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Brazilian Journal of Biology

Print version ISSN 1519-6984

Braz. J. Biol. vol.73 no.3 São Carlos Aug. 2013

http://dx.doi.org/10.1590/S1519-69842013000300020 

Biology

Ethylene synthesis inhibition effects on oxidative stress and in vitro conservation of Lippia filifolia (Verbenaceae)

Efeitos da Inibição da síntese do etileno no estresse oxidativo e na conservação in vitro de Lippia filifolia (Verbenaceae)

MR. Pimentaa 

C. Ribeirob 

CQG. Soaresa 

GC. Mendesa 

VF. Bragaa 

LB. Reisc 

WC. Otonie 

CF. Resendea 

LF. Viccinid 

PHP. Peixotoa  * 

aDepartamento de Botânica, Instituto de Ciências Biológicas – ICB, Universidade Federal de Juiz de Fora – UFJF, CEP 36036-900, Juiz de Fora, MG, Brazil

bDepartamento de Biologia Geral, Universidade Federal de Viçosa – UFV, CEP 36570-900, Viçosa, MG, Brazil

cInstituto de Ciências Biológicas e da Saúde, Universidade Federal de Viçosa – UFV, CEP 38810-000, Rio Paranaíba, MG, Brazil

dDepartamento de Biologia, Instituto de Ciências Biológicas – ICB, Universidade Federal de Juiz de Fora – UFJF, CEP 36036-900, Juiz de Fora, MG, Brazil

eDepartamento de Biologia Vegetal, Universidade Federal de Viçosa – UFV, CEP 36570-900, Viçosa, MG, Brazil


ABSTRACT

This study aimed to investigate the effects of ethylene biosynthesis inhibitors on oxidative metabolisms and the in vitro conservation of Lippia filifolia, using the lipid peroxidation index (TBARS), antioxidative enzymes and pigments as biomarkers. We found that EDTA, sodium thiosulfate (STS) and especially Co had protective effects on oxidative stress in tissues cultured in vitro, resulting in a delay of the senescence and the reduction of subcultures frequency, con-tributing to the germplasm conservation of this species.

Key words: ethylene inhibitors; TBARS; ROS; micropropagation; germplasm conservation

RESUMO

O objetivo deste estudo foi investigar os efeitos de inibidores da biossíntese do etileno no metabolismo oxidativo e na conservação in vitro de Lippia filifolia. Para isso, foram avaliados o índice de peroxidação lipídica (TBARS), a atividade de enzimas antioxidativas e o conteúdo de pigmentos fotossintéticos e de antocianinas. Os resultados evidenciaram que o EDTA, o tiossulfato de sódio (STS) e, especialmente, o Co apresentaram ação protetora sobre o estresse oxidativo nos tecidos, o que resultou em atraso no início da senescência das culturas e na redução da frequência dos subcultivos, contribuindo para a conservação do germoplasma dessa espécie.

Palavras-Chave: inibidores de etileno; TBARS; EROs; micropropagação; conservação de germoplasma

1. Introduction

One of the main diversity centres of the genus Lippia is located in Cadeia do Espinhaço, Minas Gerais state, Brazil. In this environment, some endemic species have been threatened by severe destruction, particularly caused by mining activities (Giulietti et al., 1987). Micropropagation offers an alternative method to con-ventional vegetative propagation and germplasm conser-vation, especially for endemic and endangered species. Plant tissue culture involves manipulations, and the explants respond to environmental, physiological and metabolic changes. As a consequence, there is an increase in the demand for antioxidant protection to compensate for the pro-oxidative changes that occur in parallel to metabolic and developmental transitions (Benson, 2000).

In vitro propagation has limitations, especially when the accumulation of ethylene in culture vessels is severe and/or the genotypes exhibit sensibility to this phyto-hormone (Ievinsh et al., 2000). The ethylene biosynthesis is relatively simple and different substances interfere in specific points of the pathway (Wang et al., 2002). Cobalt (Co) ions, chelating agents (EDTA, EGTA) and salicylic acid (SA) prevent or reduce aminocyclopropane carboxylic acid (ACC) conversion to ethylene (Wang et al., 2002). Silver nitrate (AgNO3) and silver thiosulfate (STS) also affect ethylene activity, reducing sensitivity and also the negative effects on plant tissues (Wang et al., 2002).

The ethylene effects on in vitro morphogenesis are not fully understood, but the role of this hormone in senescence has been widely reported (Roustan et al., 1989; Kumar et al., 1998; Wang et al., 2002). Senescence is a natural phenomenon related both to ethylene and oxidative stress (Wang et al., 2002). The reactive oxygen species (ROS) are toxic molecules naturally produced as a result of aerobic metabolism, and therefore they should be rapid and efficiently scavenging, which occurs due to different antioxidant systems (Scandalios, 1993; Anderson et al., 1995; Mittler, 2002).

The aim of this work was to investigate the effects of ethylene biosynthesis inhibitors on oxidative stress and the in vitro conservation of Lippia filifolia, using the lipid peroxidation index (TBARS), antioxidative enzymes activity and the accumulation of pigments as biomarkers.

2. Materials and Methods

2.1. Tissue culture

Apical shoots (average 10 mm) of Lippia filifolia Mart. and Schauer ex Schauer previously established in MS media (Murashige and Skoog, 1962) without growth regulators were used. The explants were inoculated on MS medium supplemented with 10 nM of α-naphtha-leneacetic acid (NAA). Additionally, different substance inhibitors of synthesis or ethylene action were added: AgNO3 (6, 12 or 18 mM), SA (80, 160 or 240 mM), Co (CoCl2.6H2O - 20, 40 or 60 mM), EDTA (45, 90 or 135 mM) or STS (6, 12 or 18 mM). The pH was adjusted to 5.7 ± 0.1 before autoclaving. Fifteen millilitres of culture medium were added into test tubes (2.5 x 15 cm). The tubes were capped with polypropylene closures and further sealed with a 9 µm PVC film. Cultures were maintained for 60 days in a growth chamber at 16 h photoperiod, 26/20 °C (day/night temperature), and an irradiance of around 36 µmol m−2 s−1.

2.2. TBARS evaluation

Thiobarbituric acid reactive substances (TBARS) in tissues were evaluated as described by Cakmak and Horst (1991). Tissues were homogenised in 4 mL of 1% (m/v) trichloroacetic acid (TCA). After purification, 1 mL of supernatant was added to 3 mL of 0.5% (m/v) thio-barbituric acid (TBA) in 20% (m/v) TCA. The test tubes were capped and incubated in a water bath at 95 °C, for 2 h. The reaction was stopped by cooling the test tubes in an ice bath. After clarification by centrifugation, supernatant absorbance was evaluated at 532 and 660 nm. The MDA-TBA (TBARS) complex formation was estimated using a molar extinction coefficient of 155 mM−1 cm−1 (Heath and Packer, 1968).

2.3. Enzymatic analysis

Enzymatic extracts to determine activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7) and polyphe-noloxidase (PPO, EC 1.10.3.2, EC 1.10.3.1, EC 1.14.18.1) were obtained by tissue homogenisation in 0.1 M potassium phosphate buffer, pH 6.8 with 0.1 mM EDTA. The homogenates were filtered and centrifuged, and the supernatants were used to perform the enzyme assays. SOD activity was measured by adding aliquots of the enzymatic extracts to the reaction mixture containing 13 mM L-methionine, 75 mM p-nitroblue tetrazolium (NBT), 100 nM EDTA and 2 µM riboflavin in 50 mM sodium phosphate buffer, pH 7.8 (Del Longo et al., 1993). The enzyme catalysis was carried out in a chamber illuminated by a 15-W fluorescent lamp for 3 min (Gianno-politis and Ries, 1977). Photoreduction of NBT to blue formazan was measured by the increase of absorbance to 560 nm. One unit of SOD is defined as the amount of enzyme necessary to inhibit the NBT photoreduction by 50% (Beauchamp and Fridovich, 1971). POD activity was assayed by adding aliquots of the enzymatic extracts to 5 mL of a reaction mixture containing 25 mM potassium phosphate buffer, pH 6.8, 20 mM pyrogallol and 20 mM H2O2 (Kar and Mishra, 1976). After 1 min, the reaction was stopped by adding 0.5 mL of H2SO4 5% (v/v). Absorbance was evaluated at 420 nm. POD activity was measured by using a molar extinction coefficient of 2.47 mM−1 cm−1 (Chance and Maehley, 1955). PPO activity was measured as described for POD (Kar and Mishra, 1976), except for the exclusion of H2O2 from the incubation media. CAT activity was measured by adding aliquots of enzyme extract to 3 mL of a mixture containing 12.5 mM H2O2 in 50 mM of potassium phosphate buffer, pH 7.0 (Havir and McHale, 1987). The enzyme activity was measured as the absorbance decreased to 240 nm, assuming a molar extinction coefficient of 36 mM−1 cm−1 (Anderson et al., 1995).

2.4. Pigment analysis

For the chlorophylls and carotenoids analysis, samples were extracted from 5.0 mL of acetone 80% (v/v). Chlorophylls a and b, total chlorophyll and carotenoids were determined according to Lichtenthaler (1987). Total anthocyanins were measured following the procedure described by Mancinelli (1990), using 5.0 mL of methanol-HCl (99:1, v/v). All extracts were clarified by centrifugation before spectrophotometric determinations.

2.5. Statistical analysis

The experiment was carried out in a completely randomised design. The ANOVA and the Scott-Knott test were performed with 5% probability, using the SAEG software 9.1.

3. Results and Discussion

3.1. Explants regeneration and senescence

The regeneration rate of the explants was 100%, regardless of the treatment. Explants which were maintained in a culture medium supplemented with EDTA or STS and, especially, with Co showed 92-95% of rooted microcuttings. Under these conditions, the senescence rate was 50% slower (150 days) than in explants cultured in medium with SA and AgNO3 (100 days), which was reinforced by a marked chlorosis and faster leaf abscission.

3.2. Lipids peroxidation (TBARS index)

In this study, it was possible to observe significant reductions in the TBARS index at the highest level of SA, in the presence of EDTA and STS, regardless of the concentration, and especially when Co was added to the culture medium (Table 1). On the other hand, lipid peroxidation was stimulated as the AgNO3 increased in the culture media. This stimuli is attributed to increased levels of ethylene in response to silver ions (Molassiotis et al., 2005), which may be caused by its toxicity or by the reduction of tissue sensitivity to ethylene. This fact can be metabolically interpreted as a deficiency of this hormone causing an increase in the biosynthesis (Theologis, 1992).

Table 1 TBAR [mmol g−1 (f.m.)] contents, SOD [10−3U g−1 (f.m.)], POD [mmol g−1 (f.m.) min−1], PPO [mmol g−1 (f.m.) min−1] and CAT [mmol g−1 (f.m.) min−1] activities, chlorophyll a+b [mg g−1 (f.m.)], carotenoids [mg g−1 (f.m.)] and anthocyanin [A530 g−1 (f.m.)] contents and chlorophyll a/b ratio in in vitro cultured Lippia filifolia. Means followed by the same letter (for each substance) are not significantly different by the Scott-Knott test at 5% probability. [(+) higher than the control; (-) smaller than the control; (=) equal to the control)]. (n = 5 replications) 

Treatment TBAR SOD POD PPO CAT Chl a + b Carot. Chla / Chlb Anthoc.
Control 0.607 0.499 0.157 0.030 1.436 0.573 0.185 2.981 3.740
AgNO3 6 µM 1.003 c+ 0.721 b= 0.209 b= 0.071 c+ 1.792 b= 0.303 a 0.212 a= 0.302 b 0.995 b
AgNO3 12 µM 1.906 b+ 1.459 a+ 0.534 a+ 0.205 a+ 4.514 a+ 0.230 b 0.153 b- 0.352 b 1.800 a
AgNO3 18 µM 2.931 a+ 0.573 b= 0.554 a+ 0.159 b+ 3.720 a+ 0.207 b 0.127 c 0.580 a 1.656 a
SA 80 µM 1.005 a+ 0.505 a= 0.246 c+ 0.087 c+ 4.836 a+ 0.235 b 0.216 a+ 0.285 b 4.020 a=
SA 160 µM 0.672 a= 0.793 a+ 0.324 a+ 0.127 a+ 3.504 b+ 0.267 a 0.220 a+ 0.427a 2.933 b
SA 240 µM 0.476 b 0.695 a= 0.272 b+ 0.108 b+ 3.618 b+ 0.283 a 0.209 a+ 0.515a 3.454 b
Co 20 µM 0.116 a 0.266 b 0.082 b 0.020 c 1.091 b 0.138 a 0.105 a 1.625 b 2.025 a
Co 40 µM 0.070 a 0.403 a 0.163 a= 0.045 a+ 1.432 a= 0.092 b 0.055 b 1.377 b 2.480 a
Co 60 µM 0.072 a 0.241 b 0.128 a= 0.034 b= 1.204 b 0.035 c 0.033 b 3.121 a= 0.529 b
EDTA 45 µM 0.313 a 0.519 a= 0.237 a+ 0.055 a+ 2.410 a+ 0.082 b 0.052 a 3.473 a+ 2.535 b
EDTA 90 µM 0.165 b 0.317 b 0.200 b+ 0.041 b+ 1.672 b+ 0.095 a 0.039 b 1.555 b 5.013 a+
EDTA 135 µM 0.152 b 0.370 a= 0.143 c= 0.037 b= 1.718 b+ 0.085 b 0.035 b 1.939 b 3.929 a=
STS 6 µM 0.286 a 0.973 a+ 0.268 a+ 0.068 a+ 2.300 a+ 0.173 a 0.042 b 1.912 c 2.621 b
STS 12 µM 0.268 a 0.560 b= 0.232 b+ 0.067 a+ 2.522 a+ 0.200 a 0.048 b 3.383 b= 2.653 b
STS 18 µM 0.216 a 0.203 c 0.156 c= 0.041 b+ 1.206 b 0.186 a 0.084 a 4.051 a+ 5.079 a=

3.3. Enzymes activities

Several enzymes are related to the protection of protoplasm and cell integrity against oxidative stresses (Anderson et al., 1995). We observed that SOD activity was reduced when Co and EDTA were added, regardless of their concentration, as well as STS at the highest level (Table 1). SOD activity is usually associated with POD or CAT activities, enzymes that use H2O2 produced in the reaction performed by SODs (Cakmak and Horst, 1991; Arora et al., 2002; Mittler, 2002). The combined action of SODs, CATs and PODs, associated to low-molecular-weight antioxidant substances, can effectively eliminate, scavenge and/or immobilise toxic oxygen species (Scandalios, 1993; Siegel, 1993).

POD activities were higher when comparing the control, especially in treatments with AgNO3 and SA (Table 1). In contrast, the enzyme activity was lower in the presence of Co. The PODs action on electron donor molecules, using H2O2 as substrate, has an important contribution on ROS detoxification (Gaspar et al., 1985).

PODs can also degrade indole-3-acetic acid (IAA) and the aminocyclopropane carboxylic acid (ACC), which are important intermediaries in the ethylene biosynthesis pathway (Wang et al., 2002). ACC oxidases are PODs associated with membranes that can regulate ethylene production (Gaspar et al., 1985; Wang et al., 2002). Gaspar et al. (1985) suggested that ethylene also regulates the activities of phenylalanine ammonia-lyases (PALs) and acid PODs, enzymes related to lignification. Sakamoto et al. (2008) observed that H2O2 is involved in abscission signalling, and also that ethephon-induced abscission is suppressed by inhibitors of H2O2 production, suggesting that H2O2 acts downstream from ethylene in in vitro senescence and abscission signaling. Therefore, ethylene could induce lipid peroxidation as a result of the increase in H2O2 production. Both PODs and PPOs catalysed the oxidation of phenolic substances and are also involved in the phenylpropanoid biosynthesis pathway (Siegel, 1993). A similar result that was observed for PODs was also observed for PPOs (Table 1).

CAT activity was higher than in the control for treatments with AgNO3 and SA (Table 1). The CAT activity observed here supports the hypothesis that AgNO3 and SA are harmful to explant quality, which was reinforced by the highest lipid peroxidation (TBARS) observed when those substances were present (Table 1). On the other hand, reduction in CAT activity was observed when Co was added to the medium (Table 1). In general, under stressed conditions an increase was observed in POD activity, while CAT activity was reduced (Cakmak and Horst, 1991; Siegel, 1993; Agarwal et al., 2005; Molassiotis et al., 2005). This result indicates that H2O2 is most highly consumed in oxidative events than removed from the metabolism. We verified an increase in CAT activity (Table 1) especially when more intensive lipid peroxidation was observed (Table 1), reinforcing the hypothesis that AgNO3 and SA increase H2O2 production (Mutlu et al., 2009). It is well known that SA may affect the hypersensitivity response as a reaction to the increase of H2O2 production (Agarwal et al., 2005), which, in contrast, can be very harmful to in vitro culture systems. The observed SOD, POD, PPO and CAT activities (Table 1) suggest that EDTA, STS and mainly Co contribute to the reduction in ethylene production and lipid peroxidation in in vitro L. filifolia explants, delaying the onset of senescence in their tissues (Meratan et al., 2009; Vatankhah et al., 2010).

3.4. Pigments contents

The photosynthetic pigment levels in the tissues as well as the relative proportions of them have been used as biomarkers to evaluate different kinds of stresses (Agarwal et al., 2005). The inclusion of Co, EDTA and STS to the culture medium reduced the total content of chlorophyll and carotenoids (Table 1). However, the chlorophyll a/b ratio was less affected in response to these substances than to AgNO3 and SA, suggesting an adjustment in stress conditions in the photosynthetic apparatus. Jeon et al. (2006) pointed out that when the chlorophyll a/b ratio is kept at levels close to the normal, damages to antenna pigments are generally reduced, suggesting a higher efficiency in the pigments readjustment.

The total anthocyanin content within the tissues varied considerably in response to different treatments (Table 1). The anthocyanin levels remained close to the control in the presence of SA, EDTA and STS, but were reduced when AgNO3 and Co were added to medium. Inhibitory effects of Co on anthocyanin biosynthesis were already reported (Dube et al., 1993). EDTA and etephon, exogenous sources of ethylene, were also associated to flavonoids biosynthesis (Elliot, 1977; Dube et al., 1993). Nagata et al. (2003) suggested that anthocyanin and others flavonoids contribute to the ROS removal. In contrast, Vanderauwera et al. (2005) observed a negative impact of H2O2 on DNA clusters transcription related to anthocyanin biosynthesis. Nevertheless, we did not observe any direct relationship here between TBARS and anthocyanin accumulation (Table 1).

Senescence is a natural phenomenon resulting from both the increase of oxidative metabolism and the decrease of antioxidant enzyme activity, and ethylene is particularly associated to these processes (Kumar et al., 1998; Benson, 2000; Ievinsh et al., 2000; Mittler, 2002; Arora et al., 2002; Wang et al., 2002; Meratan et al., 2009). Our study showed that EDTA, STS and mainly Co have protective effects against lipid peroxidation on in vitro L. filifolia explants. These protective effects occur, apparently, due to the inhibition effects of those substances on ethylene biosynthesis. The incorporation of EDTA, STS and, mainly Co to the culture medium can effectively contribute to both increasing the efficiency of L. filifolia micropropagation and reducing the frequency of subculturing, which have positive effects on in vitro germplasm conservation of this species.

Acknowledgements

The authors wish to acknowledge the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FA-PEMIG), Financiadora de Estudos e Projetos (FINEP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support.

References

AGARWAL S., SAIRAM RK., SRIVASTAVA G. and MEENA RC. 2005. Changes in antioxidant enzymes activity and oxidative stress by abscisic and salicylic acid in wheat genotypes. Biologia Plantarum, vol. 49, no. 4, p. 451-550. [ Links ]

ANDERSON MD., PRASAD TK. and STEWART CR. 1995. Changes in isozyme profiles of catalase, peroxidase, and glutathione reductase during acclimation to chilling in mesocotylus of maize seedlings. Plant Physiology, vol. 109, no. 4, p. 1247-1257. [ Links ]

ARORA A., SAIRAM RK. and SRIVASTAVA GC. 2002. Oxidative stress and antioxidative system in plants. Current Science, vol. 82, no. 10, p. 1227-1238. [ Links ]

BEAUCHAMP C. and FRIDOVICH I. 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Analytical Biochemistry, vol. 44, no. 1, p. 276-287. [ Links ]

BENSON EE. 2000. Special Symposium - In vitro plant recalcitrance. Do free radicals have a role in plant tissue culture recalcitrance? In Vitro Cellular and Developmental Biology - Plant, vol. 36, no. 3, p. 163-170. [ Links ]

CAKMAK I. and HORST JH. 1991. Effects of aluminum on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max). Physiologia Plantarum, vol. 83, no. 3, p. 463-468. [ Links ]

CHANCE B. and MAEHLEY AC. 1955. Assay of catalases and peroxidases. Methods in Enzymology, vol. 2, p. 764-775. [ Links ]

DEL LONGO OT., GONZÁLEZ CA., PASTORI GM. and TRIPPI VS. 1993. Antioxidant defenses under hyper-oxygenic and hyperosmotic conditions in leaves of two lines of maize with differential sensitivity to drought. Plant and Cell Physiology, vol. 34, no. 7, p. 1023-1028. [ Links ]

DUBE A., BHARTI S. and LALORAYA MM. 1993. Inhibition of anthocyanin synthesis in the first internode of Sorghum bicolor by cobaltous ions: the site of action of cobalt. Physiologia Plantarum, vol. 87, no. 4, p. 441-446. [ Links ]

ELLIOT DC. 1977. Induction by EDTA of anthocyanin synthesis in Spiroldela oligorriza. Australian Journal of Plant Physiology, vol. 4, no. 1, p. 39-49. [ Links ]

GASPAR T., PENEL C., CASTILLO FJ. and GREPPIN H. 1985. A two-step control of basic and acidic peroxidases and its significance for growth and development. Physiologia Plantarum, vol. 64, no. 3, p. 418-423. [ Links ]

GIANNOPOLITIS CN. and RIES SK. 1977. Superoxide dismutases. I. occurrence in higher plants. Plant Physiology, vol. 59, no. 2, p. 309-314. [ Links ]

GIULIETTI AM., MENEZES NL., PIRANI JR., MEGURO M. and WANDERLEY MGL. 1987. Flora da Serra do Cipó, Minas Gerais: caracterização e lista das espécies. Boletim de Botânica da Universidade de São Paulo, vol. 9, p. 1-151. [ Links ]

HAVIR EA. and McHALE NA. 1987. Biochemical and devel-opmental characterization of multiple forms of catalase in tobacco leaves. Plant Physiology, vol. 84, no. 2, p. 450-455. [ Links ]

HEATH RL. and PACKER L. 1968. Photoperoxidation in isolated chloroplast. I. Kinetics and stoichometry of fatty acid peroxidation. Archives of Biochemistry and Biophysics, vol. 125, no. 1, p. 189-198. [ Links ]

IEVINSH G., KRUZMANE D., RUSITE E., ARENTE G. and GERTNERE D. 2000. Modulation of Solanum tuberosum L. morphogenesis and antioxidative status in a stem explant culture by limitation of gas exchange: putative effects of ethylene. Journal of Plant Physiology, vol. 156, no. 5-6, p. 717-723. [ Links ]

JEON MW., ALI MB., HAHN EJ. and PAEK KY. 2006. Photo-synthetic pigments, morphology and leaf gas exchange during ex vitro acclimatization of micropropagated CAM Doritaenopsis plantlets under relative humidity and air temperature. Environmental and Experimental Botany, vol. 55, no. 1-2, p. 183-194. [ Links ]

KAR M. and MISHRA D. 1976. Catalase, peroxidase, and polyphenoloxidase activities during rice leaf senescence. Plant Physiology, vol. 57, no. 2, p. 315-319. [ Links ]

KUMAR PP., LAKSHMANAN P. and THORPE TA. 1998. Review: Regulation of morphogenesis in plant tissue culture by ethylene. In Vitro Cellular and Developmental Biology – Plant, vol. 34, no. 2, p. 94-103. [ Links ]

LICHTENTHALER HK. 1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods in Enzymology, vol. 148, p. 350-382. [ Links ]

MANCINELLI AL. 1990. Interaction between light quality and light quantity in photoregulations of anthocyanin production. Plant Physiology, vol. 92, no. 4, p. 1191-1195. [ Links ]

MERATAN AA., GHAFFARI SM. and NIKNAM V. 2009. In vitro organogenesis and antioxidant enzymes activity in Acanthophyllum sordidum. Biologia Plantarum, vol. 53, no. 1, p. 5-10. [ Links ]

MITTLER R. 2002. Oxidative stress antioxidants and stress tolerance. Trends in Plant Science, vol. 7, no. 9, p. 405-410. [ Links ]

MOLASSIOTIS A., DIAMANTIDIS G., THERIOS I. and DIMASSI K. 2005. Effects of salicylic acid on ethylene induction and antioxidant activity in peach rootstock regenerants. Biologia Plantarum, vol. 49, no. 4, p. 609-612. [ Links ]

MURASHIGE T. and SKOOG F. 1962. A revised media for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, vol. 15, no. 3, p. 473-497. [ Links ]

MUTLU S., ATICI Ö. and NALBANTOGLU B. 2009. Effects of salicylic acid and salinity on apoplastic antioxidant enzymes in two wheat cultivars differing in salt tolerance. Biologia Plantarum, vol. 53, no. 2, p. 334-338. [ Links ]

NAGATA T., TODORIKI S., MASUMIZU T., SUDA I., FURUTA S., DU Z. and KIKUCHI S. 2003. Levels of active oxygen species are controlled by ascorbic acid and anthocyanin in Arabidopsis. Journal of Agricultural and Food Chemistry, vol. 51, no. 10, p. 2992-2999. [ Links ]

ROUSTAN JP., LATCHE A. and FALLOT J. 1989. Stimulation of Daucus carota somatic embryogenesis by inhibitors of ethylene synthesis: cobalt and nickel. Plant Cell Reports, vol. 8, no. 3, p. 182-185. [ Links ]

SAKAMOTO M., MUNEMURA I., TOMITA R. and KO-BAYASHI K. 2008. Involvement of hydrogen peroxide in leaf abscission signaling, revealed by analysis with an in vitro abscission system in Capsicum plants. The Plant Journal, vol. 56, no. 1, p. 13-27. [ Links ]

SCANDALIOS JG. 1993. Oxygen stress and superoxide dismutases. Plant Physiology, vol. 101, no. 1, p. 7-12. [ Links ]

SIEGEL BZ. 1993. Plant peroxidase - an organismic perspective. Plant Growth Regulation, vol. 12, no. 3, p. 303-312. [ Links ]

THEOLOGIS A. 1992. One rotten apple spoils the whole bushel: the role of ethylene in fruit ripening. Cell, vol. 70, no. 2, p. 181-184. [ Links ]

VATANKHAH E., NIKNAM V. and EBRAHIMZADEH H. 2010. Activity of antioxidant enzyme during in vitro organogenesis in Crocus sativus. Biologia Plantarum, vol. 54, n. 3, p. 509-514. [ Links ]

VANDERAUWERA S., ZIMMERMANN P., ROMBAUTS S., VANDENABEELE S., LANGEBARTELS C., GRUISSEM W., INZÉ D. and BREUSEGEM FV. 2005. Genome-wide analysis of hydrogen peroxide-regulated gene expression in Arabidopsis reveals a high light-induced transcriptional cluster involved in anthocyanin biosynthesis. Plant Physiology, vol. 139, no. 2, p. 806-821. [ Links ]

WANG KLC.; LI H. and ECKER JR. 2002. Ethylene biosynthesis and signaling networks. The Plant Cell, vol. 14, no. suppl. 1, p. S131-S151. [ Links ]

Received: February 9, 2012; Accepted: August 14, 2012

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