Abstract
Cyanogenic glucosides (α-hydroxynitrile glucosides) are a well known class of chemical defence compounds that protect the plant against herbivores. If plant tissue is disrupted, cyanogenic glucosides come into contact with specific β-glucosidases and are hydrolysed, which results in the release of hydrogen cyanide gas. Non-cyanogenic hydroxynitrile glucosides, named rhodiocyanosides, have recently evolved within the Lotus genus, making it a model to study the diversification and specialisation of enzyme activities.
Using a combination of both forward- and reverse-genetics in the legume model Lotus japonicus, mutants were obtained in the β-glucosidases responsible for the bio-activation of hydroxynitrile glucoside defence compounds. This shows distinct physiological roles for two closely related β-glucosidases named BGD2 and BGD4. BGD2 is able to degrade all hydroxynitrile glucosides, while BGD4 has a specialised role in the degradation of the non-cyanogenic rhodiocyanosides. This is explained at the molecular level: protein modelling and site-directed mutagenesis combined with activity assays, shows that a single amino-acid difference is the main determinant of the difference in specificity.
The data suggest the evolutionary scenario that the ability to hydrolyse rhodiocyanosides was a latent ability of a progenitor cyanogenic β-glucosidase and that substrate specialisation towards rhodiocyanosides required no more than a single amino acid substitution. Unexpectedly, an additional cyanogenic β-glucosidase was identified, which is specifically expressed in the inner parts of the flower, suggesting a role in the interaction with floral visitors. The work contributes to the general understanding of the evolution of plant metabolic pathways and the molecular mechanisms by which new enzyme specificity is obtained.
Using a combination of both forward- and reverse-genetics in the legume model Lotus japonicus, mutants were obtained in the β-glucosidases responsible for the bio-activation of hydroxynitrile glucoside defence compounds. This shows distinct physiological roles for two closely related β-glucosidases named BGD2 and BGD4. BGD2 is able to degrade all hydroxynitrile glucosides, while BGD4 has a specialised role in the degradation of the non-cyanogenic rhodiocyanosides. This is explained at the molecular level: protein modelling and site-directed mutagenesis combined with activity assays, shows that a single amino-acid difference is the main determinant of the difference in specificity.
The data suggest the evolutionary scenario that the ability to hydrolyse rhodiocyanosides was a latent ability of a progenitor cyanogenic β-glucosidase and that substrate specialisation towards rhodiocyanosides required no more than a single amino acid substitution. Unexpectedly, an additional cyanogenic β-glucosidase was identified, which is specifically expressed in the inner parts of the flower, suggesting a role in the interaction with floral visitors. The work contributes to the general understanding of the evolution of plant metabolic pathways and the molecular mechanisms by which new enzyme specificity is obtained.
| Originalsprog | Engelsk |
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| Forlag | Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen |
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| Antal sider | 135 |
| Status | Udgivet - 2012 |