Functional characterisation of genes involved in pyridine alkaloid biosynthesis in tobacco

Suvi T. Häkkinen, S. Tilleman, S. Swiatek, V. De Sutter, Heiko Rischer, I. Vanhoutte, H. Van Onckelen, P. Hilson, D. Inzé, Kirsi-Marja Oksman-Caldentey (Corresponding Author), A. Goossens

Research output: Contribution to journalArticleScientificpeer-review

38 Citations (Scopus)

Abstract

Although secondary metabolism in Nicotiana tabacum (L.) (tobacco) is rather well studied, many molecular aspects of the biosynthetic pathways and their regulation remain to be disclosed, even for prominent compounds such as nicotine and other pyridine alkaloids. To identify players in tobacco pyridine alkaloid biosynthesis a functional screen was performed, starting from a tobacco gene collection established previously by means of combined transcript profiling and metabolite analysis. First, full-length cDNA clones were isolated for 34 genes, corresponding to tobacco transcript tag sequences putatively associated with pyridine alkaloid metabolism. Full-length open reading frames were transferred to pCaMV35S-steered overexpression vectors. The effects of plant transformation with these expression cassettes on the accumulation of nicotine and other pyridine alkaloids were assessed in transgenic tobacco Bright-Yellow 2 (BY-2) cell suspensions and hairy root cultures. This screen identified potential catalysers of tobacco pyridine metabolism, amongst which a lysine decarboxylase-like gene and a GH3-like enzyme. Overexpression of the GH3-like enzyme, presumably involved in auxin homeostasis and designated NtNEG1 (Nicotiana tabacum Nicotine-Enhancing GH3 enzyme 1), increased nicotine levels in BY-2 hairy roots significantly. This study shows how functional genomics-based identification of genes potentially involved in biosynthetic pathways followed by systematic functional assays in plant cells can be used at large-scale to decipher plant metabolic networks at the molecular level.
Original languageEnglish
Pages (from-to)2773-2785
JournalPhytochemistry
Volume68
Issue number22-24
DOIs
Publication statusPublished - 2007
MoE publication typeNot Eligible
Event50 Years of the Phytochemical Society of Europe. Highlights in the Evolution of Phytochemistry - Cambridge, United Kingdom
Duration: 11 Apr 200714 Apr 2007

Fingerprint

pyridine alkaloids
Tobacco
Biosynthesis
Alkaloids
tobacco
Genes
nicotine
biosynthesis
Nicotine
genes
lysine decarboxylase
Metabolism
Nicotiana tabacum
metabolism
biochemical pathways
Biosynthetic Pathways
Enzymes
enzymes
Indoleacetic Acids
pyridines

Keywords

  • nicotiana tabacum
  • solanaceae
  • tobacco
  • functional genomics
  • pyridine alkaloids
  • nicotine
  • lysine decarboxylase
  • GH3-like enzyme
  • jasmonate
  • auxin conjugates
  • cDNA-AFLP
  • metabolites
  • metabolomics

Cite this

Häkkinen, Suvi T. ; Tilleman, S. ; Swiatek, S. ; De Sutter, V. ; Rischer, Heiko ; Vanhoutte, I. ; Van Onckelen, H. ; Hilson, P. ; Inzé, D. ; Oksman-Caldentey, Kirsi-Marja ; Goossens, A. / Functional characterisation of genes involved in pyridine alkaloid biosynthesis in tobacco. In: Phytochemistry. 2007 ; Vol. 68, No. 22-24. pp. 2773-2785.
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abstract = "Although secondary metabolism in Nicotiana tabacum (L.) (tobacco) is rather well studied, many molecular aspects of the biosynthetic pathways and their regulation remain to be disclosed, even for prominent compounds such as nicotine and other pyridine alkaloids. To identify players in tobacco pyridine alkaloid biosynthesis a functional screen was performed, starting from a tobacco gene collection established previously by means of combined transcript profiling and metabolite analysis. First, full-length cDNA clones were isolated for 34 genes, corresponding to tobacco transcript tag sequences putatively associated with pyridine alkaloid metabolism. Full-length open reading frames were transferred to pCaMV35S-steered overexpression vectors. The effects of plant transformation with these expression cassettes on the accumulation of nicotine and other pyridine alkaloids were assessed in transgenic tobacco Bright-Yellow 2 (BY-2) cell suspensions and hairy root cultures. This screen identified potential catalysers of tobacco pyridine metabolism, amongst which a lysine decarboxylase-like gene and a GH3-like enzyme. Overexpression of the GH3-like enzyme, presumably involved in auxin homeostasis and designated NtNEG1 (Nicotiana tabacum Nicotine-Enhancing GH3 enzyme 1), increased nicotine levels in BY-2 hairy roots significantly. This study shows how functional genomics-based identification of genes potentially involved in biosynthetic pathways followed by systematic functional assays in plant cells can be used at large-scale to decipher plant metabolic networks at the molecular level.",
keywords = "nicotiana tabacum, solanaceae, tobacco, functional genomics, pyridine alkaloids, nicotine, lysine decarboxylase, GH3-like enzyme, jasmonate, auxin conjugates, cDNA-AFLP, metabolites, metabolomics",
author = "H{\"a}kkinen, {Suvi T.} and S. Tilleman and S. Swiatek and {De Sutter}, V. and Heiko Rischer and I. Vanhoutte and {Van Onckelen}, H. and P. Hilson and D. Inz{\'e} and Kirsi-Marja Oksman-Caldentey and A. Goossens",
year = "2007",
doi = "10.1016/j.phytochem.2007.09.010",
language = "English",
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Häkkinen, ST, Tilleman, S, Swiatek, S, De Sutter, V, Rischer, H, Vanhoutte, I, Van Onckelen, H, Hilson, P, Inzé, D, Oksman-Caldentey, K-M & Goossens, A 2007, 'Functional characterisation of genes involved in pyridine alkaloid biosynthesis in tobacco', Phytochemistry, vol. 68, no. 22-24, pp. 2773-2785. https://doi.org/10.1016/j.phytochem.2007.09.010

Functional characterisation of genes involved in pyridine alkaloid biosynthesis in tobacco. / Häkkinen, Suvi T.; Tilleman, S.; Swiatek, S.; De Sutter, V.; Rischer, Heiko; Vanhoutte, I.; Van Onckelen, H.; Hilson, P.; Inzé, D.; Oksman-Caldentey, Kirsi-Marja (Corresponding Author); Goossens, A.

In: Phytochemistry, Vol. 68, No. 22-24, 2007, p. 2773-2785.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Functional characterisation of genes involved in pyridine alkaloid biosynthesis in tobacco

AU - Häkkinen, Suvi T.

AU - Tilleman, S.

AU - Swiatek, S.

AU - De Sutter, V.

AU - Rischer, Heiko

AU - Vanhoutte, I.

AU - Van Onckelen, H.

AU - Hilson, P.

AU - Inzé, D.

AU - Oksman-Caldentey, Kirsi-Marja

AU - Goossens, A.

PY - 2007

Y1 - 2007

N2 - Although secondary metabolism in Nicotiana tabacum (L.) (tobacco) is rather well studied, many molecular aspects of the biosynthetic pathways and their regulation remain to be disclosed, even for prominent compounds such as nicotine and other pyridine alkaloids. To identify players in tobacco pyridine alkaloid biosynthesis a functional screen was performed, starting from a tobacco gene collection established previously by means of combined transcript profiling and metabolite analysis. First, full-length cDNA clones were isolated for 34 genes, corresponding to tobacco transcript tag sequences putatively associated with pyridine alkaloid metabolism. Full-length open reading frames were transferred to pCaMV35S-steered overexpression vectors. The effects of plant transformation with these expression cassettes on the accumulation of nicotine and other pyridine alkaloids were assessed in transgenic tobacco Bright-Yellow 2 (BY-2) cell suspensions and hairy root cultures. This screen identified potential catalysers of tobacco pyridine metabolism, amongst which a lysine decarboxylase-like gene and a GH3-like enzyme. Overexpression of the GH3-like enzyme, presumably involved in auxin homeostasis and designated NtNEG1 (Nicotiana tabacum Nicotine-Enhancing GH3 enzyme 1), increased nicotine levels in BY-2 hairy roots significantly. This study shows how functional genomics-based identification of genes potentially involved in biosynthetic pathways followed by systematic functional assays in plant cells can be used at large-scale to decipher plant metabolic networks at the molecular level.

AB - Although secondary metabolism in Nicotiana tabacum (L.) (tobacco) is rather well studied, many molecular aspects of the biosynthetic pathways and their regulation remain to be disclosed, even for prominent compounds such as nicotine and other pyridine alkaloids. To identify players in tobacco pyridine alkaloid biosynthesis a functional screen was performed, starting from a tobacco gene collection established previously by means of combined transcript profiling and metabolite analysis. First, full-length cDNA clones were isolated for 34 genes, corresponding to tobacco transcript tag sequences putatively associated with pyridine alkaloid metabolism. Full-length open reading frames were transferred to pCaMV35S-steered overexpression vectors. The effects of plant transformation with these expression cassettes on the accumulation of nicotine and other pyridine alkaloids were assessed in transgenic tobacco Bright-Yellow 2 (BY-2) cell suspensions and hairy root cultures. This screen identified potential catalysers of tobacco pyridine metabolism, amongst which a lysine decarboxylase-like gene and a GH3-like enzyme. Overexpression of the GH3-like enzyme, presumably involved in auxin homeostasis and designated NtNEG1 (Nicotiana tabacum Nicotine-Enhancing GH3 enzyme 1), increased nicotine levels in BY-2 hairy roots significantly. This study shows how functional genomics-based identification of genes potentially involved in biosynthetic pathways followed by systematic functional assays in plant cells can be used at large-scale to decipher plant metabolic networks at the molecular level.

KW - nicotiana tabacum

KW - solanaceae

KW - tobacco

KW - functional genomics

KW - pyridine alkaloids

KW - nicotine

KW - lysine decarboxylase

KW - GH3-like enzyme

KW - jasmonate

KW - auxin conjugates

KW - cDNA-AFLP

KW - metabolites

KW - metabolomics

U2 - 10.1016/j.phytochem.2007.09.010

DO - 10.1016/j.phytochem.2007.09.010

M3 - Article

VL - 68

SP - 2773

EP - 2785

JO - Phytochemistry

JF - Phytochemistry

SN - 0031-9422

IS - 22-24

ER -