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

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.

The seco-iridoid pathway from Catharanthus roseus

“…G8O antisense probes19 and SGD antisense probes46 were used as internal phloem-associated parenchyma and epidermis markers, respectively. Paraffin-embedded serial longitudinal sections of young developing leaves were hybridized with digoxigenin-labelled riboprobes and localized with antidigoxigenin-alkaline phosphatase-conjugated antibodies47.…”

Folivory elicits a strong defense reaction in Catharanthus roseus: metabolomic and transcriptomic analyses reveal distinct local and systemic responses

“…Interestingly, only a moderate increase of strictosidine (1.8 times more accumulated; p  = 0.02, Wilcoxon rank sum test) was observed in newly emerged leaves that might reflect the consumption of this compound to allow the dramatic increase in the biosynthesis of the downstream MIAs including catharanthine, vindoline or anhydrovinblastine. Based on these results, one could speculate that C. roseus set up two distinct responses to herbivory, a local and quickly induced one relying on strictosidine accumulation which may cross link proteins following its deglucosylation catalyzed by SGD after membrane leakage3, and a systemic and long-term mechanism involving a higher accumulation of toxic MIAs in newly developing organs.…”

“…A good example of such a role relies on the proposed phytoanticipin function of strictosidine which upon leaf attack and membrane leakage can be enzymatically deglucosylated to form a highly reactive aglycone. This conversion induces a massive protein reticulation that was suggested to limit aggressor attacks, the so-called “nuclear time-bomb” process3. Other studies have also established the toxicity of several MIAs against pests or herbivores but essentially by feeding experiments using high concentrations of selected MIAs or total leaf extracts456.…”

Identification of five B-type response regulators as members of a multistep phosphorelay system interacting with histidine-containing phosphotransfer partners of Populus osmosensor

“…This led to the production of two distinct fusion proteins for HPt2, HPt7, HPt9, RR13, RR16 and RR19 which each type of fusion including YFP N -HPt2 and YFP C -HPt2 as described for HPt2. Transient transformation of C. roseus cells by particle bombardment and YFP imaging were performed using the CFP nucleus marker according to Guirimand et al [56] with adaptation for BiFC assays [57]. …”