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Glutathione metabolism and antioxidant responses during Eleutherococcus senticosus somatic embryo development in a bioreactor. Authors; Authors and ...

Plant Cell Tiss Organ Cult (2007) 89:121–129 DOI 10.1007/s11240-007-9220-9

ORIGINAL PAPER

Glutathione metabolism and antioxidant responses during Eleutherococcus senticosus somatic embryo development in a bioreactor A. M. Shohael Æ M. B. Ali Æ E. J. Hahn Æ K. Y. Paek

Received: 20 March 2006 / Accepted: 1 March 2007 / Published online: 13 June 2007  Springer Science+Business Media B.V. 2007

Abstract Compared to non-embryogenic callus, proembryonic mass, globular, and heart-shaped embryos of Eleutherococcus senticosus had higher levels of endogenous reduced glutathione (GSH). GSH content declined during the course of the embryo development (torpedo and cotyledon). Similarly, glutathione reductase that is involved in the recycling of GSH providing a constant intracellular level of GSH was also higher in globular and heartshaped embryos. The transient increase in GSH contents also correlated with the changes in measured c-glutamylcysteine synthetase activity over the same period. The endogenous levels of oxidized glutathione showed similar trend during development of the somatic embryos, whereas it declined in maturing somatic embryos. A pronounced increase in glutathione-S-transferase, glutathione peroxidase, catalase, and guaiacol peroxidase activity was observed during somatic embryo maturation. Ascorbate-glutathione

A. M. Shohael  E. J. Hahn  K. Y. Paek Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju, Korea M. B. Ali (&) Metabolic Regulation Laboratory, Food Biotechnology Division, National Food Research Institute, Tsukuba, Ibaraki, Kannondai 305-8642, Japan e-mail: [email protected]

cycle enzymes (ascorbate peroxidase; dehydroascorbate reductase and monodehydroascorbate reductase) activities also induced indicated that antioxidant enzymes played an important role during embryo development. These results suggested that the coordinated up-regulations of the antioxidant enzymes and glutathione redox system provide protection during somatic embryo development in E. senticosus. Antioxidant responses through alterations of the glutathione redox systems, have been described in the present studies have a significant role in somatic embryo development. Keywords Bioreactor  Eleutherococcus senticosus  Enzymes  c-glutamylcysteine synthetase  Glutathione  Somatic embryogenesis Abbreviations APX Ascorbate peroxidase (EC 1.11.1.11) CAT Catalase (EC 1.11.1.6) 2, 4 D 2, 4-dichlorophenoxyaceticacid DHAR Dehydroascorbate reductase (EC 1.8.5.1) DHA Dehydroascorbate c-GCS Glutamylcysteine synthetase GST Glutathione-S-transferase (EC 2.5.1.18) GPx Glutathione peroxidase (EC 1.11.1.12) GR Glutathione reductase (EC 1.6.4.2) G-POD Guaiacol peroxidase (EC 1.11.1.7)

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122

MDHAR GSH GSSG

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Monodehydroascorbate reductase (EC 1.6.5.4) Reduced glutathione Oxidized glutathione

Introduction Glutathione is the most abundant low-molecular weight thiol in most biological systems and functions primarily as a reductant to maintain-SH groups in a reduced state. Reduced glutathione (GSH) and its oxidized form (GSSG) have been shown to participate in a variety of diverse physiological events in both animals and plants (Gardiner et al. 1998; Smith et al. 2000). The GSH : GSSG ratio has been shown to coordinate with gene expression and specific events during plant growth and development (Wingate et al. 1988; Nocter and Foyer 1998) with a high GSH : GSSG ratio often correlating with rapid cell growth (Smith et al. 2000; de Pinto et al. 1999; Tommasi et al. 2001). Experiments conducted with Arabidopsis roots demonstrated that the endogenous levels of GSH were high in rapidly dividing cells, and low in quiescent cells (Sanchez-Fernandez et al. 1997). Redox state plays an important role during somatic embryo development and in higher plants a reduced culture environment is required during the initial embryogenic events, characterized by active cell proliferation (Belmonte et al. 2005). On the other hand, oxidized state is another relevant factor, which is also required for the completion of embryogenesis (De Gara et al. 2003; Yeung and Belmonte 2004). It has been observed that manipulations of the redox state in white spruce somatic embryogenesis culture medium, affected by applications of either reduced (GSH) or oxidized (GSSG) glutathione, have profound effects on embryo yield and quality. Glutathione limits the lifespan of reactive oxygen species (ROS), lipid peroxide content, alters calcium signaling (Gomez et al. 2004) and also participates in the calcium-dependent pathways of ROS-signal transduction (Rentel and Knight 2004). The characteristics and functional importance of thiolic groups ROS metabolism and associated redox-signaling cascades have recently been discussed (Foyer and Noctor 2005). Plant also developed a highly efficient ascorbate-glutathione cycle enzymes, comprising

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ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), providing protection against different stress factors, which has become an interesting research subject in recent years (Mittler et al. 2004). The main non-enzymatic antioxidant molecules are ascorbate and glutathione, which are also integrated in the cycle mentioned above, and a-tocopherol, b-carotene and flavonoids (Noctor and Foyer 1998; Halliwell and Gutteridge 2000). The ascorbate-glutathione cycle is an important antioxidant protection system against H2O2 generated in different cell compartments. Its occurrence has been reported in chloroplasts, mitochondria, peroxisomes and cytoplasm (Jime´nez et al. 1997; Noctor and Foyer 1998; Asada 2000). Catalase (CAT), guaiacol peroxidase (G-POD) together with glutathione peroxidase (GPx) and glutathione-Stranseferase (GST) can also participate in improving the defense systems. Eleutherococcus senticosus is a small, woody medicinal plant belongs to the family Araliaceae native to Northeastern Asia. Many species of Eleutherococcus are considered as an endangered species because of excessive commercial harvest from their natural habitat. Eleutherococcus species contain eleutherosides as main compound including eleutheroside A, B, C, D, E, F, G and complex polysaccharides as an active ingredients from roots and leaves. The plants are used as a tonic and a sedative as well as to treat rheumatism and diabetes (Davydov and Krikorian 2000). Conventional propagation via seeds is very difficult because of low-germination rate of seeds and delayed rooting of seedlings curtails its propagation through seeds. In vitro propagation through somatic embryogenesis has been reported in several plants (Kumar et al. 2002; Martin 2003). Somatic embryogenesis offers an excellent experimental system to study the physiological and biochemical aspects of embryo development. To our knowledge little is known about the glutathione and other enzymes metabolism during somatic embryogenesis of E. senticosus in bioreactor. During optimal conditions, the balance between ROS formation and consumption is tightly controlled by an array of antioxidant enzymes and redox metabolites (Noctor and Foyer 1998). Exposure of plants to many environmental stress factors leads to oxidative stress characterized by the generation of ROS in plant

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tissues (Mittler et al. 2004). Various reports have shown that ROS, especially H2O2 acts as a signaling molecule in response to various abiotic stresses (Desikan et al. 2001; Neill et al. 2002). To gain a better understanding on the role of glutathione and antioxidant on somatic embryogenesis, the main objective of this work was to monitor the changes in glutathione levels and other enzymes in relation to embryo morphology during development in E. senticosus.

Materials and methods Plant material and in vitro culture condition In vitro seedlings of E. senticosus were maintained on solid MS medium (Murashige and Skoog 1962) without plant growth regulators. Young leaves (2 cm in length) were collected from subcultured plants every 3 weeks. Leaves were cut into 5 · 5 mm pieces and were placed on semisolid MS basal medium supplemented with 1 mg l1 2,4-dichlorophenoxyaceticacid (2,4-D), with 3% sucrose and 0.2% gelrite for callus induction. The medium pH was adjusted to 5.8 prior to addition of gelrite and sterilized at 1218C for 15 min and distributed into 15 · 140 mm Petri dishes (15 ml of medium). Cultures were maintained in darkness at 258C and evaluated for somatic embryogenesis after 12 weeks. For proliferation of embryogenic cultures (pro embryonic mass), friable embryogenic calluses were cultured on the same medium as described above and subcultured every 2 weeks. Compact and yellowish brown callus which failed to proliferate in the fresh medium were considered as non-embryogenic callus.

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transferred to 100 ml MS liquid medium without 2, 4-D in 300 ml Erlenmeyer flasks. The cultures were incubated at 100 rpm on a gyratory shaker and globular embryos were observed after 28–35 days of culture. The culture room temperature was maintained at 258C in darkness. Bioreactor culture Ten grams of embryos were collected and transferred to a 3-l balloon type bubble bioreactor (Paek et al. 2005) containing 2-l MS medium supplemented with 3% sucrose. The volume of input air in bioreactor was adjusted to 0.1 vvm (air volume/culture volume, min). The air temperature in bioreactor was controlled at 22 ± 18C using an air control system. Complete development of the different embryo stages was achieved after 28 days. At the end of the development phase, the content of each bioreactor was passed through different stainless steel sieves to separate different stages of embryos ( > 800 mm = cotyledonary; 600 mm = torpedo; 420 mm = heart; < 420 mm = globular). Measurements of GSH and GSSG For glutathione, 0.5 g embryos from different development stages and non-embryogenic callus were ground in liquid nitrogen and homogenized in 1 ml 6.67% (w/v) sulfosalicylic acid. Extracts were centrifuged at 20,000 · g for 15 min at 48C. Reduced glutathione and oxidized glutathione was determined following the method of Griffith (1980). The total glutathione contents were calculated from a standard curve of GSH. GSH was determined by subtracting GSSG, as GSH equivalents, from the total glutathione content.

Embryogenic cell suspension culture Determination of antioxidant enzymes activity Embryogenic cells of E. senticosus were transferred to MS liquid medium supplemented with 1 mg l1 2, 4-D. The suspension cultures were subcultured every 15 days. To induce somatic embryos, 2-week-old embryogenic cell clumps were filtered through a 120–212 mm stainless steel sieve to remove the larger clumps in sterile condition. The suspension was allowed to settle for 5 min, for easier removal of the used medium. About 500 mg of cell clumps was

For determination of antioxidant enzyme activities, 0.5 g of different developmental stages of somatic embryo or non-embryogenic callus was homogenized in 1.5 ml of respective extraction buffer in a prechilled mortar and pestle by liquid nitrogen. The homogenate was filtered through four layers of cheesecloth and centrifuged at 19,000 · g for 20 min at 48C. The supernatant was centrifuged

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again at 19,000 · g for 20 min at 48C for determination of antioxidant enzyme activities. The preparation was applied to a column of sephadex G-25, equilibrated with the same buffers and kept in an ice bath until the assays were completed. Protein concentration of the enzyme extract was determined according to Bradford (1976). c-GCS activity (c-glutamylcysteine synthetase) c-glutamylcysteine synthetase activity assay was adapted from the indirect method of Volohonsky et al. (2002), which utilizes the coupled reaction of pyruvate kinase (PK) and lactate dehydrogenase (LDH) to determine the rate of formation of ADP by c-GCS through the oxidation of NADH. Reaction mixture contained 0.1 M Tris–HCl buffer, pH 8, 150 mM KCl, 2 mM EDTA, 20 mM MgCl2, 5 mM ATP, 2 mM phosphoenolpyruvate, 10 mM L-glutamate, 10 mM L-a-aminobutyrate, 0.2 mM NADH, 7 U ml1 PK, and 10 U ml1 LDH. Enzyme activity was evaluated by following the decrease in the absorbance of NADH at 340 nm at 258C. Assay of GR, GST, GPx, CAT, and G-POD activity For determination of GR, GST, GPx, CAT, and G-POD activities, embryos or non-embryogenic callus were homogenized in 100 mM sodium phosphate buffer (pH 7.0) containing 1 mM EDTA under liquid nitrogen. GR activity was assayed by following the reduction of 5, 50 -dithiobis (2-nitro benzoic acid) at 412 nm [extinction coefficient (e), 13.6 mM1 cm1] as described by Smith et al. (1988). The assay mixture (1 ml) contained 100 mM K-phosphate buffer (pH 7.5), 1 mM oxidized glutathione, 0.1 mM NADPH, and 100 ml of enzyme extract. GST activity was determined by measuring the increase in absorbance at 340 nm (e, 9.6 mM1 cm1), incubating reduced glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB) as substrates, according to Droter et al. (1985). The 1 ml reaction mixture contained 100 mM K-phosphate buffer, pH 6.25 and 0.8 mM CDNB. GPx activity was assayed by the oxidation of NADPH at 340 nm (e, 6.22 mM1 cm1) as described by Pagila and Valentine (1967). The reaction mixture constituted of 50 mM K-phosphate buffer, pH 7.0, containing

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1 mM EDTA, 0.24 U GR (Sigma-Aldrich, St. Louis, MO, USA), 10 mM GSH, 0.20 mM NADPH, and 1 mM sodium azide. After addition of enzyme, test tubes were incubated at 378C for 10 min. The reaction was initiated by the addition of 50 ml of 1 mM H2O2. CAT activity was measured by the disappearance of H2O2 following the method of Aebi (1984). G-POD activity was measured by following the change of absorption at 436 nm due to guaiacol oxidation (e, 6.39 mM1 cm1) following Pu¨tter (1974). The activity was assayed for 5 min in a reaction solution composed of 50 mM K-phosphate buffer (pH 7.0), 20.1 mM guaiacol, 12.3 mM H2O2, and required amount of enzyme extract. Assay of APX, MDHAR, and DHAR activity For APX, MDHAR, and DHAR activities, embryos were homogenized in 100 mM sodium phosphate buffer (pH 7.0) containing 5 mM ascorbate, 10% glycerol, 2% PVP, and 1 mM EDTA. APX activity was measured in a 1 ml reaction volume containing 50 mM K-phosphate buffer (pH 7.0), 0.1 mM H2O2, and 0.2 mM ascorbate (Chen and Asada 1989). The activity was recorded as a decrease in absorbance at 290 nm for 3 min (e, 2.8 mM1 cm1). MDHAR activity was assayed by monitoring the change in absorbance at 340 nm due to NADH oxidation (e, 6.2 mM1 cm1) for 4 min in a 1 ml reaction mixture containing 90 mM K-phosphate buffer (pH 7.0), 0.0125% Triton · 100, 0.2 mM NADH, 2.5 mM L-ascorbic acid, 0.25 U ascorbate oxidase and enzyme extract (Hossain et al. 1984). One unit of ascorbate oxidase is defined by the manufacturer (Sigma Chemical Company, Mississauga ON, Canada) as the amount that causes the oxidation of 1 mmol of ascorbate to monodehydroascorbate per minute. DHAR activity was determined by measuring the reduction of dehydroascorbate at 265 nm for 4 min (Doulis et al. 1997). The reaction mixture consisted of 90 mM K-phosphate buffer (pH 7.0), 1 mM EDTA, 5.0 mM GSH, and enzyme extract. The reaction was initiated by the addition of 0.2 mM dehydroascorbate (e, 14 mM1 cm1). Statistics The bioreactor experiment had three replicates and was repeated twice. The data were initially compared

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by analysis of variance and differences between means were detected using the Dancans Multiple Range Tests using SAS program (Version 6.12, SAS Institute Inc., Cary, IL, USA).

Results and discussion Embryo development, glutathione and glutathione dependent enzymes Development of E. senticosus somatic embryos occurs through a precise series of morphogenic events (non-embryogenic callus, proembryonic mass, globular, heart, torpedo, and cotyledonary, Fig. 1), described in detail in Shohael et al. (2005). Pronounced changes in the glutathione pools were observed in different stages of somatic embryos (Table 1). Proembryonic mass, globular, and heartshaped embryos had higher levels of endogenous GSH in comparison to non-embryogenic callus. GSH content declined during the course of the development (torpedo and cotyledon), but non-embryogenic callus had higher levels of GSH and GSSG in comparison to torpedo and cotyledonary stage. Higher amounts of GSH and GSSG in dry embryos compared to germinating seeds were also noted which prevents further germination process (Tommasi et al. 2001). Fahey et al. (1980) also suggested that presence of GSSG in dormant wheat embryos block

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protein synthesis. These reports strongly supports our results that higher amounts of GSH and GSSG in non-embryogenic callus may be due to protein synthesis blockage preventing further development leading to yellowish brown callus. However, this higher level of GHS content is not in agreement with Belmonte et al. (2005) who observed low level of GSH at an early stage of embryo development. GR (Fig. 2a) that is involved in the recycling of GSH providing a constant intracellular level of GSH was also higher in globular and heart shaped embryos (Table 1). The transient increase in GSH contents also correlated with the changes in measured c-GCS activity over the same period (Fig. 2b). The endogenous levels of GSSG showed similar trend during development of the somatic embryos, whereas it declined in maturing somatic embryos. Compared to non-embryogenic callus, the GSSG/GSH ratio increased steadily during early somatic embryos development, reaching maximum values in heart shaped somatic embryos, and declined sharply in torpedo and cotyledon embryos (Table 1). Under normal physiological conditions, the ratio of oxidized glutathione to reduced glutathione is low. Maintenance of this state by regeneration of GSH is catalyzed in many tissues by an NADPH-dependent GR, and the accumulation of GSSG is therefore commonly correlated with oxidative stress (Noctor and Foyer 1998). When biological systems dehydrate, the resulting loss in activity of enzymes such as GR

Fig. 1 Somatic embryogenesis of Eleutherococcus senticosus growing in a bioreactor. Non-embryogenic callus (a). Proembryonic mass (b). Globular embryos (c). Heart stage embryos (d). Torpedo embryos (e), and cotyledon embryos (f)

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Table 1 Reduced glutathione (GSH), oxidized glutathione (GSSG), ratio of GSSG/GSH during embryo development stage in Eleutherococcus senticosus in a bioreactor Development stage NE

PM

GL

H

T

Cot

GSH

0.72ba

0.78a

0.80a

0.84a

0.66c

0.66c

GSSG

0.13b

0.16a

0.18a

0.22a

0.11b

0.09c

GSSG/GSH

0.19b

0.20b

0.22a

0.26a

0.16b

0.12c

a Mean separation within rows by Duncan’s multiple range test at p = 0.05. Glutathione and GGSG contents are presented as nmol g 1 FW except ratios of GSH/GSSG

120 *

**

** *

90 60 30

NE PM GL 30

H

T

25 ** 20

** **

*

*

15 10 5 0 PM

GL

H

T

Development stages

and NADPH generating pathways leads to an increase in the oxidative environment (Noctor and Foyer 1998). Under such conditions, the formation and accumulation of disulfides (GSSG) and mixed disulfides between glutathione and other thiols such as protein (PSSG) can be expected to occur. Several studies have demonstrated that changes in the endogenous glutathione redox ratio, especially during periods of water deficit, are important indicators of water stress. However, their importance during embryogenesis is less resolved (De Gara et al. 2003; Zhang and Kirkham 1996). Our study shows

250

COT

(B)

200

**

150

*

**

**

*

T

COT

100 50 0

COT

(C)

NE

123

-1 -1 γ GCS (µmol min mg protein)

(A)

NE PM GL

GPx (µmol min-1 mg-1 protein)

150

0

GST (µmol min-1 mg-1 protein)

Fig. 2 Activities of GR (a), c-GCS (b), GST (c), and GPX (d) on different stages of somatic embryos in compare with nonembryogenic callus (NE non-embryogenic callus, PM proembryonic mass, GL globular embryos, H heart stage embryos, T torpedo embryos and COT cotyledon stage embryos). Asterisk sign * and ** on each bar indicates means are significantly different from the non-embryogenic callus at P < 0.05 and P < 0.01, respectively. Mean values of all investigated parameters are presented ± SE (n = 3)

GR (µmol min-1 mg-1 protein)

NE non-embryogenic callus, PM proembryonic mass, GL globular embryo, H heart stage embryo, T torpedo embryo, Cot cotyledon embryo

100

H

(D)

80 ** 60 40

**

**

**

20 0 NE PM GL

H

T

COT

Development stages

that in somatic embryos, the endogenous glutathione pool slowly shifts towards the oxidized state as development progresses, reaching the lowest GSSG/ GSH ratio in cotyledonary embryos. It has been reported that a high-GSSG/GSH ratio promotes the synthesis of specific glutathione-conjugated proteins important for seed development (Rhazi et al. 2003). GSTs are multifunctional proteins involved in such diverse intracellular events as primary and secondary metabolism, signaling, conjugating natural plant products, and stress metabolism. Significant increase in GST (Fig. 2c) and GPx (Fig. 2d) activities were

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Ascorbate-glutathione cycle enzymes, catalase, and peroxidase activities The activities of enzymes in the ascorbate-glutathione cycle were affected during somatic embryogenesis (Fig. 3). During embryo development, both MDHAR (Fig. 3b) and DHAR (Fig. 3c) activities progressively increased with the maximum occurring at cotyledonary stage. APX activity was high at the PM and GL stages but decreased during later development when activity was similar to the control (Fig. 3a). MDHAR and DHAR generate reduced ascorbate preventing the accumulation of MDHA radical and dehydroascorbate that can interfere negatively with embryo development (De Gara and Tommasi 1999). The increased MDHAR and DHAR activities suggested that there may be the generation of reduced ascorbate which maintained the higher APX activity during embryo development as APX requires ascorbate as substrate. It also suggested that ascorbate generated by MDHAR and DHAR was immediately consumed by the developing embryo and that may be reason for low-APX activity at the

(A)

160

**

** *

120 80 40 0 NE PM GL

H

T

(C) 20

** 15

**

5

** *

*

0

(B)

60

**

50 **

40 30

*

*

PM

GL

*

*

*

20 10 0 NE

25

10

70

COT CAT (µmol min-1 mg-1 protein)

APX (µmol min-1 mg-1 protein)

200

DHAR (µmol min-1 mg-1 protein)

Fig. 3 Activities of APX (a), MDHAR (b), DHAR (c), and CAT (d) on different stages of somatic embryos in compare with embryogenic and nonembryogenic callus (NE non-embryogenic callus, PM proembryonic mass, GL globular embryos, H heart stage embryos, T torpedo embryos, and COT cotyledon stage embryos). Asterisk sign * and ** on each bar indicates means are significantly different from the non-embryogenic callus at P < 0.05 and P < 0.01, respectively. Mean values of all investigated parameters are presented ± SE (n = 3)

MDHAR (µmol min-1 mg-1 protein)

later stage of developing embryo. Ascorbate is greatly involved in cell defense reactions against stress (Noctor and Foyer 1998) and plays an important role in preventing senescence (Van Engelen and de Vries 1992). CAT is an important enzyme responsible for the removal of H2O2 produced under various stress conditions and prevents from the oxidative-stress-related damage (Willekens et al. 1995). CAT activity increased at the globular and torpedo stages compared to other stages have suggested that ROS scavenging were higher might be involved in the stress tolerance during somatic embryogenesis. Bewley and Black (1994) reported that in oily seeds, CAT removes H2O2 produced during b-oxidation of the fatty acids in the early phase of seedling growth. These facts strongly suggest that antioxidant enzymes are deeply involved in the metabolism during somatic embryogenesis. Within the seed, embryo development occurs in a low-osmoticum environment (Yeung and Brown 1982), which may slow the embryonic growth and allow a normal histodifferentiation pattern (Yeung 1995). Thus, besides its protective function, the stress response in developing embryos may have a developmental role. Organized development in cultured tissues, including somatic embryogenesis is often

observed, suggesting a role for these enzymes in antioxidant metabolism during embryo development.

0.9

H

(D)

T

* *

*

0.6

COT

0.3

0 NE PM GL

H

T

Development stages

COT

NE

PM GL H T Development stages

COT

123

Plant Cell Tiss Organ Cult (2007) 89:121–129

References

20 **

16

-1

-1

G-POD (µmol min mg protein)

128

12 **

8

**

* 4

*

0 NE

PM GL H T Development stages

COT

Fig. 4 Activities of G-POD on different stages of somatic embryos in compare with embryogenic and non-embryogenic callus (NE non-embryogenic callus, PM proembryonic mass, GL globular embryos, H heart stage embryos, T torpedo embryos, and COT cotyledon stage embryos). Asterisk sign * and ** on each bar indicates means are significantly different from the non-embryogenic callus at P < 0.05 and P < 0.01, respectively. Mean values of all investigated parameters are presented ± SE (n = 3)

promoted by the imposition of stress treatments (Aderkas and Bonga 2000). A pronounced increase in G-POD activity was observed during somatic embryo maturation (Fig. 4). Cordewener et al. (1991) suggested that presence of peroxidases is essential for maintaining the size and shape of protoderm cells during somatic embryogenesis. Peroxidase plays an important role for lignin formation and G-POD would seem to keep the germinating embryos cell wall rigid (Whetten et al. 1998). Antioxidant responses, through alterations of the antioxidant enzymes, ascorbate-glutathione redox systems, have been described in several systems, including embryo development (Yeung 1995; Huang et al. 2001). In conclusion, this study demonstrates that E. senticosus somatic embryo development is associated with marked changes in glutathione metabolism supported by other antioxidant enzyme activity. Acknowledgments This work was financially supported by the Ministry of Education and Human Resources Development (MOE), the Ministry of commerce, Industry and Energy (MOCIE) and the Ministry of Labour (MOLAB), Republic of Korea through the fostering project of the lab of Excellency. One of the authors (MBA) wishes to acknowledge the Japanese Society for the Promotion of Science (JSPS) for providing financial assistance.

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