The subcellular organization of strictosidine biosynthesis in Catharanthus roseus epidermis highlights several trans-tonoplast translocations of intermediate metabolites.

Catharanthus roseus synthesizes a wide range of valuable monoterpene indole alkaloids, some of which have recently been recognized as functioning in plant defence mechanisms. More specifically, in aerial organ epidermal cells, vacuole-accumulated strictosidine displays a dual fate, being either the precursor of all monoterpene indole alkaloids after export from the vacuole, or the substrate for a defence mechanism based on the massive protein cross-linking, which occurs subsequent to organelle membrane disruption during biotic attacks. Such a mechanism relies on a physical separation between the vacuolar strictosidine-synthesizing enzyme and the nucleus-targeted enzyme catalyzing its activation through deglucosylation. In the present study, we carried out the spatial characterization of this mechanism by a cellular and subcellular study of three enzymes catalyzing the synthesis of the two strictosidine precursors (i.e. tryptamine and secologanin). Using RNA in situ hybridization, we demonstrated that loganic acid O-methyltransferase transcript, catalysing the penultimate step of secologanin synthesis, is specifically localized in the epidermis. A combination of green fluorescent protein imaging, bimolecular fluorescence complementation assays and yeast two-hybrid analysis enabled us to establish that both loganic acid O-methyltransferase and the tryptamine-producing enzyme, tryptophan decarboxylase, form homodimers in the cytosol, thereby preventing their passive diffusion to the nucleus. We also showed that the cytochrome P450 secologanin synthase is anchored to the endoplasmic reticulum via a N-terminal helix, thus allowing the production of secologanin on the cytosolic side of the endoplasmic reticulum membrane. Consequently, secologanin and tryptamine must be transported to the vacuole to achieve strictosidine biosynthesis, demonstrating the importance of trans-tonoplast translocation events during these metabolic processes.

Spatial organization of the vindoline biosynthetic pathway in Catharanthus roseus.

Vindoline constitutes the main terpenoid indole alkaloid accumulated in leaves of Catharanthus roseus, and four genes involved in its biosynthesis have been identified. However, the spatial organization of the tabersonine-to-vindoline biosynthetic pathway is still incomplete. To pursue the characterization of this six-step conversion, we illustrated, with in situ hybridization, that the transcripts of the second biosynthetic enzyme, 16-hydroxytabersonine 16-O-methyltransferase (16OMT), are specifically localized to the aerial organ epidermis. At the subcellular level, by combining GFP imaging, bimolecular fluorescence complementation assays and yeast two-hybrid analysis, we established that the first biosynthetic enzyme, tabersonine 16-hydroxylase (T16H), is anchored to the ER as a monomer via a putative N-terminal helix that we cloned using a PCR approach. We also showed that 16OMT homodimerizes in the cytoplasm, allowing its exclusion from the nucleus and thus facilitating the uptake of T16H conversion product, although no T16H/16OMT interactions occur. Moreover, the two last biosynthetic enzymes, desacetoxyvindoline-4-hydroxylase (D4H) and deacetylvindoline-4-O-acetyltransferase (DAT), were shown to operate as monomers that reside in the nucleocytoplasmic compartment following passive diffusion to the nucleus allowed by the protein size. No D4H/DAT interactions were detected, suggesting the absence of metabolic channeling in the vindoline biosynthetic pathway. Finally, these results highlight the importance of the inter- and intracellular translocations of intermediates during the vindoline biosynthesis and their potential regulatory role.

Two distinct intracellular Ca2+-release components act in opposite ways in the regulation of the auxin-dependent MIA biosynthesis in Catharanthus roseus cells.

Calcium-mediated signalling is ubiquitous in both animals and plants. Changes in cytoplasmic free Ca(2+) concentration couple diverse arrays of stimuli to their specific responses, the specificity of the stimulus being determined by integrated actions between multiple Ca(2+) mobilization pathways. In this work, a pharmacological approach is reported, aimed at deciphering the role of calcium as a second messenger in the transduction pathway leading to the inhibitory effect of 2,4-dichlorophenoxyacetic acid (2,4-D), in regulating monoterpene indole alkaloid (MIA) biosynthesis in Catharanthus roseus cells. It is demonstrated here that auxin-dependent MIA biosynthesis is differentially regulated by two distinct calcium release components from internal stores in C. roseus showing pharmacological profiles similar to those displayed by animal RyR and IP3 channels. MIA biosynthesis is stimulated by caffeine (Ca(2+)-release activator through RyR channels) and by heparin and TMB8 (Ca(2+)-release inhibitors of IP3 channels) whereas MIA biosynthesis is inhibited by mastoparan (Ca(2+)-release activator of IP3 channels) and by ruthenium red and DHBP (Ca(2+)-release inhibitors of RyR channels). Furthermore, calcium, as 2,4-D, acts on MIA biosynthesis by regulating the monoterpene moiety of the MIA biosynthesis pathway since calcium channel modulators preferentially modulate g10h expression, the gene encoding the enzyme of the secoiridoid monoterpene pathway, that is the major target of 2,4-D action. In addition, the simultaneous use of caffeine (an activator of RyR channel in animals) and TMB8 (an inhibitor of the IP3 channel) in 2,4-D treated cells triggers a synergistic effect on MIA accumulation. This finding suggests an opposite and co-ordinated action of multiple Ca(2+)-release pathways in 2,4-D signal transduction, adding a new level of complexity to calcium signalling in plants and questioning the existence of RyR and IP3 channels in plants.

Competition between elastase and related proteases from human neutrophil for binding to alpha1-protease inhibitor.

The protease-antiprotease imbalance that is characteristic of most inflammatory lung disorders depends on the spatial-temporal regulation of active inhibitor and protease concentrations in lung secretions. We have studied the competition between the three main serine proteases from human neutrophil primary granules in their binding to alpha1-Pi, the main serine proteases inhibitor in lung secretions. Elastase was the only target of alpha1-Pi when identical molar amounts of purified inhibitor and the three proteases were tested together. The other two proteases were only inhibited once elastase was saturated. Elastase remained the preferred target of inhibitors when bronchoalveolar lavage fluids from patients with lung pneumonia and acute respiratory distress syndrome were used as the source of inhibitors, in spite of the presence of additional inhibitors in lung secretions. Since neutrophil proteases are expressed at the neutrophil surface, we also measured residual activities of membrane-bound proteases after purified neutrophils were incubated with bronchoalveolar fluids. Again, elastase was the preferred target of the inhibitors. We conclude that protease 3 and cathepsin G are not controlled as efficiently as elastase in lung secretions, a feature that must be taken into account when developing inhibitor-based anti-inflammatory therapies.