STRESS IN CALLUS OF Hippocratea excelsa: CATALASE ACTIVITY, HYDROGEN PEROXIDE CONTENT AND CANOPHYLLOL ACCUMULATION

Josefina Herrera-Santoyo. Biologist, M.Sc, PhD Student, Universidad Nacional Autónoma de México (UNAM). Departamento de Ecología y Recursos Naturales, Facultad de Ciencias. UNAM. Ciudad Universitaria, C.P. 04510 Coyoacán Mexico D.F., Mexico. e-mail: jhs@hp.fciencias.unam.mx.

Humberto Antonio López-Delgado. Biologist UNAM, México. M.Sc. Colegio de Posgraduados. Ph.D. University of Wales, United Kingdom. Programa Nacional de Papa, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP). Mexico.

Martha Elena Mora-Herrera. Biologist UNAM, Mexico. M.Sc. Colegio de Posgraduados. Ph.D. Student UNAM, México. Programa Nacional de Papa, INIFAP. Mexico.

SUMMARY

Stress factors often induce oxidative stress in plant cells, generally leading to the synthesis of signal molecules that activate a range of signal transduction pathways and antioxidants. One of the signal molecules produced under stress is H₂O₂, which has been associated to production in vitro of secondary metabolites (SM) and to antioxidant activity. Canophyllol production by Hippocratea excelsa was used as an example to show the enhanced production of SM under stress. Seeds obtained from fruits were cultured in vitro. Two stresses were applied: nutritional, by reducing the amount of N₂, and osmotic, using polyethylene glycol. Canophyllol, H₂O₂ content and catalase activity were measured. Nutritional stress caused a different response than osmotic stress for catalase activity and canophyllol accumulation, whereas H₂O₂ contents increased under both stresses. The highest amount of canophyllol (8 times the control) was obtained under the nutritional stress, in the treatment with 25% of control N₂ strength. This work demonstrates that the responses of canophyllol production and catalase activity in H. excelsa callus are differentially signaled by the different stress factors.

RESUMEN

RESUMO

Nas células vegetais alguns fatores adversos induzem freqüentemente estresse oxidativo, o qual gera síntese de moléculas que ativam uma gama de sinais nas rotas de transdução e antioxidantes. Uma molécula-sinal produzida sob estresse é o H₂O₂, o qual tem sido associado à produção de metabólitos secundários in vitro e a atividade antioxidante das células vegetais. Neste trabalho se utilizou a produção de canofilol em calos de Hippocratea excelsa como um exemplo para demonstrar o incremento na produção de metabólitos secundários sob estresse. Sementes obtidas de frutos foram cultivadas in vitro para a obtenção do calo. Dois diferentes tipos de estresse foram aplicados: nutricional, reduzindo a quantidade de N₂ no meio, e osmótico, usando polietilenglicol. Mediu-se o conteúdo de canofilol e de H₂O₂, assim como a atividade da enzima catalase. O estresse nutricional causou uma resposta diferente a do estresse osmótico na acumulação de canofilol e na atividade da catalase, enquanto que o conteúdo de H₂O₂ aumentou sob ambos tipos de estresse. A maior quantidade de canofilol (8 vezes o controle) se obteve sob estresse nutricional no tratamento de 25% de N₂. Este trabalho mostra que as respostas na produção de canofilol e a atividade de catalase em calos de H. excelsa são sinalizadas diferencialmente nos dois diferentes tipos de estresse aplicados.

The expression of many pathways of secondary metabolites (SM) in plant cells is altered by external factors such as nutrient levels, stress factors, light and growth regulators. Many of the constituents of culture media are important determinants of SM biosynthesis and accumulation (Rao and Ravishankar, 2002). Biotic and abiotic stress factors trigger changes in the plant cell, which leads to a cascade of reactions ultimately resulting in the formation and accumulation of SM, helping the plant to overcome the stress factors (Rakhmankulova et al., 2003). Stress factors often induce oxidative stress in the plant cell, generally leading to the biosynthesis of signal molecules (Dat et al., 2000). These activate a range of signal transduction pathways and antioxidants (Ganesan and Thomas, 2001). One of the signal molecules produced under stress is H₂O₂ (Neill et al., 2002), which has been associated to SM production in vitro and antioxidant activities (Chong et al., 2004).

Hippocratea excelsa (Hippocrateaceae) is a native species of Mexico and Central America. The root bark of this plant, known as "cancerina", is used in the Mexican traditional medicine for treating peptic ulcers, skin ailments, kidney disease and menstruation disorders. In rural México this plant is known as mata piojo (louse killer); as this name suggests, the pesticide properties are valued by farmers and ranchers (Palacios et al., 1989). Previous chemical reports on this plant have detailed the isolation of friedelanes and triterpenoid quinone methides (Calzada et al., 1991). Here, canophyllol production by H. excelsa was used as an example to show the enhanced production of SM under stress.

Seeds obtained from fruits were placed on MS medium (Murashige and Skoog, 1962) supplemented with 1.5mg·l^-1 naphthalene acetic acid; 2.0mg·l^-1 benzyl amino purine; 30g·l^-1 sucrose; 20mg·l^-1 ascorbic acid; and 2.5g·l^-1 phytagel. The cultures were incubated at 25 ±2°C in darkness for 21 days. The calluses obtained were subcultured for 14 months in order to homogenize the culture before experiments.

H. excelsa calli (4g) were transferred to MS liquid medium (125ml) with the different stress treatments.

Nutritional. The concentration of total N₂ in the standard MS medium was modified to apply nutritional stress. Three levels were tested: a) 100% MS concentration; 0.021M NH₄NO₃ and 0.019M KNO₃ (control); b) 50% MS; 0.0105M NH₄NO₃ and 0.0095M KNO₃; and c) 25%MS; 0.005M NH₄NO₃ and 0.0048M KNO₃.

Osmotic. Four levels of polyethylene glycol (PEG 8000) were tested in the medium, namely 0 (control), 1, 2 and 3% w/v. Experiments were carried out in triplicate.

In both stress treatments, yield extracts, canophyllol accumulation, catalase activity and H₂O₂ content, were measured each in triplicate.

Fresh and dry weight. The fresh weight was obtained after filtration of the medium and then the calluses were freeze dried to obtain the dry weight.

Canophyllol extraction and quantification. In order to obtain a terpenoid fraction, ground freeze-dried calluses of H. excelsa from the different treatments were extracted 3 times with hexane, for 3 days under agitation. The extracts were evaporated to dryness and the dry matter weighed. Chromatographic analysis of the hexane extracts was performed on 20×10cm silica gel 60 F₂₅₄ plates (used after activation for 1h at 110°C). The hexane extract (1.0mg) was dissolved in CHCl₃ (1.0ml). Aliquots of 50μl from each sample plus 10μl of the standard solutions were applied to the plates as bands by means of a CAMAG Automatic TLC sampler 4. Samples of three independent experiments were run on the same plate. When the spots were dried, separation was performed in a saturated chamber at room temperature using hexane:ethyl acetate (EtAcO) 8:2 v/v as mobile phase. The development distance was 90mm. After development, the plates were dried in a fume cupboard at room temperature for ~15min, and derivatized by spraying with the Liebermann-Burchard reagent (3-4ml) followed by heating at 90°C for 15min on a hot plate. After heating, plates were scanned immediately using a densitomer (CAMAG TLC Scanner 3) operated by the software winCATS ver. 1.1.3.0 running on a personal computer, under the following measurement conditions: type- remission and mode- absorption (tungsten lamp source). The wavelength of maximum absorption of canophyllol (Rf= 0.47) was found to be 450nm. Canophyllol was purified from roots of H. excelsa as standard, according to Calzada et al (1991) and its identity confirmed from its melting point and ¹H nuclear magnetic resonance (NMR) spectrum. A reference solution in chloroform (1mg·ml^-1), was utilized for the analysis. The concentration of canophyllol in the samples was determined from the peak area in reference to a standard calibration curve (1-5μg). Standard calibration graphs were calculated using the densitometer by linear regression and sample areas interpolated automatically under computer control. The interpolated weight of canophyllol in a zone, extract reconstitution volume, volume applied to the plate and sample weight tissue were used in appropriate equations to calculate the concentration of canophyllol in the samples.

H₂O₂ content. Was measured according to Warm and Laties (1982), using a luminometer (MGM instruments Optocomp-p).

Statistical Analysis. Significance of data was determined by Tukey´s Multiple Range Test.

Yield extracts. Reducing the N₂ strength of the medium significantly affected the fresh weight/dry weight (FW/DW) ratio of the callus as a result of the induced stress (Table I). The lowest N₂ concentration showed the highest FW/DW ratio. A similar effect was observed in the hexane extractable matter (Table I).

Canophyllol quantification. The canophyllol concentration increased under N₂ stress compared to the control (Table II). Incubation in 50% N₂ showed a 1.4 fold increment with respect to the control, whereas incubation in 25% N₂, caused a 7.97 fold increment in canophyllol (Table II). In the chromatography profiles an increment of band intensity was observed in extracts from cultures grown in both 50 and 25% N₂ strength (Figure 1).

Catalase activity. This was found not to be significantly different in any of the treatments in contrast to the control (data not shown).

Yield extracts. The FW/DW ratio was not affected by this stress (Table III).

Canophyllol quantification. The levels of osmotic stress applied in this work did not induce a significant change in the canophyllol concentration in contrast with the control callus (data not shown).

H₂O₂ content. Similarly to the nutritional stress, the osmotic stress significantly enhanced H₂O₂ production, especially in the more severe treatment (3% PEG), in contrast to the control (Figure 3).

Catalase activity. A significant increase of this antioxidant activity was observed in the 3% PEG treatment (Figure 4).

Both stresses induced an oxidative stress, confirmed by a significant increment of the H₂O₂ levels in the cells in the more severe treatments. This compound has been proposed as a positive signal molecule in the production of secondary metabolites (SM), as reported for the beta-thujaplicin production in Cupressus lusitanica (Zhao et al., 2005).

Even though the H₂O₂ content was higher under osmotic stress (Figure 3) than under nutritional stress (Figure 2), canophyllol was accumulated only under nutritional stress. In the chromatographic analysis no qualitative differences were observed in the hexane extracts regarding the presence of compounds between the two different stresses applied. Only quantitative differences were observed. This response agrees with other reports where reduced levels of total N₂ enhaced SM production, such as capsaicin in Capsicum frutescens, anthraquinones in Morinda citrifolia and anthocyanins in Vitis species (Rao and Ravishankar, 2002).

N₂ deficiency affects callus growth and it is during the low growth rate phase of the cellular cultures that the highest SM production has been reported (Kakegawa et al., 1995). Under nutritional stress the FW/DW ratio increased in the 25% N₂ treatment, which yielded the highest canophyllol accumulation. This treatment led to a low growth rate, possibly due to the stress reflected in a significant increase of H₂O₂, which might be a signal for the canophyllol production.

Stress affects growth rate (Chapin, 1991). However, in this work neither the FW/DW rate nor the canophyllol production were affected under osmotic stress, even though the accumulation of H₂O₂ suggested a stress condition of the culture. Perhaps different stress conditions could enhance the canophyllol production, since it has been suggested that stress duration and intensity are important for eliciting SM accumulation (Kim et al., 2004). The results indicated that the utilization of the densitometry for quantification of canophyllol is a good choice for a simple analysis. For the quantitative analyses of active compounds in a crude plant extract, the utilization of specific derivatization reagents shows advantages (visual comparison, the detection by dipping reagents enables specific colour reactions and the consumption of organic solvents, as well as the analysis time, are lower) over the high performance liquid chromatography (HPLC). The use of the Liebermann-Burchard reagent in the scanning was quite reliable. Spraying the plates each time with a fixed volume of reagent is fundamental for obtaining reproducible results.

Different catalase activity was observed in the different stress treatments. Under nutritional stress no significant differences were observed, whereas with osmotic stress a correlation between catalase activity and H₂O₂ content was observed. Catalase activity as well as the H₂O₂ content increased significantly in the 3% PEG treatment. Perhaps both osmotic and nutritional stresses act through different signaling pathways, affecting enzymatic activity and the signal cascade responses leading to SM production. Many types of stimuli play a role in gene regulation and trigger activation of the pathways leading to in situ production of a number of SMs (Sudha and Ravishankar, 2002). This work demonstrates that the responses of canophyllol production and catalase activity in H. excelsa callus are differentially signaled by the different stress factors.

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