Strictosidine activation in Apocynaceae: towards a "nuclear time bomb"?
© Guirimand et al; licensee BioMed Central Ltd. 2010
Received: 5 May 2010
Accepted: 19 August 2010
Published: 19 August 2010
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 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.
In 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.
A 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 first committed step in MIA biosynthesis is carried out by strictosidine synthase (STR; EC: 220.127.116.11) which catalyses the condensation of the indolic precursor tryptamine with the glucosylated secoiridoid precursor secologanin to produce strictosidine (Figure 1). Subsequently, strictosidine β-D-glucosidase (SGD; EC: 18.104.22.168) 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). Following an uncomplete C. roseus STR sequence description , the full cDNA encoding these enzymes have been isolated from both C. roseus [1, 12] and R. serpentina [2, 13].
Glycoside hydrolysis by specific sequestrated glycosidases activates many glycosylated secondary metabolites leading to plant defence strategies against herbivores  such as those observed in the so-called "mustard oil bomb" glucosinolate-myrosinase defence systems in Brassicaceae [15–18]. Although the differential compartmentation has not been elucidated in every model, the accumulating glucosylated metabolites must be physically separated (either at the cellular level or at the subcellular level) from the activating β-glucosidases . The activation of toxic or repulsive metabolites occurs following enzyme-substrate reunion during herbivore feeding . 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 . However, no formal demonstration of such a process has been published so far. Interestingly, studies on Ligustrum obtusifolium leaves showed that an unidentified sequestrated β-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 .
In this work, we evaluated the feasibility of such an enzymatic activation mechanism for strictosidine with the production of the dialdehyde intermediate by SGD. Our efforts were focused mainly on the C. roseus model and to a lesser extent on the R. serpentina enzymes. Electrophoretic-mobility shift assays (EMSA) clearly show that the strictosidine deglucosylation product(s) has/have in vitro protein cross-linking and precipitating properties that strictosidine does not have. We therefore carefully studied, using in situ hybridisation and GFP-imaging approaches, the cellular and subcellular localisation of STR and SGD to ascertain the physical separation of both enzymes. Our results reveal a common localisation of both gene products in the C. roseus epidermis, with STR being sequestrated in the vacuole whereas SGD intriguingly accumulated as highly stable supramolecular aggregates within the nucleus. The results are discussed both in terms of physiological and ecophysiological perspectives.
Results and discussion
The strictosidine deglucosylation product(s) promote(s) in vitroprotein cross-linking and precipitation
CrSTR and CrSGDare co-expressed in the epidermis of aerial organs
CrSTR is targeted to the vacuole through the secretory pathway allowing vacuolar strictosidine accumulation
CrSGD is targeted to the nucleus using a bipartite NLS sequence and adopts a multimerised organisation relying on an accessible C-terminus sequence
The conserved differential localisation of STR and SGD from R. serpentina and C. roseussuggests a common strictosidine activation mechanism in Apocynaceae
The CrSGD multimerisation is a specific and autonomous mechanism that increases the CrSGD proteolysis resistance
One point that remained to be explored was the significance of the SGD multimerisation. It has previously been reported that the aggregation pattern of some plant-defence-related β-glucosidases helps to stabilise their activity [14, 40, 41]. We first studied whether the SGD nuclear aggregation was unique among MIA-related β-glucosidases by analysing the targeting pattern of raucaffricine b-D-glucosidase (RsRGD), another R. serpentina MIA-related β-glucosidase (Figure 1). Interestingly, RsRGD was also targeted to the nucleus (Figure 9m-t) in agreement with the predicted C-terminal bipartite NLS (521-VKRSIREDDEEQVSSKR L RK-540) attributing additional intriguing importance of this organelle during the MIA biosynthetic pathway. However, both orientations of the fusion (RsRGD-GFP and GFP-RsRGD) displayed a diffuse nuclear fluorescence pattern suggesting that SGD aggregation may be unique among MIA-related β-glucosidases. Therefore, it would be of interest to study the subcellular localisation and potential aggregation of two new β-glucosidases closely related to RGD (and to lesser extend to SGD) that have been implicated in terpenoid-isoquinoline alkaloid biosynthesis in Psychotria ipecacuanha  and for which a predicted C-terminal NLS has been identified (data not shown).
Physiological and ecophysiological implications
In C. roseus young leaves, a strictosidine pool constitutively exists in the mM range and is 10-fold increased following hormonal treatment mimicking herbivore and/or necrotrophic microorganism attack whereas slight decrease is observed for vindoline and catharanthine, positioned downstream along the MIA pathway (Figures 1 and 5). This is in agreement with a potential important triggering role for the strictosidine vacuolar pool during herbivore and/or pathogen attack. The second part of this model constitutes a so-called "nuclear time bomb" (Figure 11b). We propose that the massive activation of the strictosidine vacuolar pool by the nuclear SGD complex could occur following cellular disruption, for instance during herbivore feeding or necrotrophic pathogen attack, and that the induced protein cross-linking and precipitation could be a mean for the plant to deter some herbivores from their feeding habit in a similar manner to the Ligustrum/oleuropein system. The exact role of protein cross-linking and precipitation in the defence mechanism is here not fully understood partially due to the lack of knowledge on Apocynaceae aggressors. However, induced protein cross-linking and precipitation could be either deleterious for the herbivore/necrotrophic pathogen enzymatic activities and/or could lead to decrease of nutritive value of the food as previously reported for the Ligustrum/oleuropein system . In the latter case, the larva of Brahmaea wallichii, a Ligustrum specialist herbivore has been shown to avoid the plant defence strategy by an adaptive evolution. In this instance, very high concentration of free glycine are found in the larval digestive juice which quench the protein-cross linking effect of the activated oleuropein aglycon . To our knowledge, no such specialist herbivore has been described in C. roseus and R. serpentina . Such an activation of strictosidine is probably only a part of the plant defence strategies developed in these species given their metabolomic high complexity including numerous directly toxic compounds [7–9, 55].
CrSTR (GeneBank accession CAA43936), CrSGD (AAF28800), RsSTR (CAA44208), RsSGD (CAC83098) and RsRGD (AAF03675) cDNAs were amplified using Pfu high fidelity DNA polymerase (Promega) from reverse-transcribed total RNA and were fully sequenced. All primers and plasmids used for cloning are listed in Additional file 5.
Recombinant CrSGD production
The CrSGD full-length open reading frame was amplified with the pQE-SGD-Bam and pQE-SGD-Hind oligonucleotides and further cloned in pQE-30 (Qiagen) (Additional file 6). The recombinant protein was expressed in E. coli JM109 strain during 4 h at 30°C following addition of 1 mM isopropyl-β-D-galactoside during the exponential growth step of the bacterial cell culture. The SGD-expressing and the control (empty vector) E. coli cells were collected by centrifugation (15 min, 5,000×g) and resuspended in activity buffer [100 mM sodium phosphate buffer pH 6.55, PMSF (1 mM), EDTA-free protease inhibitor cocktail at working concentration (Roche, Meylan, France)]. The recombinant CrSGD-enriched soluble and the control protein fractions were recovered following a sonication-mediated E. coli disruption and centrifugation (15 min, 25,000×g).
In vitroassays of strictosidine activation and BSA cross-linking
All the assays (60 μl) were conducted under continuous shaking during 2 h at 30°C. The reaction mixture consisted in activity buffer and 0.05% (w/v) BSA (Fraction V, Sigma A7906) to which was either added 10 mM strictosidine (Phytoconsult, Leiden, The Netherlands); 10 mM strictosidine and 18.5 μg of protein extract from E. coli control cells; 10 mM strictosidine and 18.5 μg of protein extract from SGD-expressing E. coli cells; 10 mM tryptamine (Sigma); 10 mM secologanin (Phytoconsult, Leiden, The Netherlands); 10 mM glutaraldehyde (Euromedex, Mundolsheim, France). The tubes were photographed and centrifuged during 1 min at 10,000×g. Ten μl of the supernatants were analysed by SDS-PAGE (3% stacking gel, 10% resolving gel) and Coomassie blue staining to monitor BSA cross-linking and precipitation. In the three strictosidine-containing conditions, 1 μl of the supernatant was mixed in 388 μl of methanol for subsequent HPLC monitoring of strictosidine using a previously described system  with the following modifications. The solvent system was aqueous phosphoric acid (0.1% w/v; eluent A) and acetonitrile (eluent B), running as a gradient from 10% B to 27% B within 15 min, to 40% B within 45 min: the flow-rate was 0.5 ml/min and detection at 220 nm. Strictosidine was identified and quantified according to its UV spectrum and retention time by comparison with authentic standard (Phytoconsult, NL).
Monitoring of strictosidine, vindoline and catharanthine contents in young leaves following hormonal treatments
Periwinkle seeds (C. roseus cv. 'Pacifica pink') were surface sterilized by incubation in ethanol (70% v/v) for 2 min, in NaOCl (2.5%) for 20 min at 200 rpm and then washed three times in sterile bi-distilled water. Seeds were subsequently incubated for 24 h in sterile water under darkness at 25 ± 2°C, transferred on static Murashige & Skoog (MS) culture medium for 3 d under darkness at 25 ± 2°C and then exposed to cool white fluorescent light (45 μmol m-2 s-1) with a 16 h/8 h photoperiod at 25 ± 2°C. Six-week-old seedlings were transferred singly to sterile magenta box with 50 ml of solid MS culture medium diluted 1:10. After 13 weeks of culture, the plants were transferred to six well plates containing in each well 5 ml of liquid MS culture medium diluted 1:10. The hydroponic plants were treated with methyljasmonate (200 μM) and ethephon (100 μM) at the root level. Each treatment was tested in two independent experiments, each consisting of three replicates. Lyophilized leaves were ground with pillar and mortar and 10 mg were extracted in 400 μl methanol. Plant material and solvent were shaken at 1200 rpm for 1 h and centrifuged at 18,000×g for 5 min. The supernatant was used for subsequent HPLC analyses as described above. The three analysed MIA were identified and quantified according to their UV spectra and retention times by comparison with authentic standards (Strictosidine, Phytoconsult, NL; vindoline and catharanthine, Gedeon Richter ltd., Budapest, HU).
A pBluescript II SK+ vector containing the 1.9 Kb CrSGD full length cDNA (pBS-CrSGD)  was used for the in vitro transcription of digoxigenin-labelled CrSGD riboprobes. Transcription of the antisense CrSGD riboprobe was realised with the T7 RNA polymerase (Promega) and a BamHI linearised plasmid as a template using digoxigenin-UTP according to the manufacturer's instruction (Roche, Meylan, France) whereas the sense CrSGD riboprobe was similarly transcribed using the T3 RNA polymerase (Promega) and a XhoI-linearised pBS-CrSGD. CrSTR and CrD4H riboprobes were described previously . The hydrolysis of riboprobes, the whole in situ hybridisation protocol and the microscope analysis were realised as previously described [21, 25, 27].
GFP and YFP constructs for CrSTR and CrSGD localisation studies
Plasmids expressing GFP and/or YFP fusion proteins of CrSTR and CrSGD including their orientation, truncated or mutated variants were constructed using the plasmids and the procedures previously described . Details on primers and on cloning procedures are listed in Additional file 5.
YFP constructs for BiFC studies of CrSGD interactions
For BiFC assays, the CrSGD coding sequence was amplified with primers SGD-GFP-C-S and SGD-GFP-C-AS (Additional file 5) and cloned via SpeI in frame with the 5' or 3' ends of the coding sequence of the N-terminal (YFPN, amino acids 1-173) and C-terminal (YFPC, amino acids 156-239) fragments of YFP. The pSPYNE(R)173 and pSPYCE(MR) plasmids  were used to generate constructs for YFPN-SGD and YFPC-SGD expression, respectively. The split-YFP coding sequence and Nos terminator of the pSPYNE173 and pSPYCE(M) plasmids  were removed by a SpeI/EcoRI digestion and cloned into the pSCA-cassette YFPi plasmid  pre-digested by SpeI/EcoRI to remove the full length coding sequence of YFP and the terminator. The resulting pSCA-SPYNE173 and pSCA-SPYCE(M) plasmids were used for the expression of SGD-YFPN and SGD-YFPC fusion proteins, respectively.
GFP constructs for RsSTR, RsSGD and RsRGD localisation studies
For studying the subcellular localisation of RsSTR, RsSGD and RsRGD, the coding sequence of the three enzymes were cloned in the pSCA-cassette-GFPi plasmid to express a GFP-fused protein (Additional file 7).
A set of organelle markers was used for co-transformation studies with the STR and SGD constructs. "ER"-mcherry (CD3-960), "ER"-YFP (CD3-958) and "plastid"-CFP (CD3-994) markers  were obtained from the ABRC http://www.arabidopsis.org. The YFP-GUS cytoplasmic marker was previously described . The CFP-GUS cytoplasmic marker and the CFP nucleocytoplasmic marker were generated following amplification of the CFP coding sequence using primers CFP-for and CFP-rev (Additional file 8). The YFP coding sequence of the pSCA-cassette YFPi and pSCA-cassette YFP-GUS plasmids  was then substituted by the CFP coding sequence via a BglII/NheI cloning step to create the cytoplasmic and nucleocytoplasmic markers, respectively. Using the same procedure, a pSCA-cassette mcherry-GUS was also created after amplification of the mcherry coding sequence with primers mcherry-for and mcherry-rev (Additional file 8). To construct the "nucleus"-mcherry-GUS and "nucleus"-CFP-GUS markers, the sequence of the nucleoplasmin bipartite NLS  has been added at the N-terminal end of both mcherry-GUS and CFP-GUS fusion proteins following annealing of the NLS-nucleo-for and NLS-nucleo-rev primers (Additional file 8) performed as previously described , and cloning of the resulting product into BglII/SpeI-linearised pSCA-cassette-mcherry-GUS and pSCA-cassette-CFP-GUS plasmids. Cell wall (cellulose) staining was performed with calcofluor (0.1 mg/ml). For BiFC assays, the bZIP-YFPN and bZIP-YFPC expressing plasmids  were obtained from Jörg Kudla http://www.uni-muenster.de/Biologie.Botanik/agkudla/Plasmids.html.
Biolistic-mediated transient transformation of C. roseus cells, C. roseusleaves and onion epidermis
GFP fusion constructs were either co-transformed with mcherry or YFP organelle markers and cells transformed with single GFP fusion constructs were further stained with Calcofluor. YFP fusion constructs were co-transformed with CFP organelle markers. BiFC constructs were additionally co-transformed with the "plastid"-CFP marker used as a transformation control. The whole detailed protocol of C. roseus cell cultures (co)transformation has been previously optimised and extensively detailed . A similar procedure has been applied for transient transformation of C. roseus young leaves (5-10 mm) that were platted onto solid Gamborg B5 medium one hour prior to particle bombardment with abaxial epidermis face-up. For the transient transformation of onion cells, internal epidermis of fresh onion were peeled and placed on solid vitamin-free MS medium and bombarded following the protocol of C. roseus cell transformation.
Brefeldin A treatments
For studying the vacuolar route of CrSTR, the ER-to-cis golgi anterograde endomembrane transport has been inhibited by transferring C. roseus platted cells onto a solid Gamborg B5 medium containing 40 μM brefeldin A (Invitrogen) one hour before bombardment. The observations were performed 24 h post-transformation.
An Olympus BX51 epifluorescence microscope equipped with the Olympus DP50 digital camera and the Cell* imaging software (Soft Imaging System, Olympus) was used for image capture and for merging false-coloured images of transiently (co)transformed XFP expressing cells. Details on the combinations of filter sets used for each application are given in Additional file 9. The morphology of the transformed cells was observed with differential interference contrast (DIC).
Anti-GFP western blotting
The fusion protein content of CrSGD-GFP and GFP-CrSGD stably transformed C. roseus calli, obtained following the previously described protocol , was analyzed by immunoblot detection with anti-GFP polyclonal antibodies (Molecular Probes) used at 1/1000 dilution.
Native PAGE and SDS-PAGE-Coomassie blue staining and native PAGE-β-glucosidase zymograms to study CrSGD aggregation and stability
To study the recombinant CrSGD in vitro aggregation, 40 μg of protein extract from E. coli control cells and SGD-expressing E. coli cells were mixed with SDS-loading buffer (50 mM Tris-HCl, pH 6.8, 0.5% (v/v) β-mercaptoethanol, 5% (w/v) SDS, 10% (v/v) glycerol, 0.01% (w/v) bromophenol blue) and boiled when specified before analysis on 8% SDS-PAGE with subsequent Coomassie blue staining. The β-glucosidase activity of these protein extracts was analysed by subjecting the same amount of protein to 8% native PAGE after addition of native-loading buffer (50 mM Tris-HCl, pH 6.8, 10% (v/v) glycerol, 0.01% (w/v) bromophenol blue). The β-glucosidase zymograms were conducted by equilibrating the gels in 50 mM citrate/100 mM phosphate buffer (pH 5.8) during 10 min with further incubation for 10 min in a developing solution containing 1 mM 4-methylumbelliferyl-β-D-glucoside (MUGlc) as previously described . β-glucosidase activity was visualised under UV irradiation (365 nm). The resistance of CrSGD aggregates and the stability of the β-glucosidase activity towards proteolysis were assayed by incubating 40 μg of protein extract from CrSGD-expressing E. coli cells (without EDTA-free protease inhibitor cocktail) with increasing concentrations of proteinase K (Invitrogen) up to 1 μg/μl in a 50 μl reaction mixture. After a one hour-incubation at 37°C, the mixture was subjected to 8% native PAGE either followed by Coomassie blue staining and β-glucosidase zymogram.
Vincent Courdavault is the author to whom material demand should be addressed.
This research was financially supported by the Ministère de l'Enseignement Supérieur et de la Recherche and by a grant from the University of Tours (Appel à projets Orléans-Tours 2009) and the Région Centre (AMC2B). We acknowledge Prof. Dr. J. Kudla (University of Münster, Germany) for providing us the BiFC constructs. We also thank Dr. J Memelink (University of Leiden, The Netherlands) for providing us the pBS-CrSGD clone and Dr. AJ Simkin (EA2106, University of Tours) for careful revision of the manuscript.
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