Altered HBK3 expression affects glutathione and ascorbate metabolism during the early phases of Norway spruce (Picea abies) somatic embryogenesis

Plant Physiology and Biochemistry 47 (2009) 904–911

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Research article

Altered HBK3 expression affects glutathione and ascorbate metabolism during the early phases of Norway spruce (Picea abies) somatic embryogenesis Mark. F. Belmonte, Claudio Stasolla* Dept. Plant Science, University of Manitoba, Winnipeg, R3T 2N2 Manitoba, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 January 2009 Accepted 27 May 2009 Available online 12 June 2009

Plant homeobox genes play an important role in plant development, including embryogenesis. Recently, the function of a class I homeobox of knox 3 gene, HBK3, has been characterized in the conifer Picea abies (L.) Karst (Norway spruce) [8]. During somatic embryogenesis, expression of HBK3 is required for the proper differentiation of proembryogenic masses into somatic embryos. This transition, fundamental for the overall embryogenic process, is accelerated in sense lines over-expressing HBK3 (HBK3-S) but precluded in antisense lines (HBK3-AS) where the expression of this gene is experimentally reduced. Altered HBK3 expression resulted in major changes of ascorbate and glutathione metabolism. During the initial phases of embryogeny the level of reduced GSH was higher in the HBK3-S lines compared to their control counterpart. An opposite profile was observed for the HBK3-AS lines where the glutathione redox state, i.e. GSH/GSH þ GSSG, switched towards its oxidized form, i.e. GSSG. Very similar metabolic fluctuations were also measured for ascorbate, especially during the transition of proembryogenic masses into somatic embryos (7 days into hormone-free medium). At this stage the level of reduced ascorbate (ASC) in the HBK3-AS lines was about 75% lower compare to the untransformed line causing a switch of the ascorbate redox state, i.e. ASC/ASC þ DHA þ AFR, towards its oxidized forms, i.e. DHA þ AFR. Changes in activities of several ascorbate and glutathione redox enzymes, including dehydroascorbate reductase (EC 1.8.5.1), ascorbate free radical reductase (EC 1.6.5.4) and glutathione reductase (GR; EC 1.6.4.2) were responsible for these metabolic differences. Data presented here suggest that HBK3 expression might regulate somatic embryo yield through alterations in glutathione and ascorbate metabolism, which have been previously implicated in controlling embryo development and maturation both in vivo and in vitro. Ó 2009 Elsevier Masson SAS. All rights reserved.

Keywords: Ascorbic acid Embryo development Glutathione HBK3 Picea glauca Redox state

1. Introduction Norway spruce somatic embryo development is a well characterized culture system often used as a model system for studying plant embryogeny. A desirable feature of this system is the clear progression of embryo development (Fig. 1) which can be regulated through manipulations of the culture environment [18,19]. The embryogenic potential is maintained through proliferation of proembryogenic masses (PEMs) in the presence of supplied plant growth regulators (PGRs) auxin and cytokinin. Proembryogenic masses are sub-cultured every 7 days into fresh medium for continual proliferation. Removal of PGRs decreases cell proliferation and induces the differentiation of PEMs into somatic embryos, which can then continue their developmental program in the presence of supplied abscisic acid [18]. A key step of the overall embryogenic program is the transition of proembryogenic masses

* Corresponding author. Tel.: þ1 204 474 6098; fax: þ1 204 474 2578. E-mail address: [email protected] (C. Stasolla). 0981-9428/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2009.05.011

into somatic embryos triggered by the removal of PGRs. Several studies have shown that this transition, which fails to occur in developmentally arrested lines [18] is accompanied by several physiological events including programmed cell death [11,19] and changes in gene expression [36,39]. KNOTTED-like homeobox (KNOX) genes encode transcription factors which regulate important events of plant growth and development [13]. Sequence analyses showed six conserved features including a long homeobox domain implicated in DNA binding mechanisms and a KNOX and ELK domains [26]. Functional studies have revealed the participation of some of these genes in processes related to division, differentiation, and cell fate acquisition. A well characterized KNOX gene in Arabidopsis is SHOOTMERISTEMLESS, which is expressed in the apical pole where it regulates the balanced between cell division and differentiation within the shoot meristem [28]. Three KNOTTED-like homeobox (KNOX) genes from Norway spruce have been identified and found to be expressed during somatic embryogenesis [25,37,38]. Recently we demonstrated that one of these genes, HBK3, plays a key role during embryogenesis, especially during the transition of PEMs into

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into somatic embryos through changes in ascorbate and glutathione metabolism.

2. Materials and methods PEMs Day

1

4 PGR +

Somatic embryos 7

1

4

2.1. Plant material

Cotyledonary embryos

The Norway spruce line (95:88:22, [17]) was kindly provided by Prof. von Arnold. Methods for transforming the embryogenic cells were previously described in detail [8]. Maintenance of proembryogenic masses (PEMs) and initiation of embryo development were carried out exactly as described in [11]. Proembryogenic masses were cultured using half-strength LP medium (modified after [18]) with the inclusion of PGR, auxin (9.0 mM 2,4-dichlorophenoxyacetic acid, 2,4-D) and cytokinin (4.4 mM N6-benzy ladenine, BA). Proliferation of PEMs was sustained by weekly subculturing the cells into fresh medium containing PGRs (PGRþ). Transdifferentiation of PEMs into somatic embryos was performed by transferring cells into half-strength LP medium devoid of PGR [hormone free (PGR) medium] for a week. For this experiment we chose three lines: a control line transformed with an empty vector [referred to as wild type (WT) line], and two transformed lines having respectively the highest and the lowest HBK3 expression level among a total of 8 transformed lines generated in previous studies [8]. The selected sense line with the highest HBK3 level was referred to as HBK3-S1, whereas the antisense line exhibiting the lowest HBK3 expression level was named HBK3-AS2 [8]. The level of HBK3 transcripts in the three lines was measured by Northern analyses (see Fig. 1 in [8]). Tissue was collected at days 1,4, and 7 in the presence of PGR (þPGR) and days 1, 4, and 7 after the withdrawal of PGRs (PGRs) (see Fig. 1) for analysis on ascorbate and glutathione metabolism.

7

PGR(hormone free)

ABA

Fig. 1. The process of somatic embryo formation in Picea abies (Norway spruce). In media containing the hormones auxin and cytokinin (PGRþ), proembryogenic masses (PEMs) proliferate. After transfer onto hormone free (PGR) medium, somatic embryos are formed from PEMs [although sometimes a very small number of somatic embryos can also be observed in the (PGR)-medium]. Continuation of embryo development is achieved in the presence of exogenously supplied abscisic acid (ABA). Days in culture represent harvesting points used in our experiment. Dark arrow marks the transition from PEMs into somatic embryos. Diagram modified from [18].

somatic embryos (Fig. 1). Using a transformation approach it was shown that this transition is accelerated in those sense lines overexpressing HBK3 (HBK3-S), whereas it is precluded in antisense lines (HBK3-AS) with reduced KBK3 expression [8]. The physiological mechanisms underlying these differences remain unknown. Antioxidant responses are considered to be important modulators of embryo development and have been described in many plant and animal systems [5,7,12,14–16,22,29]. Both ascorbate and glutathione have been shown to guide the cell cycle and promote differentiation based on the redox status of each antioxidant. Studies on vitro systems have revealed that the balance between the reduced (ASC and GSH) and oxidized forms (DHA þ AFR and GSSG) (Fig. 2) affects morphogenesis. A switch of the redox status towards a more reduced environment (high ASC/DHA þ AFR and GSH/GSSG ratios) was observed during the early days in culture in white spruce lines with high embryogenic potential [6,34]. As the embryos mature, however, the redox status slowly becomes more oxidized [6,9]. These metabolic changes did not occur in white spruce cell lines unable to produce somatic embryos [34]. Based on the above it is the objective of the current study to examine if HBK3 expression regulates the differentiation of PEMs

2.2. Glutathione and ascorbate metabolism Determinations of both reduced and oxidized forms of ascorbate and glutathione were carried out exactly as reported in [41]. Enzymatic activity was examined following the methods of Arrigoni et al. [3] as modified by Belmonte et al. [6]. Each extraction was replicated a minimum of three times.

ASC GSSG

NADP+

NAD(P)+

H2O2 AFRR

APX H2O

GR

DHAR

NADPH+H+ AFR

GSH

NAD(P)H

DHA Fig. 2. Schematic representation of the ascorbate and glutathione metabolism. Reduced ascorbate (ASC) is oxidized to ascorbate free radicals (AFR) and ultimately to dehydroascorbate (DHA) by ascorbate peroxidase (APX). Reduction of AFR and DHA back to ASC is catalyzed by ascorbate free radical reductase (AFRR) and dehydroascorbate reductase (DHAR). Reduced glutathione (GSH) and oxidized glutathione (GSSG) are recycled through the enzymes DHAR and glutathione reductase (GR).

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Table 1 Percentage composition of P. abies somatic embryos in a WT line, a line overexpressing HBK3 (HBK3-S1) and a line down-regulating HBK3 (HBK3-AS2). Cells were harvested for analysis at different days in the proliferation (PGRþ) and hormone free (PGR) media (see Fig. 1). Numbers represent the percent composition of somatic embryos formed in relation to proembryogenic masses (PEMs). Genotype

(day 7)PGRþ

(day 4) PGR

(day 7)PGR

WT HBK3-S1 HBK3-AS2

2 14* 4

21 23 5*

37 63* 12*

coefficient 6.2 mM1 cm1). The reaction mixture contained 1 mM DHA, 2 mM GSH, and 100 mM potassium phosphate buffer, pH 6.3. GR (EC 1.6.4.2) activity was determined following the NADPHdependent oxidation of GSSG at 340 nm (extinction coefficient 14 mM1 cm1). The reaction mixture contained 0.1 M Tris–HCl, pH 7.8, 2 nM EDTA, and 0.5 mM GSSG. The reaction was initiated with the addition of 50 mM NADPH. 2.4. Fluorescent labelling of GSH

*indicates values are significantly different from control values (P  0.05) at the same sampling time.

2.3. Enzyme activity measurements Activities of ascorbate peroxidase (APX), ascorbate free radical reductase (AFRR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in developing embryos were analyzed following homogenization of tissues at 4  C in medium containing 50 mM Tris–HCl, pH 7.2, 0.3 M mannitol, 1 mM EDTA, 0.1% BSA, 0.05% cysteine, and 2% (w/v) polyvinylpyrrolidone. The homogenate was then centrifuged at 4  C for 20 min at 16,000g, and the supernatant collected for the analysis of enzymatic activities as reported by Arrigoni et al. [3]. APX (EC 1.11.1.11) activity was estimated by measuring the hydrogen peroxide-dependant oxidation of ASC (extinction coefficient 2.8 mM1 cm1) following the decrease in absorbance at 265 nm. Only the cytosolic component of APX activity was measured since no ASC was added to the grinding medium [1]. The reaction mixture contained 50 mM ASC, 90 mM H2O2, and 50 mM potassium phosphate buffer, pH 6.5. AFRR (EC 1.6.5.4) activity was measured following the rate of NADH oxidation at 340 nm (extinction coefficient 6.0 mM1 cm1). The reaction mixture contained 0.2 mM NADH, 1 mM ascorbate, and 0.1 M Tris–HCl, pH 7.2. The reaction was initiated with the addition of 0.5 units of ascorbate oxidase (EC 1.10.3.3). DHAR (EC 1.8.5.1) activity was determined by following the GSH-dependent production of ascorbate at 265 nm (extinction

Reduced glutathione (GSH) was visualized in developing Norway spruce somatic embryos following incubation of cultures with monochlorobimane (Molecular Probes, Eugene OR), according to the method described in [27]. Embryos were incubated in freshly made 10 mM monochlorobimane for 30 min and washed briefly in hormone free media and mounted on a microscope depression slide to avoid cell compression. The glutathione S-bimane fluorescence was visualized using a filter with excitation at 442 nm and emission at 563 nm using. Light microscopic images of the same cells were taken simultaneously to better localize the fluorescent signal within the cellular aggregates. 2.5. Statistical analysis Significant differences between the means were calculated according to the Student’s t-test. 3. Results 3.1. Effect of HBK3 on growth of Norway spruce somatic embryos Changes in the growing medium resulted in profound alterations in culture composition (Fig. 1). At the end of the proliferation period (day 7 PGRþ) only 2% of the culture aggregates were composed of somatic embryos in the WT line. This percentage increased rapidly upon removal of the plant growth regulators (Table 1). In the HBK3-S1 line 14% of the cellular aggregates were

0.75 WT HBK3(S) 0.7

HBK3(AS)

* 0.65

* 0.6

0.55 1

3

5

7

PGR+ Fig. 3. Dry weight:fresh weight ratio (DW:FW) of PEMs of Norway spruce in WT, HBK3-S1 and HBK3-AS2 lines. Cells were collected and dried in an oven for 72 h every two days during the seven day proliferation period and compared to the initial fresh weight of the tissue. Values  SE are means of three independent experiments, each with three replicates. * indicates values that are significantly different from control (P  0.05).

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represented by somatic embryos at day 7 (PGRþ). In the PGRmedium the percentage composition of somatic embryos increased to a maximum value of 63% (almost double the value of the WT line). Compared to WT, the percentage of somatic embryos produced by the HBK3-AS2 line was always significantly lower in the PGR- medium (Table 1). During the proliferation stage of development (PGRþ), the DW:FW ratios were markedly different among the cell lines, especially at the end of this culture period. The DW:FW ratio failed to increase in the HBK3-AS2 line compared to that of WT cells and cells over-expressing HBK3 (Fig. 3). 3.2. Developmental changes in the ascorbate and glutathione pools Picea abies transformed with HBK3 in the sense (S) or antisense (AS) orientation showed remarkable differences in glutathione content (Fig. 4). In WT cells the cellular level of reduced glutathione (GSH) declined slowly in PGR þ medium before increasing upon the removal of plant growth regulator (PGR). This pattern was also observed in the HBK3-S1 line although the level of GSH was always higher at any day in culture (Fig. 4). No major fluctuations were observed in the GSH level of the HBK3-AS2 line. Compared to the WT line, GSH content in the HBK3-AS2 line was much lower at the end of the culture period. Levels of GSSG were almost similar among the three lines at all stages of development, with the exception of day 7 PGRþ, where the HBK3-S1 line showed the highest GSSG content. These changes in metabolite levels resulted in alterations of the GSH/GSH þ GSSG ratio which increased in both control and HBK3-S1 lines in the hormone free (PGR) medium, whereas it remained constant in the HBK3-AS2 line (Fig. 4). Localization studies showed a predominant accumulation of GSH in the meristematic cells of WT and HBK3-S1 lines. Signal of GSH in the HBK3-AS2 lines was always week and diffuse (Fig. 5). In WT cells and in cells over-expressing HBK3 the level of reduced ascorbate (ASC) increased during development. This trend was however more pronounced in the latter cells. The ASC content did not change in the HBK3-AS2 cells which showed the lowest values at any day in culture (Fig. 6). The only differences in the levels of the oxidized forms of ascorbate (DHA þ AFR) were observed at day 7 PGR þ where HBK3-S1 and HBK3-AS2 cells showed the highest values (Fig. 6). Upon removal of plant growth regulators (PGR-) the ascorbate redox ratio (ASC/ASC þ DHA þ AFR) increased in both WT cells and cells over-expressing HBK3. This increase was not observed in the HBK3-AS2 line (Fig. 6).

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the end of the culture period (day 7 in PGR medium) in the HBK3S1 line (Fig. 7). 4. Discussion A key step during the in vitro progression of embryo development in Norway spruce is the transdifferentiation of PEMs into somatic embryos [11,18,19]. Several studies have described the physiological and molecular events accompanying this transition and revealed that programmed cell death [11,19] and precise changes in gene expression [36,40] are required for the formation of somatic embryos. Our previous work showed that HBK3, a class I KNOX gene, is directly implicated in the PEM-somatic embryo transition [8]. Besides inducing major morphological changes, especially within the shoot apical pole [8], HBK3 expression

3.3. Changes in the activity of the enzymes involved in ascorbate and glutathione metabolism In the WT line, the activity of APX, the enzyme responsible for the removal and detoxification of H2O2, remained steady in the presence of PGRs before declining upon removal of the plant growth regulators [hormone free (PGR-)] medium. In general the activity of APX was lower in both transformed lines in the PGR þ medium (Fig. 7). Fluctuations in the activity of DHAR, the enzyme that catalyzes the reduction of DHA to ASC, were more pronounced in the HBK3-S1 line. The activity of this enzyme declined sharply upon transfer onto the PGR medium but increased upon further development. This increase was not observed in the other two cell types (Fig. 7). The activity of ascorbate free radical reductase (AFRR) was always highest in the HBK3S1 line, especially at days 1 and 4 in the PGR þ medium, and days 4 and 7 in the PGR- medium. Small fluctuations in the activity of GR, the enzyme responsible for the reduction of GSSG to GSH, were observed in all cell lines during the proliferation medium (PGR þ medium). A sharp increase in GR activity was observed at

Fig. 4. Changes in glutathione metabolism in WT and HBK3 transformed lines (A) Endogenous levels of reduced glutathione (GSH) and its oxidized form (GSSG) in control, HBK3-S1 and HBK3-AS2 cell lines (B) Ratio of GSH/GSH þ GSSG in the three cell lines. Cells were harvested at days 1, 4, and 7 in the proliferation (PGRþ) medium and hormone free (PGR) medium. Values are mean  SE of three independent experiments. *indicates values that are significantly different from control (P  0.05) at the same day in culture.

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Fig. 5. Localization of intracellular GSH in developing Norway spruce somatic embryos. Wild type (WT) cells and cells of the HBK3-S1 and HBK3-AS2 lines were incubated with monochlorobimane (see Material and methods) and examined using light (left panels) and fluorescent (right panel) microscopy. Bar ¼ 240 mm.

regulates the number of somatic embryos produced in culture. Compared to control, differentiation of PEMs into somatic embryos is accelerated in lines over-expressing HBK3 and delayed in those lines with reduced HBK3 expression (Table 1). The present work shows a clear relationship between HBK3 transcript levels and glutathione and ascorbate metabolism and provides a plausible explanation for the role of HBK3 in regulating the embryogenic process in spruce. Removal of PGRs from the culture medium promotes the differentiation of somatic embryos from PEMs (Table 1) and induces a significant increase in endogenous GSH level (Fig. 4). Accumulation of GSH seems to be under the control of HBK3 since it is increased in cells with higher HBK3 expression and decreased in cells with reduced expression of this gene. In the over-expressing (HBK3-S1) line, GSH level is higher at any stage in culture reaching a maximum value at the end (day 7) of the hormone free (PGR-) medium. At this stage the high GSH content may be the result of

increased GR activity, which recycles GSH from GSSG (Figs. 2,4, and 7). A high activity of this enzyme has been reported previously especially in young and rapidly growing tissues [10,39]. High GSH levels, which we observed mainly in the meristematic cells of developing embryos (Fig. 5) may be required to promote the growth of the embryo heads, by inducing meristematic cell proliferation, as well as creating a favourable environment for proper cellular differentiation. The necessity of high levels of GSH to support cell proliferation in both animal and plant systems has been well documented [21,32]. A clear relationship between GSH content and rate of cell division was described in epidermal and cortical initials of Arabidopsis roots [21,31]. Possible modes of action of GSH during cell division have been provided by independent studies suggesting the involvement of this metabolite in the G1-S transition of the cell cycle [30] as well as in nucleotide and nucleic acid synthesis [4]. In white spruce (Picea glauca) GSH applications increased the

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Fig. 6. Changes in ascorbate metabolism in WT and HBK3 transformed lines (A) Endogenous levels of reduced ascorbate (ASC) and ascorbate free radical and dehydroascorbate (AFR þ DHA) in control, HBK3-S1 and HBK3-AS2 cell lines (B) Ratio of ASC/ASC þ DHA þ AFR in the three cell lines. Cells were harvested at days 1, 4, and 7 in the proliferation (PGRþ) medium and hormone free (PGR) medium. Values are mean  SE of three independent experiments. * indicates values that are significantly different from control (P  0.05) at the same day in culture.

incorporation of preformed bases, i.e. adenine and adenosine, into nucleotides, mainly ATP, and nucleic acids [4]. Besides favouring cell proliferation, GSH might be also required for cell and tissue differentiation. Progression of PEMs into somatic embryos is in fact accompanied by marked structural changes (Fig. 1) involving the dismantling and elimination of specific cells via programmed cell death (PCD, [11]) and the formation of new tissues, e.g. protoderm. Recent evidence in both animal and plants indicates that GSH is required for PCD and cell fate acquisition. Henmi et al. [23] have shown that the PCD events leading to

tracheary element differentiation in cultured mesophyll cells of Zinnia elegans are suppressed if GSH is experimentally reduced by applications of L-buthionine sulfoximine (BSO). In spruce high levels of GSH might be required for proper execution of PCD, which several studies have revealed to be an obligatory event for somatic embryo formation. Initially Filonova et al. [19] found a positive correlation between the number of somatic embryos produced from PEMs and the number of cells within the PEMs undergoing PCD. Secondly it was discovered that inhibition of PCD through buffering of the culture medium reduced the frequency of PEM-somatic

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cells; Fig. 4). Based on the above evidence we suggest that HBK3 expression regulates PEM-somatic embryo differentiation through modulation of the glutathione metabolism resulting in elevated levels of GSH. Such changes fail to occur in lines with reduced HBK3 expression thereby compromising the formation of somatic embryos. Glutathione metabolism is closely linked to ascorbate through the Halliwell–Asada cycle (Fig. 2, [20]). Like GSH, the ASC pool is generally larger in cells over-expressing HBK3 especially after the transfer onto the hormone free (PGR-) medium (Fig. 6). This increase is possibly due to the activities of the two ASC recycling enzymes DHAR and AFRR which are also higher in cell with high HBK3 expression (Fig. 7). High levels of cellular ACS have been measured during the early phases of embryogeny in vivo [2,6] and in vitro [34] where they are mainly implicated in detoxification processes, by removing reactive oxygen species produced by oxidative metabolism of growing embryos, as well as cell division events. Previous studies have documented the high levels of ASC in tissues undergoing fast growth. De Gara et al. [14] reported that in pea stem the content of ASC is high in the meristematic region and it gradually declines in older tissues. Similarly, exogenous ASC applications induce cell proliferation in several systems including Allium cepa roots [3], cambial cells of Lupinun albus roots [3], and meristematic cells of spruce [37]. During white spruce (P. abies) embryogenesis a switch of the total ascorbate pool towards its reduced form, i.e. ASC, was observed in those lines able to produce embryos, whereas it was precluded in a developmentally arrested line unable to do so [33–35]. The observation that the ascorbate redox (ASC/ASC þ DHA þ AFR) fails to switch towards a reduced state in cells down-regulating HBK3 and unable to produce somatic embryos at high frequency suggests that this gene regulates early embryogeny through changes is ascorbate metabolism. In conclusion, results from this study indicate that the control of HBK3 on somatic embryo development might be exercised through the modulation of the glutathione–ascorbate metabolism. High HBK3 expression increases the level of both GSH and ASC, through changes in activity of their respective recycling enzymes, which are both required for the initiation of the embryogenic process at the PEM-somatic embryo transition. The delay in this transition observed in the lines down-regulating HBK3 is associated to a failure of the cells to switch the glutathione and ascorbate pool towards a reduced state. Results of this study, which further our knowledge associating the effects of a KNOTTED gene to specific metabolic changes of the cellular antioxidant system, are valuable for the future design of culture conditions enhancing in vitro embryogenesis. Acknowledgements Fig. 7. Enzyme activities of APX, DHAR, AFRR and GR in the WT, HBK3-S1 and HBK3AS2 cell lines. APX: 1 unit ¼ 1 nmol ASC oxidized mg1 protein min1; AFRR: 1 unit ¼ 1 nmol NADH oxidized mg1 protein min1; DHAR: 1 unit ¼ 1 nmol ASC reduced mg1 protein min1. Cells were harvested at days 1, 4, and 7 in the proliferation (PGRþ) medium and hormone free (PGR) medium. Values are mean  SE of three independent experiments. * indicates values that are significantly different from control (P  0.05) at the same day in culture.

This research was supported by the Natural Sciences and Engineering Research Council of Canada Research Grants to CS and an NSERC PGS-D to MFB. The assistance of Mr. Bert Luit is also greatly appreciated. References

embryo differentiation [11]. Evidence that GSH controls embryonic cellular differentiation also appears in animal studies which show the role of this metabolite in modulating the early phases of embryonic development (reviewed in [24]). A close relationship between cellular GSH level and somatic embryo production is also evident from the observation that in the PGRþ proliferation medium, which inhibits the formation of somatic embryos, embryo production is significantly increased in the HBK3-S1 line exhibiting high levels of GSH (more than double those of WT

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