Strictosidine activation in Apocynaceae: towards a "nuclear time bomb"?

Grégory Guirimand,Nathalie Blanc,Arnaud Lanoue,Benoit St-Pierre,Samira Mahroug,Anthony Guihur,Vincent Courdavault,Nathalie Giglioli-Guivarc'H,Vincent Burlat

doi:10.1186/1471-2229-10-182

Grégory Guirimand, Nathalie Blanc + Show 7 more

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https://doi.org/10.1186/1471-2229-10-182

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Abstract

BackgroundThe first two enzymatic steps of monoterpene indole alkaloid (MIA) biosynthetic pathway are catalysed by strictosidine synthase (STR) that condensates tryptamine and secologanin to form strictosidine and by strictosidine β-D-glucosidase (SGD) that subsequently hydrolyses the glucose moiety of strictosidine. The resulting unstable aglycon is rapidly converted into a highly reactive dialdehyde, from which more than 2,000 MIAs are derived. Many studies were conducted to elucidate the biosynthesis and regulation of pharmacologically valuable MIAs such as vinblastine and vincristine in Catharanthus roseus or ajmaline in Rauvolfia serpentina. However, very few reports focused on the MIA physiological functions.ResultsIn this study we showed that a strictosidine pool existed in planta and that the strictosidine deglucosylation product(s) was (were) specifically responsible for in vitro protein cross-linking and precipitation suggesting a potential role for strictosidine activation in plant defence. The spatial feasibility of such an activation process was evaluated in planta. On the one hand, in situ hybridisation studies showed that CrSTR and CrSGD were coexpressed in the epidermal first barrier of C. roseus aerial organs. However, a combination of GFP-imaging, bimolecular fluorescence complementation and electromobility shift-zymogram experiments revealed that STR from both C. roseus and R. serpentina were localised to the vacuole whereas SGD from both species were shown to accumulate as highly stable supramolecular aggregates within the nucleus. Deletion and fusion studies allowed us to identify and to demonstrate the functionality of CrSTR and CrSGD targeting sequences.ConclusionsA spatial model was drawn to explain the role of the subcellular sequestration of STR and SGD to control the MIA metabolic flux under normal physiological conditions. The model also illustrates the possible mechanism of massive activation of the strictosidine vacuolar pool upon enzyme-substrate reunion occurring during potential herbivore feeding constituting a so-called "nuclear time bomb" in reference to the "mustard oil bomb" commonly used to describe the myrosinase-glucosinolate defence system in Brassicaceae.

Highlights

The first two enzymatic steps of monoterpene indole alkaloid (MIA) biosynthetic pathway are catalysed by strictosidine synthase (STR) that condensates tryptamine and secologanin to form strictosidine and by strictosidine b-D-glucosidase (SGD) that subsequently hydrolyses the glucose moiety of strictosidine
The 66 kDa BSA band disappeared when BSA was incubated with strictosidine and a protein extract from CrSGD-overexpressing E. coli cells in a similar way to BSA incubated with glutaraldehyde which is used as a protein cross-linking control (Figure 2b)
By incubating recombinant CrSGD enriched protein extracts with increasing amount of proteinase K (PK), we clearly demonstrated the low susceptibility of multimerised CrSGD to protein hydrolysis attack, since PK treatment only caused a slight decrease of the multimer size without affecting its apparent activity (Figure 10c-d)

Summary

Introduction

The first two enzymatic steps of monoterpene indole alkaloid (MIA) biosynthetic pathway are catalysed by strictosidine synthase (STR) that condensates tryptamine and secologanin to form strictosidine and by strictosidine b-D-glucosidase (SGD) that subsequently hydrolyses the glucose moiety of strictosidine. Some species-specific MIAs such as vinblastine and vincristine in Catharanthus roseus or ajmaline in Rauvolfia serpentina are well known for their highly valuable pharmaceutical properties [4,5,6] (Figure 1). Strictosidine b-D-glucosidase (SGD; EC: 3.2.1.105) hydrolyses the strictosidine glucose moiety producing an unstable aglycon that is rapidly converted into a dialdehyde intermediate and further into cathenamine [1,2,6,10] (Figure 1). The activation of toxic or repulsive metabolites occurs following enzyme-substrate reunion during herbivore feeding [14] Such an activation mechanism has been proposed for strictosidine with the formulated hypothesis that upon cell damage, SGD would rapidly convert strictosidine into an aglycon [1,2,9], which has been shown to have antimicrobial activity [9]. Studies on Ligustrum obtusifolium leaves showed that an unidentified sequestrated b-glucosidase was able to activate a compound chemically related to strictosidine, i.e. the phenolic secoiridoid glucoside oleuropein, leading to the production of an highly reactive dialdehyde that acts as a strong protein cross-linker with a potent chemical defence role [19]

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