Interaction between abscisic acid and nitric oxide in PB90‐induced catharanthine biosynthesis of catharanthus roseus cell suspension cultures

Elicitations are considered to be an important strategy to improve production of secondary metabolites of plant cell cultures. However, mechanisms responsible for the elicitor‐induced production of secondary metabolites of plant cells have not yet been fully elucidated. Here, we report that treatment of Catharanthus roseus cell suspension cultures with PB90, a protein elicitor from Phytophthora boehmeriae, induced rapid increases of abscisic acid (ABA) and nitric oxide (NO), subsequently followed by the enhancement of catharanthine production and up‐regulation of Str and Tdc, two important genes in catharanthine biosynthesis. PB90‐induced catharanthine production and the gene expression were suppressed by the ABA inhibitor and NO scavenger respectively, showing that ABA and NO are essential for the elicitor‐induced catharanthine biosynthesis. The relationship between ABA and NO in mediating catharanthine biosynthesis was further investigated. Treatment of the cells with ABA triggered NO accumulation and induced catharanthine production and up‐regulation of Str and Tdc. ABA‐induced catharanthine production and gene expressions were suppressed by the NO scavenger. Conversely, exogenous application of NO did not stimulate ABA generation and treatment with ABA inhibitor did not suppress NO‐induced catharanthine production and gene expressions. Together, the results showed that both NO and ABA were involved in PB90‐induced catharanthine biosynthesis of C. roseus cells. Furthermore, our data demonstrated that ABA acted upstream of NO in the signaling cascade leading to PB90‐induced catharanthine biosynthesis of C. roseus cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:994–1001, 2013     

Catharanthus roseus: micropropagation and in vitro techniques

Different methods of in vitro culture of Catharanthus roseus provide new sources of plant material for the production of secondary metabolites such as indole alkaloids. Callus, cell suspension, plantlets, and transgenic roots cultured in the bioreactor are used in those experiments. The most promising outcomes include the production of the following indole alkaloids: ajmalicine in unorganised tissue, catharanthine in the leaf and cell culture in the shake flask and airlift bioreactor, and vinblastine in shoots and transformed roots. What is very important, enzymatic coupling of monomeric indole alkaloids, vindoline and catharanthine, is possible to form vinblastine in cell cultures. The method of catharanthine and ajmalicine production in the suspension culture in bioreactors has been successful. In this method, elicitation may be used acting on different metabolic pathways. Also of interest is the method of obtaining arbutin from the callus culture of C. roseus conducted with hydroquinone. The transformed root culture seems to be the most promising for alkaloid production. The genetically transformed roots, obtained by the infection with Agrobacterium rhizogenes, produce higher levels of secondary metabolites than intact plants. Also, whole plants can be regenerated from hairy roots. The content of indole alkaloids in the transformed roots was similar or even higher when compared to the amounts measured in studies of natural roots. The predominant alkaloids in transformed roots are ajmalicine, serpentine, vindoline and catharanthine, found in higher amounts than in untransformed roots. Transformed hairy roots have been also used for encapsulation in calcium alginate to form artificial seeds.     

Increased accumulation of indole alkaloids by some cell lines of Catharanthus roseus in response to addition of vanadyl sulphate

Vanadyl sulphate (10–500 mg/l), when added to cell suspension cultures of Catharanthus roseus stimulated increased intracellular accumulation of catharanthine and ajmalicine. This response was demonstrated in both flask and fermenter (30 litre) systems. The response varied, and depended upon cell line, concentration of vanadyl sulphate and the stage of the growth phase at which the cells were treated. This process has the potential to increase the yield and reduce the production time for commercially useful secondary plant metabolites.Abbreviations Ajm ajmalicine - Cath catharanthine - CAS ceric ammonium sulphate - VOSO₄ vanadyl sulphate - FW fresh weight - n.d. not detected     

Elicitor-induced nitric oxide burst is essential for triggering catharanthine synthesis in Catharanthus roseus suspension cells

Elicitor prepared from the cell walls of Penicillium citrinum induced multiple responses in Catharanthus roseus suspension cells, including rapid generation of nitric oxide (NO), sequentially followed by enhancement of catharanthine production by C. roseus cells. Elicitor-induced catharanthine biosynthesis was blocked by NO-specific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and nitric oxide synthase (NOS) inhibitor S,S-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea (PBITU). PBITU also strongly inhibited elicitor-induced NO generation by C. roseus suspension cells. The inhibiting effect of PBITU on elicitor-induced catharanthine production was reversed by external application of NO via the NO-donor sodium nitroprusside. The results strongly suggested that NO, generated by NOS or NOS-like enzymes in C. roseus suspension cells when treated with the fungal elicitor, was essential for triggering catharanthine synthesis.     

Gas composition strategies for the successful scale-up of Catharanthus roseus cell cultures for the production of ajmalicine

Ajmalicine, serpentine, catharanthine, and vindoline are monoterpenoid indole alkaloids (MIAs) of commercial interest which are produced by the Catharanthus roseus plant. Cultures of C. roseus have been investigated as a potential source of these pharmaceutically important compounds since the early 1960s. In addition, their production from C. roseus cultures has served as a model system for investigating secondary metabolism and for evaluating production-enhancing strategies. Initially, this review will survey (1) the MIAs of interest for large-scale production from plant cell cultures and (2) the volumetric productivities of a specific MIA, ajmalicine, achieved and projected using plant cell cultures. To meet the need for these valuable compounds, the production of these MIAs from plant cell cultures must be successfully reproduced in large-scale aerated and agitated reactors. While the large-scale cultivation of plant cell cultures is currently feasible, initial attempts at scale-up may yield results that differ from that optimized in flasks. To bridge the jump between production in flasks and production in large-scale bioreactors, changes introduced with scale-up such as gas composition must be identified and rationally manipulated to reproduce or even improve growth and secondary metabolite production. Hence, this review will (1) identify the effects of gas composition (i.e., O₂, CO₂, ethylene, or other endogenous volatile compounds) on growth and secondary metabolism and (2) draw operating strategies for optimizing the gas composition for growth of C. roseus cultures and the production of ajmalicine.     

Different shake flask closures alter gas phase composition and ajmalicine production in Catharanthus roseus cell suspensions

The type of closure chosen for plant cell cultures can significantly alter the headspace gas composition of a culture, leading to major differences in the production of secondary metabolites. In cell suspension cultures of Catharanthus roseus, ethylene accumulated in cultures with limited gas exchange and appeared to inhibit the production of ajmalicine. The variability in product yields between replicates can also be attributed to gas composition differences.     

The Use of Organic and Inorganic Compounds to Increase the Accumulation of Indole Alkaloids in Catharanthus roseus (L.) G. Don Cell Suspension Cultures

Smith, J. I, Smart, N. J., Kurz, W. G. W. and Misawa, M. 1987.The use of organic and inorganic compounds to increase the accumulationof indole alkaloids in Catharanthus roseus (L.) G. Don cellsuspension cultures.—J. exp. Bot. 38: 1501–1506. The addition of sodium chloride, potassium chloride or sorbitolto 5–d–old cell suspension cultures of Catharanthusroseus stimulated an increase in the intracellular accumulationof catharanthine and other indole alkaloids within 48–72h. The magnitude of the response depended upon the concentrationof the compound added. The use of such inexpensive and readilyavailable compounds to increase the yields and reduce the requiredculture times has considerable potential for the productionof useful secondary metabolites from cell cultures of C. roseusand other plant species.     

A differential response to chemical elicitors in Catharanthus roseus in vitro cultures

The effects of methyl jasmonate, salicylic acid and ethylene on alkaloid accumulation in in vitro cell suspension, hairy roots and rootless shoot cultures of Catharanthus roseus were analyzed. Ajmalicine, but not catharanthine, accumulation was promoted by jasmonate and ethylene treatments in cell suspensions. In hairy roots, jasmonate induced the accumulation of both alkaloids, whereas ethylene only induced catharanthine accumulation. In shoot cultures, positive effects of jasmonate and ethylene were recorded only in vindoline accumulation. Ethylene diminished catharanthine accumulation in these cultures. No effect of salicylic acid was observed in any of the studied in vitro culture systems. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of plant growth regulators on the biosynthesis of vinblastine,vindoline and catharanthine in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Catharanthuse roseus is a well-known medicinal plant for its two valuable anticancer compounds: vinblastine and vincristine, which belongs to terpenoid indole alkaloids. Great efforts have been made to study the principles of its secondary metabolic pathways to regulate the alkaloids biosynthesis. In this article, different plant growth regulators were shortly applied to Catharanthus roseus plants during the blooming period to study their effects on the biosynthesis of vinblastine, vindoline and catharanthine. Salicylic acid and ethylene (ethephon) treatments resulted in a significant increase of vinblastine, vindoline and catharanthine while abscisic acid and gibberellic acid had a strongly negative influence on the accumulation of the three important alkaloids. Methyl jasmonate showed no great effect on the production of these valuable alkaloids. Chlormequat chloride highly enhanced the accumulation of vinblastine but greatly decreased the contents of vindoline and catharanthine.     

A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus

The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle (Catharanthus roseus). Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003–0.01% yields. Metabolic engineering efforts to either improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. A VIGS method was developed herein to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro.     

The development of a single-stage growth and indole alkaloid production medium for Catharanthus roseus (L.) G. Don suspension cultures

By modification of a standard Murashige and Skoog plant tissue culture maintenance medium, a system has been developed for Catharanthus roseus cell suspension cultures in which both growth and indole alkaloid accumulation can occur in a single-stage culture of 14–21 days. Precise optimization of the medium depends upon the cell line under investigation, but the inclusion of lactose as the carbohydrate source, and NAA and kinetin as growth regulators, will generally increase yields of the indole alkaloid catharanthine. Treatment of cells growing in this optimized medium with agents that stimulate the accumulation of secondary metabolites both increases the yield of catharanthine and reduces the time required for production. We believe that this process could be useful for the commercial production of plant secondary metabolites.     

Stimulation of anthocyanin synthesis in grape (<Emphasis Type="Italic">Vitis vinifera</Emphasis>) cell cultures by pulsed electric fields and ethephon

Anthocyanin from grape cell cultures can be used as a natural alternative to synthetic dyes; particularly due to their reported health-promoting properties. In this study, production of anthocyanin in cell suspension culture of Vitis vinifera was evaluated following treatment with either ethephon and/or pulsed electric fields (PEF). Overall, total production of anthocyanin increased in treated cells compared to untreated cells. Treatment of cell suspension with PEF at day 14 of culture resulted in 1.7-fold increase (1.42 mg/g DW) in anthocyanin content when compared to control cells; while, treatment with ethephon resulted in 2.3-fold increase (1.99 mg/g DW) in anthocyanin content. When cells were treated with both ethephon and PEF, 2.5-fold increase in anthocyanin content (2.2 mg/g DW) was observed. These findings demonstrate that PEF induces a defense response in plant cells, and it may also alter the dielectric properties of cells and/or cell membranes, and would serve as a viable elicitor of secondary metabolites in plant cell cultures.     

Stimulation of ajmalicine production and excretion from Catharanthus roseus: effects of adsorption in situ,elicitors and alginate immobilization

Summary More efficient bioreactors for the production and recovery of secondary metabolites from plant cell cultures are needed. Three factors that have the potential to increase productivity are adsorption in situ, elicitors, and cell immobilization. The effects of these factors on ajmalicine production from Catharanthus roseus are reported in this paper. Elicitation using autoclaved cultures of the mold, Phytophthora cactorum, stimulates a 60% increase in ajmalicine production. The response time to elicitor addition was under 11 h. Adsorption of ajmalicine from the extracellular medium with the neutral resin, Amberlite XAD-7, greatly enhanced the release of ajmalicine (less than 10% extracellular to 40%) with a 40% increase in total productivity. Immobilization in Caalginate beads resulted in a significant increase in the accumulation of ajmalicine in the medium. The effects of elicitation, adsorption and immobilization were synergistic. For a 23-day culture period the amount of ajmalicine in the medium for cells subjected to all three treatments was 90 mg/L compared to 2 mg/L for suspension cultures cultured under otherwise identical conditions. These results suggest that immobilized cell bioreactors may be feasible for continuous production of products normally stored intracellularly in vacuoles in plant cells.     

Traditional and non-traditional plant growth regulators alters phytochemical constituents in Catharanthus roseus

The effect of different plant growth regulators (PGR) and elicitor treatments on the alkaloid profile variation of Catharanthus roseus was investigated in the present study. The PGR used were paclobutrazol (PBZ), gibberellic acid (GA₃) and Pseudomonas fluorescens elicitors (PF Elicitors). The estimated alkaloids were ajmalicine, catharanthine, tabersonine, serpentine and vindoline. In roots, the ajmalicine content increased significantly under all the treatments on all sampling days. In roots, the catharanthine contents increased with the age in control and growth regulator treatments, but the increase was not prominent and significant in PGR treatments when compared to controls. The serpentine contents of the plant increased with PGR treatments, but the increase was more prominent in PBZ treatments when compared to other treatments. The increase was in the order PBZ > PF Elicitors > GA₃. C. roseus never showed any significant increase in tabersonine contents in the roots under GA₃ treatments, but it increased significantly under PBZ and PF Elicitors when compared to control plants. The root vindoline contents increased with PBZ and PF Elicitors treatments but the decreased under GA₃ treatments when compared to control plants. Our results have good significance, as these increases the secondary metabolites of this traditional medicinal plant.     

Promotion of indole alkaloid production in Catharanthus roseus cell cultures by rare earth elements

Production of the indole alkaloids, ajmalicine or catharanthine, in cell suspension cultures of Catharanthus roseus was enhanced by cerium (CeO₂ and CeCl₃), yttrium (Y₂O₃) and neodymium (NdCl₃). The yield of ajmalicine in these treated-cultures reached 51 mg l^–1 (CeO₂), 40 mg l^–1 (CeCl₃), 41 mg l^–1 (Y₂O₃) and 49 mg l^–1 (NdCl₃) while catharanthine production reached to 36 mg l^–1 (CeO₂) and 31 mg l^–1 (CeCl₃). A major portion of increased alkaloids was released into medium in these treatments. But Sm₂O₃, SmCl₃, La₂O₃, LaCl₃, complex of chromium (III)-titanium (IV) and NaSeO₄ treatments had little effect on alkaloid production of C. roseus cell cultures.     

Enhanced catharanthine and vindoline production in suspension cultures of Catharanthus roseus by ultraviolet-B light

Suspension cultures of Catharanthus roseus were used to evaluate ultraviolet-B (UV-B) treatment as an abiotic elicitor of secondary metabolites. A dispersed cell suspension culture from C. roseus leaves in late exponential phase and stationary phase were irradiated with UV-B for 5 min. The stationary phase cultures were more responsive to UV-B irradiation than late exponential phase cultures. Catharanthine and vindoline increased 3-fold and 12-fold, respectively, on treatment with a 5-min UV-B irradiation.