Print version ISSN 0100-4042
Quím. Nova vol.30 no.8 São Paulo 2007
Paulo Sérgio Pereira*; Fábio Kiss Ticli, Suzelei de Castro França; Camila Moniz de Souza Breves; Miriam Verginia Lourenço
Unidade de Biotecnologia, Universidade de Ribeirão Preto, 14096-900 Ribeirão Preto - SP, Brazil
Cell suspension cultures of Tabernaemontana catharinensis were treated with autoclaved homogenates of Candida albicans, Fusarium oxysporum, Penicillium avelanium and Saccharomyces cerevisiae. The effects caused by the concentration, exposure time and the type of elicitor on the accumulation of pentacyclic triterpenes were monitored. When exposed to biotic elicitors for longer periods, some cell lines redoubled the production of those triterpenes. Saccharomyces cerevisiae homogenate was the best elicitor of triterpenes in all cell lines investigated.
Keywords: Tabernaemontana catharinensis;plant cell culture;elicitation.
Tabernaemontana catharinensis (A.DC.) Miers (Syn. Peschiera catharinensis, T. hilariana, T. affinis, T. australis, T. acuminata, T. hibrida, P. albidiflora and T. salicifolia) belongs to the Apocynaceae family1. Besides indole alkaloids several pentacyclic triterpenoids were isolated from Tabernaemontana species: lupeol, a- and b- amirin, baurenil acetate, sitosterol, and others2,3. Acid triterpenes are of great interest due to the diversity of pharmacological activities they display: anti-inflammatory, hepatoprotective, antitumoral, among others4-7.
Fungal elicitation has been an effective tool to enhance the yield of secondary metabolites8, also helping in the elucidation of mechanisms of plant responses to biotic stress agents9. Most fungal elicitation strategies utilize fairly undefined mixtures such as autoclaved fungal homogenates10,11 or fungal culture filtrates9.
Several reports have shown that the use of yeast and fungi as elicitors provoked an accumulation of triterpenoid phytoalexins12-16. In elicited cultures of Catharanthus roseus (L.), the qualitative profile of terpenoid products detected was similar to that found in control cultures15, but different from the observation with Tabernaemontana divaricata14 and Uncaria tomentosa16.
In this study, 4 different types of biotic elicitors, yeast and fungi, were tested to stimulate triterpene production. We report here the effects of concentration and exposure time of those elicitors on T. catharinensis cell lines.
Callus induction and maintenance
Callus cultures were induced from disinfested leaf explants and maintained on solid MS medium supplemented with 30 g sucrose l-1 in two different hormone combination: T43 (1.0 mg 2,4-dichlorophenoxyacetic acid l-1 and 1.0 mg kinetin l-1) and T44 (1.0 mg 2,4-dichlorophenoxyacetic acid l-1 and 0.1 mg kinetin l-1). Two different cell lines - chlorophyllated cells and non-chlorophyllated cells were selected from cultures carried out in T44 medium while only one cell line was obtained in T43 medium. Cells were subcultured every 30 days and maintained at 28 ± 2 ºC under 16-hour-day photoperiod.
Six-year-old callus cultures were inoculated into a 250 mL erlenmeyer flask containing 100 mL of MS medium with 30 g sucrose L-1. The suspension cultures were established using different callus lines: calli previously cultured on T43 semisolid MS medium were inoculated into T43 liquid MS medium (Culture 2); non-chlorophyllated calli cultivated on T44 semisolid MS medium were inoculated into both T43 liquid MS medium (Culture 1) and T44 liquid MS medium (Culture 4); and chlorophyllated calli cultured on T44 solid MS medium were inoculated into T44 liquid MS medium (Culture 3). Cultures were subcultured every 30 days and maintained in a growth room, under agitation (110 rpm) on orbital shaker, at 28 ± 2 ºC exposed to a 16-hour-day photoperiod.
Preparation of biotic agents for experiments of elicitation
Candida albicans, Fusarium oxysporum and Penicilium avelanium were cultured in liquid potato/dextrose medium under agitation (110 rpm) on orbital shaker at 28 ºC. After 96 h of incubation the cultures were autoclaved and the micelia were collected by filtration and then dried. C. albicans, F. oxysporum, P. avelanium and Saccharomyces cerevisiae micelia (5, 10 and 50 mg mL-1) were homogenized in deionized water and autoclaved. Fungal homogenates were added separately to the suspension cultures either at the early (14 days) or late exponential phase of growth (20 days). No elicitor was added to the control cultures. All experiments were conducted with four T. catharinensis suspension lines (Culture 1 to 4). For a time course study, untreated and elicited suspension cultures were harvested at different time intervals (24, 48 and 72 h) by vacuum filtration. Triplicate flasks were run for each treatment and controls.
Dry and powered material (200 mg) was extracted overnight, successively with chloroform and methanol (5 mL each) at room temperature. Chloroform and methanol extracts were grouped and the solvent evaporated at room temperature. Crude extract was then resuspended in water and partitioned three times with ethyl acetate. The combined organic extracts were evaporated under vacuum, residues were dissolved in 1 mL of methanol and analysed by HPLC.
HPLC analysis conditions: Shimadzu LC10ADvp system equipped with Supelco LC18 column (250 x 4.6 mm), coupled to a diode array detector. Samples were eluted with methanol:H2O (Acetic acid, 0.1%), 85:15, at 1 mL min-1 under isocratic condition and monitored at 210 nm. 20 mL of each solution from oleanolic acid, ursolic acid and samples were used for quantitative analysis. The identification of oleanolic and ursolic acid was done by comparing their retention times and spectral data with those of standard compounds. All quantifications were performed in triplicate the independent experiments: elicited and control cultures. The quantification was evaluated using external calibration curves. Calibration curves with theirs respective standards were developed with diluted samples of standard compounds within the range of 1.0 to 0.01 mg/mL. Authentic ursolic acid and oleanolic acid were purchased from Aldrich Co.
Callus culture was initiated from axenic leaf explants of T. catharinensis inoculated on MS solid medium supplemented with two combinations of 2,4-D and kinetin (1:1 and 1:0.1). Calli cultured on T43 medium developed homogenous friable of yellow pigmented biomass and calli maintained on T44 medium developed heterogenous friable biomass with chlorophyllated and non-chlorophyllated cells. Chlorophyllated and non-chlorophyllated cells were subcultured in T44 liquid medium. Thereafter, all callus and suspension cultures were maintained and subcultured in T44 or T43 media.
Induction of ursolic and oleanolic acid production by elicitation
As a response to the biotic stress caused by microorganisms, the T. catharinensis cells was stimulated increasing the biosynthesis of triterpenoids (Table 1 to 4). The experiments were carried out with T. catharinensis cells either in the early or late exponential phase of growth (14 or 20-day-old). Cultures were homogenous in appearance, but when stressed with fungal homogenates there was a rapid change in their color.
The effects of various elicitors on triterpene accumulation in T. catharinensis suspension cultures are shown in Tables 1 to 4. Addition of cell wall homogenates, no matter the concentration or type of microorganism source tested, resulted in increased triterpene levels. Cultures 2 and 3 were more susceptible to fungal homogenate treatments showing higher yields of oleanolic and ursolic acids. Among the elicitors tested, S. cerevisae showed better results in terms of triterpene accumulation, producing 5 and 7 mg g-1 dw, of ursolic and oleanolic acids respectively, followed by C. albicans, which produced 2 and 3 mg g-1 dw, and P. avelanium 1 to 2 mg g-1.Triterpene accumulation in cultured cells treated with F. oxysporum homogenates was slight stimulated. In most experiments, the maximum production of triterpenes was achieved around 72 h of culture in an elicitor dose related accumulation. When fungal homogenates were added to the suspension cultures at the early exponential phase, 14 days of culture, the production of triterpenes was not significant compared to control cells. This situation was verified twice in the third and sixth subcultures (Experiments 1 and 2).
Several reports have shown that secondary metabolism in plants cell culture were stimulated or inhibited with fungal homo-genates8,9,11. In our study elicitors induced a rapid stimulation of the secondary metabolism pathway of T. catharinensis cells increasing the biosynthesis of triterpenoids. The rapid change in suspension cultures with fungal homogenates was also reported in elicitation experiments with other plant cells12-16.
Obtained results corroborate the theory that components of the cell wall homogenates act as signaling molecules of triterpenoid biosynthetic pathway, triggering the production of oleanolic acid and ursolic acid by treated cells. When the extracts of T. catharinensis unelicited cells were quantified, triterpenes were almost not detected. The regulation and enzymology of pentacyclic triterpenoid phytoalexin biosynthesis in cell suspension cultures of T. divaricata has been investigated. In T. divaricata, C. albicans elicitor preparations resulted in the production of considerable amounts of ursolic acid type pentacyclic triterpenoids while alkaloid production was blocked under such conditions13,14. A similar regulatory mechanism would be expected in T. catharinensis cells. Neither TLC nor HPLC analysis were performed in this work for alkaloid production.
The procedures described in this work may be employed in strategies for enhancement in productivity of secondary metabolites and for investigating the complex secondary metabolite pathways in plant tissue cultures.
F. K. Ticli thanks FAPESP for his scholarship reward (Nº 99/11073-0).
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Recebido em 1/9/06; aceito em 11/5/07; publicado na web em 25/10/07