Characterization of the plastidial geraniol synthase from Madagascar periwinkle which initiates the monoterpenoid branch of the alkaloid pathway in internal phloem associated parenchyma
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Characterization of the plastidial geraniol synthase from Madagascar periwinkle which initiates the monoterpenoid branch of the alkaloid pathway in internal phloem associated parenchyma. / Simkin, Andrew J.; Miettinen, Karel; Claudel, Patricia; Burlat, Vincent; Guirimand, Grégory; Courdavault, Vincent; Papon, Nicolas; Meyer, Sophie; Godet, Stéphanie; St-Pierre, Benoit; Giglioli-Guivarc'H, Nathalie; Fischer, Marc J.C.; Memelink, Johan; Clastre, Marc.
In: Phytochemistry, Vol. 85, 2013, p. 36-43.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Characterization of the plastidial geraniol synthase from Madagascar periwinkle which initiates the monoterpenoid branch of the alkaloid pathway in internal phloem associated parenchyma
AU - Simkin, Andrew J.
AU - Miettinen, Karel
AU - Claudel, Patricia
AU - Burlat, Vincent
AU - Guirimand, Grégory
AU - Courdavault, Vincent
AU - Papon, Nicolas
AU - Meyer, Sophie
AU - Godet, Stéphanie
AU - St-Pierre, Benoit
AU - Giglioli-Guivarc'H, Nathalie
AU - Fischer, Marc J.C.
AU - Memelink, Johan
AU - Clastre, Marc
N1 - Funding Information: The research leading to these results has received funding from the European Union Seventh Framework Programme FP7/2007-2013 under grant agreement number 222716 – SMARTCELL. We thank “ Le STUDIUM ” (Agency for Research and Hosting Foreign associated Researchers in the Centre region, France) for the financial support of Andrew J. Simkin.
PY - 2013
Y1 - 2013
N2 - Madagascar periwinkle (Catharanthus roseus [L.] G. Don, Apocynaceae) produces monoterpene indole alkaloids (MIAs), secondary metabolites of high interest due to their therapeutic value. A key step in the biosynthesis is the generation of geraniol from geranyl diphosphate (GPP) in the monoterpenoid branch of the MIA pathway. Here we report on the cloning and functional characterization of C. roseus geraniol synthase (CrGES). The full-length CrGES was over-expressed in Escherichia coli and the purified recombinant protein catalyzed the conversion of GPP into geraniol with a Km value of 58.5 μM for GPP. In vivo CrGES activity was evaluated by heterologous expression in a Saccharomyces cerevisiae strain mutated in the farnesyl diphosphate synthase gene. Analysis of culture extracts by gas chromatography-mass spectrometry confirmed the excretion of geraniol into the growth medium. Transient transformation of C. roseus cells with a Yellow Fluorescent Protein-fusion construct revealed that CrGES is localized in plastid stroma and stromules. In aerial plant organs, RNA in situ hybridization showed specific labeling of CrGES transcripts in the internal phloem associated parenchyma as observed for other characterized genes involved in the early steps of MIA biosynthesis. Finally, when cultures of Catharanthus cells were treated with the alkaloid-inducing hormone methyl jasmonate, an increase in CrGES transcript levels was observed. This observation coupled with the tissue-specific expression and the subcellular compartmentalization support the idea that CrGES initiates the monoterpenoid branch of the MIA biosynthetic pathway.
AB - Madagascar periwinkle (Catharanthus roseus [L.] G. Don, Apocynaceae) produces monoterpene indole alkaloids (MIAs), secondary metabolites of high interest due to their therapeutic value. A key step in the biosynthesis is the generation of geraniol from geranyl diphosphate (GPP) in the monoterpenoid branch of the MIA pathway. Here we report on the cloning and functional characterization of C. roseus geraniol synthase (CrGES). The full-length CrGES was over-expressed in Escherichia coli and the purified recombinant protein catalyzed the conversion of GPP into geraniol with a Km value of 58.5 μM for GPP. In vivo CrGES activity was evaluated by heterologous expression in a Saccharomyces cerevisiae strain mutated in the farnesyl diphosphate synthase gene. Analysis of culture extracts by gas chromatography-mass spectrometry confirmed the excretion of geraniol into the growth medium. Transient transformation of C. roseus cells with a Yellow Fluorescent Protein-fusion construct revealed that CrGES is localized in plastid stroma and stromules. In aerial plant organs, RNA in situ hybridization showed specific labeling of CrGES transcripts in the internal phloem associated parenchyma as observed for other characterized genes involved in the early steps of MIA biosynthesis. Finally, when cultures of Catharanthus cells were treated with the alkaloid-inducing hormone methyl jasmonate, an increase in CrGES transcript levels was observed. This observation coupled with the tissue-specific expression and the subcellular compartmentalization support the idea that CrGES initiates the monoterpenoid branch of the MIA biosynthetic pathway.
KW - Alkaloid
KW - Catharanthus roseus
KW - Geraniol synthase
KW - Internal phloem associated parenchyma
U2 - 10.1016/j.phytochem.2012.09.014
DO - 10.1016/j.phytochem.2012.09.014
M3 - Journal article
C2 - 23102596
AN - SCOPUS:84871290284
VL - 85
SP - 36
EP - 43
JO - Phytochemistry
JF - Phytochemistry
SN - 0031-9422
ER -
ID: 280017896