Alternative splicing creates a pseudo-strictosidine ß-d-glucosidase modulating alkaloid synthesis in Catharanthus roseus.

Deglycosylation is a key step in the activation of specialized metabolites involved in plant defense mechanisms. This reaction is notably catalyzed by ß-glucosidases of the glycosyl hydrolase 1 (GH1) family such as strictosidine ß-d-glucosidase (SGD) from Catharanthus roseus. SGD catalyzes the deglycosylation of strictosidine, forming a highly reactive aglycone involved in the synthesis of cytotoxic monoterpene indole alkaloids (MIAs) and in the crosslinking of aggressor proteins. By exploring C. roseus transcriptomic resources, we identified an alternative splicing event of the SGD gene leading to the formation of a shorter isoform of this enzyme (shSGD) that lacks the last 71-residues and whose transcript ratio with SGD ranges from 1.7% up to 42.8%, depending on organs and conditions. Whereas it completely lacks ß-glucosidase activity, shSGD interacts with SGD and causes the disruption of SGD multimers. Such disorganization drastically inhibits SGD activity and impacts downstream MIA synthesis. In addition, shSGD disrupts the metabolic channeling of downstream biosynthetic steps by hampering the recruitment of tetrahydroalstonine synthase in cell nuclei. shSGD thus corresponds to a pseudo-enzyme acting as a regulator of MIA biosynthesis. These data shed light on a peculiar control mechanism of ß-glucosidase multimerization, an organization common to many defensive GH1 members.

Identifying Genes Involved in alkaloid Biosynthesis in Vinca minor Through Transcriptomics and Gene Co-Expression Analysis.

The lesser periwinkle Vinca minor accumulates numerous monoterpene indole alkaloids (MIAs) including the vasodilator vincamine. While the biosynthetic pathway of MIAs has been largely elucidated in other Apocynaceae such as Catharanthus roseus, the counterpart in V. minor remains mostly unknown, especially for reactions leading to MIAs specific to this plant. As a consequence, we generated a comprehensive V. minor transcriptome elaborated from eight distinct samples including roots, old and young leaves exposed to low or high light exposure conditions. This optimized resource exhibits an improved completeness compared to already published ones. Through homology-based searches using C. roseus genes as bait, we predicted candidate genes for all common steps of the MIA pathway as illustrated by the cloning of a tabersonine/vincadifformine 16-O-methyltransferase (Vm16OMT) isoform. The functional validation of this enzyme revealed its capacity of methylating 16-hydroxylated derivatives of tabersonine, vincadifformine and lochnericine with a Km 0.94 ± 0.06 µM for 16-hydroxytabersonine. Furthermore, by combining expression of fusions with yellow fluorescent proteins and interaction assays, we established that Vm16OMT is located in the cytosol and forms homodimers. Finally, a gene co-expression network was performed to identify candidate genes of the missing V. minor biosynthetic steps to guide MIA pathway elucidation.

Vacuole-Targeted Proteins: Ins and Outs of Subcellular Localization Studies.

Accurate and efficient demonstrations of protein localizations to the vacuole or tonoplast remain strict prerequisites to decipher the role of vacuoles in the whole plant cell biology and notably in defence processes. In this chapter, we describe a reliable procedure of protein subcellular localization study through transient transformations of Catharanthus roseus or onion cells and expression of fusions with fluorescent proteins allowing minimizing artefacts of targeting.

Virus-induced gene silencing in Rauwolfia species.

Elucidation of the monoterpene indole alkaloid biosynthesis has recently progressed in Apocynaceae through the concomitant development of transcriptomic analyses and reverse genetic approaches performed by virus-induced gene silencing (VIGS). While most of these tools have been primarily adapted for the Madagascar periwinkle (Catharanthus roseus), the VIGS procedure has scarcely been used on other Apocynaceae species. For instance, Rauwolfia sp. constitutes a unique source of specific and valuable monoterpene indole alkaloids such as the hypertensive reserpine but are also well recognized models for studying alkaloid metabolism, and as such would benefit from an efficient VIGS procedure. By taking advantage of a recent modification in the inoculation method of the Tobacco rattle virus vectors via particle bombardment, we demonstrated that the biolistic-mediated VIGS approach can be readily used to silence genes in both Rauwolfia tetraphylla and Rauwolfia serpentina. After establishing the bombardment conditions minimizing injuries to the transformed plantlets, gene downregulation efficiency was evaluated at approximately a 70% expression decrease in both species by silencing the phytoene desaturase encoding gene. Such a gene silencing approach will thus constitute a critical tool to identify and characterize genes involved in alkaloid biosynthesis in both of these prominent Rauwolfia species.

Silver nanoparticle-embedded polymersome nanocarriers for the treatment of antibiotic-resistant infections.

The rapidly diminishing number of effective antibiotics that can be used to treat infectious diseases and associated complications in a physician's arsenal is having a drastic impact on human health today. This study explored the development and optimization of a polymersome nanocarrier formed from a biodegradable diblock copolymer to overcome bacterial antibiotic resistance. Here, polymersomes were synthesized containing silver nanoparticles embedded in the hydrophobic compartment, and ampicillin in the hydrophilic compartment. Results showed for the first time that these silver nanoparticle-embedded polymersomes (AgPs) inhibited the growth of Escherichia coli transformed with a gene for ampicillin resistance (bla) in a dose-dependent fashion. Free ampicillin, AgPs without ampicillin, and ampicillin polymersomes without silver nanoparticles had no effect on bacterial growth. The relationship between the silver nanoparticles and ampicillin was determined to be synergistic and produced complete growth inhibition at a silver-to-ampicillin ratio of 1 : 0.64. In this manner, this study introduces a novel nanomaterial that can effectively treat problematic, antibiotic-resistant infections in an improved capacity which should be further examined for a wide range of medical applications.

Evaluation of a method using high performance liquid chromatography with ultraviolet detection for the determination of statins in macromycetes of the genus Pleurotus cultivated by fermentation processes.

The applicability of high-performance liquid chromatography with ultraviolet light (HPLC-UV) for the determination of the presence of statins in macromycetes of the genus Pleurotus was analyzed. The fungi were obtained by liquid-state fermentation (LSF) using unconventional sources of carbon as substrates and solid-state fermentation (SSF) employing agro industrial wastes. Five statins were used as standards: lovastatin and simvastatin in the lactone form (LOVL and SIML), their corresponding hydro-acidic forms (LOVH and SIMH) and pravastatin (PRA). The following measures were evaluated: the linearity, accuracy and precision, detection limit (DL) and quantification limit (QL). The results demonstrated HPLC-UV to be an effective tool for detecting the presence of statins in extracts of LSF and SSF products. Likewise, it was hypothesized that the strains that were used for the study do not produce statins. This finding highlights the importance of continuing to evaluate other strains of the same genus by using techniques such as HPLC to first separate sufficient quantities of the compounds that were detected using the standard technique but that did not match the retention time (tR) of any of the standards used.