6

Chemical structure: 7-dehydrocholesterol. (CDX 3 kb)

Supplementary File 7

Chemical structure: 24-ethylidenelophenol. (CDX 3 kb)

Supplementary File 8

Chemical structure: 24-methylenecholesterol. (CDX 3 kb)

Supplementary File 9

Chemical structure: 24-methylenecycloartanol. (CDX 3 kb)

Supplementary File 10

Chemical structure: 24-methylenelophenol. (CDX 3 kb)

Supplementary File 11

Chemical structure: 31-nor-24,25-dihydrolanosterol. (CDX 3 kb)

Supplementary File 12

Chemical structure: 31-norcycloartano. (CDX 3 kb)

Supplementary File 13

Chemical structure: α-chaconine. (CDX 3 kb)

Supplementary File 14

Chemical structure: α-solanine. (CDX 4 kb)

Supplementary File 15

Chemical structure: α-tomatine. (CDX 5 kb)

Supplementary File 16

Chemical structure: β-sitosterol. (CDX 3 kb)

Supplementary File 17

Chemical structure: β-amyrin. (CDX 3 kb)

Supplementary File 18

Chemical structure: campesterol. (CDX 3 kb)

Supplementary File 19

Chemical structure: cholesta-7-en-3β-ol. (CDX 3 kb)

Supplementary File 20

Chemical structure: cholesterol. (CDX 3 kb)

Supplementary File 21

Chemical structure: cycloartanol. (CDX 3 kb)

Supplementary File 22

Chemical structure: cycloartenol. (CDX 3 kb)

Supplementary File 23

Chemical structure: cycloeucalenol. (CDX 3 kb)

Supplementary File 24

Chemical structure ( Delta 5,7 avenasterol) (CDX 3 kb)

Supplementary File 25

chemical structure(Delta 5, Episterol) (CDX 3 kb)

Supplementary File 26

Chemical structure (structure 2): 4,4-dimethylcholesta-8,24-dien-3β-ol (CDX 3 kb)

Supplementary File 27

Chemical structure: δ-7-avenasterol. (CDX 3 kb)

Supplementary File 28

Chemical structure: episterol. (CDX 3 kb)

Supplementary File 29

Chemical structure: esculeoside A. (CDX 7 kb)

Supplementary File 30

Chemical structure: isofucosterol. (CDX 3 kb)

Supplementary File 31

Chemical structure: lanosterol. (CDX 3 kb)

Supplementary File 32

Chemical structure: obtusifoliol. (CDX 3 kb)

Supplementary File 33

Chemical structure: stigmasterol. (CDX 3 kb)

Supplementary File 34

Chemical structure (structure 1): 4,4-dimethylcholesta-8,14(15),24-trien-3β-ol. (CDX 3 kb)

Supplementary File 35

Chemical structure (structure 3): cholesta-8,24-dien-3β-ol. (CDX 3 kb)

Supplementary File 36

Chemical structure (structure 4): cholesta-7,24-dien-3β-ol. (CDX 3 kb)

Supplementary File 37

Chemical structure (structure 5): 7-dehydrodesmosterol. (CDX 3 kb)

Supplementary File 38

Chemical structure (structure 6): desmosterol. (CDX 3 kb)

Supplementary File 39

Chemical structure: uttroside B. (CDX 7 kb)

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Sonawane, P., Pollier, J., Panda, S. et al. Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism. Nature Plants 3, 16205 (2017). https://doi.org/10.1038/nplants.2016.205

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