Production of Plant Bioactive Triterpenoid Saponins: Elicitation Strategies and Target Genes to Improve Yields

Triterpenoid saponins are a class of plant secondary metabolites with structure derived from the precursor oxidosqualene in which one or more sugar residues are added. They have a wide range of pharmacological applications, such as antiplatelet, hypocholesterolemic, antitumoral, anti-HIV, immunoadjuvant, anti-inflammatory, antibacterial, insecticide, fungicide and anti-leishmanial agents. Their accumulation in plant cells is stimulated in response to changes mediated by biotic and abiotic elicitors. The enhancement of saponin yields by methyl jasmonate in plants and cell cultures in several species indicates the involvement of these metabolites in plant defence mechanisms. The elucidation of their biosynthesis at the molecular level has advanced recently. Most studies to date have focused on the participation of early enzymes in the pathway, including oxidosqualene cyclase, squalene synthase and dammarenediol synthase, as well as in isolating and characterizing genes that encode β-amyrin synthase. Yields of bioactive saponins in various plant species and experimental systems have been successfully increased by treating cells and tissues with jasmonate or by exposing these to oxidative stress. These elicitation and molecular studies are consolidating a robust knowledge platform from which to launch the development of improved sources for commercial supply of bioactive saponins.     

Distinct modes of adventitious rooting in Arabidopsis thaliana

The literature describes different rooting protocols for Arabidopsis thaliana as models to study adventitious rooting, and results are generally perceived as comparable. However, there is a lack of investigations focusing on the distinct features, advantages and limitations of each method in the study of adventitious rooting with both wild-type (WT) ecotypes and their respective mutants. This investigation was undertaken to evaluate the adventitious rooting process in three different experimental systems, all using A. thaliana, analysing the same rooting parameters after transient exposure to auxin (indole-3-acetic acid) and control conditions: excised leaves, de-rooted plants and etiolated seedlings. The founding tissues and sites of origin of roots differed depending on the system used, whereas all rooting patterns were of the direct type (i.e., without callus formation). None of the systems had an absolute requirement for exogenous auxin, although rooting was enhanced by this phytohormone, with the exception of de-rooted plants, which had adventitious rooting strongly inhibited by exogenous auxin. Root elongation was much favoured in isolated leaves. Auxin-overproducing mutants could not be used in the detached leaf system due to precocious senescence; in the de-rooted plant system, these mutants had a WT-like rooting response, whereas the expression of the 'rooty' phenotype was only evident in the etiolated seedling system. Adventitious rooting of etiolated WT seedlings in the presence of exogenous auxin was inhibited by exogenous flavonoids, which act as auxin transport inhibitors; surprisingly, the flavonoid-deficient mutant chs had a lower rooting response compared to WT. Although Arabidopsis is an excellent model system to study adventitious rooting, physiological and developmental responses differed significantly, underlining the importance of avoiding data generalisation on rooting responses derived from different experimental systems with this species.     

Specialized Plant Metabolism Characteristics and Impact on Target Molecule Biotechnological Production

Plant secondary metabolism evolved in the context of highly organized and differentiated cells and tissues, featuring massive chemical complexity operating under tight environmental, developmental and genetic control. Biotechnological demand for natural products has been continuously increasing because of their significant value and new applications, mainly as pharmaceuticals. Aseptic production systems of plant secondary metabolites have improved considerably, constituting an attractive tool for increased, stable and large-scale supply of valuable molecules. Surprisingly, to date, only a few examples including taxol, shikonin, berberine and artemisinin have emerged as success cases of commercial production using this strategy. The present review focuses on the main characteristics of plant specialized metabolism and their implications for current strategies used to produce secondary compounds in axenic cultivation systems. The search for consonance between plant secondary metabolism unique features and various in vitro culture systems, including cell, tissue, organ, and engineered cultures, as well as heterologous expression in microbial platforms, is discussed. Data to date strongly suggest that attaining full potential of these biotechnology production strategies requires being able to take advantage of plant specialized metabolism singularities for improved target molecule yields and for bypassing inherent difficulties in its rational manipulation.     

Valepotriates accumulation in callus, suspended cells and untransformed root cultures of Valeriana glechomifolia

Summary Valeriana glechomifolia is an endemic species of southern Brazil, capable of accumulating, in all of its organs, the terpene derivatives known as valepotriates, the presumed sedative components of the roots of pharmaceutically used species of Valeriana. In vitro cultures of the plant were established and the accumulation of acevaltrate, didrovaltrate, and valtrate in callus, cell suspension, and untransformed root cultures was studied. Leaves of in natura plants and roots of micropropagated plantlets were used as the explants for callus induction and root culture establishment, respectively, on Gamborg B5 basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or with kinetin (KIN). Culture growth and secondary metabolite yields were enhanced with 2,4-D (4.52μM) and KIN (0.93μM). Maximum valepotriate contents, quantified by HPLC, of acevaltrate (ACE) 2.6mg g⁻¹ DW, valtrate (VAL) 10.2mgg⁻¹ DW, and didrovaltrate (DID) 2.9mg g⁻¹ DW were observed in root cultures after 7–8wk of culture.     

Control of development and valepotriate production by auxins in micropropagated Valeriana glechomifolia

Valeriana glechomifolia is a plant species endemic to southern Brazil that accumulates valepotriates, which are terpene derivatives, in all of its organs. Valepotriates are the presumed sedative generic components of the pharmaceutically used species of Valeriana. The influence of various concentrations of the auxins indole-3-acetic acid, indole-3-butyric acid and -naphthaleneacetic acid on the growth of micropropagated V. glechomifolia was investigated under conditions of transient and continuous exposure. Changes in the development of roots and shoots as well as the production of the valepotriates acevaltrate, valtrate and didrovaltrate (analyzed by high-performance liquid chromatography) were evaluated. The best performance in valepotriate production, growth and survival under ex vitro conditions following plant acclimatization was achieved in the continuous presence of 5.71 M IAA. When cultured in medium containing IAA plants produced stable levels of valepotriates throughout the entire cultivation period.Abbreviations ACE Acevaltrate - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - DW Dry weight - DID Didrovaltrate - NAA -Naphthaleneacetic acid - PVP Polyvinylpyrrolidone - VAL Valtrate     

N,beta-D-Glucopyranosyl vincosamide, a light regulated indole alkaloid from the shoots of Psychotria leiocarpa

From leaves of Psychotria leiocarpa, an indole alkaloid was isolated to which the structure N,beta-D-glucopyranosyl vincosamide (1) was assigned. This represents the first report of an N-glycosylated monoterpenoid indole alkaloid. In field-grown plants highest amounts of 1 were found in the leaves (2.5% of dry wt) and fruit pulp (1.5% dry wt). Lower amounts were found in the stems (0.2% dry wt) and the seeds (0.1% of dry wt), whereas the alkaloid was not detected in the roots. The accumulation of 1 in aseptic seedlings was also restricted to the shoots and increased with plant age and light exposure, independent of the supply of sucrose in the culture medium.     

Improved nutrient medium for biomass and valepotriate production in extended period stock cultures of Valeriana glechomifolia

Valeriana glechomifolia, a southern Brazilian endemic species commonly known as Valerian, accumulates the bioactive terpene derivatives valepotriates in all of its organs. In vitro growth of V. glechomifolia on solid Murashige and Skoog (MS) without phytohormones at full, 75% (MS 75), or on a modified formulation (M Δ) was compared in stock cultures kept for up to 9 mo. without subculture. Changes in biomass accumulation, development of roots and shoots, and the production of the valepotriates acevaltrate, didrovaltrate, and valtrate were monthly evaluated. The highest biomass accumulation and root development was observed in plants grown on M Δ, whereas better leaf development was detected in M-Δ- and MS-medium-grown plants after 8 and 9 mo. of culture, respectively. Maximal didrovaltrate and valtrate yields were observed in M-Δ-grown plants harvested after 5 and 6 mo. of culture, respectively, whereas acevaltrate concentration was highest on M-Δ- and MS-75-grown plants after 7 mo. of culture. Plants grown for 6 mo. without subculture in M Δ were successfully propagated, showing stable growth and valepotriate yields three- to sixfold higher that those observed in field-grown plants. The results showed a positive effect of combined moderate reduction in salt concentration and increases in selected micronutrients and myo-inositol amounts on both growth and valepotriate yields of extended period stock cultures of V. glechomifolia.     

Organ-specific and environmental control of accumulation of psychollatine,a major indole alkaloid glucoside from Psychotria umbellata

Psychollatine is an unusual indole alkaloid produced by Psychotria umbellata Vell, an Atlantic Forest understorey tree. Leaves, young inflorescences and fruit pulp displayed high amounts of psychollatine (from 2.5 to 4.5% dry wt). Seeds accumulated lower quantities (0.2% dry wt), and roots, trace amounts (0.06% dry wt). Alkaloid accumulation was not seasonally affected and leaf content was not significantly enhanced by wounding, exposure to salicylic acid, hydrogen peroxide, or UV. The alkaloid content in leaves decreased upon cutting exposure to auxin. Under strong UV, the amount of chlorophylls did not decrease compared to control contents at 48 and 72 h. Post-harvest psychollatine content in leaves was stable in different temperatures, except at 65 °C. Psychollatine was an effective singlet oxygen, superoxide, and peroxide quencher. These antioxidant properties and the constitutively high amount of psychollatine indicate a role for the alkaloid in oxidative stress responses.     

Alternative Inactivated Poliovirus Vaccines Adjuvanted with Quillaja brasiliensis or Quil-A Saponins Are Equally Effective in Inducing Specific Immune Responses

Inactivated polio vaccines (IPV) have an important role at the final stages of poliomyelitis eradication programs, reducing the risks associated with the use of attenuated polio vaccine (OPV). An affordable option to enhance vaccine immunogenicity and reduce costs of IPV may be the use of an effective and renewable adjuvant. In the present study, the adjuvant activity of aqueous extract (AE) and saponin fraction QB-90 from Quillaja brasiliensis using poliovirus antigen as model were analyzed and compared to a preparation adjuvanted with Quil-A, a well-known saponin-based commercial adjuvant. Experimental vaccines were prepared with viral antigen plus saline (control), Quil-A (50 µg), AE (400 µg) or QB-90 (50 µg). Sera from inoculated mice were collected at days 0, 28, 42 and 56 post-inoculation of the first dose of vaccine. Serum levels of specific IgG, IgG1 and IgG2a were significantly enhanced by AE, QB-90 and Quil-A compared to control group on day 56. The magnitude of enhancement was statistically equivalent for QB-90 and Quil-A. The cellular response was evaluated through DTH and analysis of IFN-γ and IL-2 mRNA levels using in vitro reestimulated splenocytes. Results indicated that AE and QB-90 were capable of stimulating the generation of Th1 cells against the administered antigen to the same extent as Quil-A. Mucosal immune response was enhanced by the vaccine adjuvanted with QB-90 as demonstrated by increases of specific IgA titers in bile, feces and vaginal washings, yielding comparable or higher titers than Quil-A. The results obtained indicate that saponins from Q. brasiliensis are potent adjuvants of specific cellular and humoral immune responses and represent a viable option to Quil-A.