JXB Advance Access originally published online on July 4, 2005
Journal of Experimental Botany 2005 56(419):2355-2364; doi:10.1093/jxb/eri228
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RESEARCH PAPER |
Alterations of the glutathione redox state improve apical meristem structure and somatic embryo quality in white spruce (Picea glauca)
1Department of Plant Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada
2Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada
* To whom correspondence should be addressed. Fax: +1 204 474 2578. E-mail: stasolla{at}ms.umanitoba.ca
Received 28 February 2004; Accepted 15 May 2005
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Abstract |
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In white spruce, an improvement of somatic embryo number and quality can be achieved through experimental manipulations of the endogenous levels of reduced (GSH) and oxidized (GSSG) glutathione. An optimal protocol for embryo production included an initial application of GSH in the maturation medium, followed by replacement with GSSG during the remaining maturation period. Under these conditions, the overall embryo population more than doubled, and the percentage of fully developed embryos increased from 22% to almost 70%. These embryos showed improved post-embryonic growth and conversion frequency. Structural studies revealed remarkable differences between embryo types, especially in storage product deposition pattern and organization of the shoot apical meristem (SAM). Compared with their control counterparts, glutathione-treated embryos accumulated a larger amount of starch during the early stages of development, and more protein and lipid bodies during the second half of development. Differences were also noted in the organization of SAMs. Shoot meristems of control embryos were poorly organized and were characterized by the presence of intercellular spaces, which caused separation of the subapical cells. Glutathione-treated embryos had well-organized meristems composed of tightly packed cells which lack large vacuoles. The improved organization of the shoot apical meristems in treated embryos was ascribed to a lower production of ethylene. Differences in meristem structure between control and treated embryos were also related to the localization pattern of HBK1, a shoot apical meristem ‘molecular marker’ gene with preferential expression to the meristematic cells of the shoot pole. Expression of this gene, which was localized to the apical cells in control embryos, was extended to the subapical cells of treated embryos. Overall, it appears that meristem integrity and embryo quality are under the direct control of the glutathione redox state.
Key words: Embryo conversion, ethylene, glutathione, meristem, Picea glauca, somatic embryogenesis, spruce
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Optimizing the propagation of tree species through somatic embryogenesis remains one of the main objectives in sustaining superior quality genotypes for reforestation strategies (Grossnickle, 2000
). Despite increasing efforts towards the design of optimal conditions, variations in the quality of embryos produced in culture can often be observed among genotypes. Studies conducted on white spruce have revealed that the quality of the embryos grown in vitro correlates with the organization of the shoot apical meristems (SAMs), which are responsible for the formation of a functional above-ground vegetative system after germination. Developing embryos with reduced post-embryonic performance are generally characterized by the presence of poorly organized SAMs, which are disrupted by the presence of intercellular spaces caused by accumulation of ethylene (Kong and Yeung, 1994). Improvements in meristem quality have been obtained through manipulations of the culture conditions, resulting in a decrease in the endogenous ethylene level (Kong and Yeung, 1994; El Meskaoui and Tremblay, 2001).
The redox environment of the culture medium is another relevant factor which influences the somatic embryogenic process. In both angiosperms and gymnosperms, a reduced culture environment is required during the initial embryogenic events, characterized by active cell proliferation, whereas an oxidized environment is beneficial for the completion of embryogenesis (Earnshaw and Johnson, 1987; Arrigoni et al., 1992; De Gara et al., 2003; Belmonte and Yeung, 2004). During white spruce somatic embryogenesis, manipulations of the redox state of the culture medium, effected by applications of either reduced (GSH) or oxidized (GSSG) glutathione, have profound effects on embryo yield and quality. The addition of GSH has been shown to promote proliferation of the embryogenic tissue, with a 25% increase in fresh weight (Belmonte et al., 2003). This growth, which was accompanied by a high production of ATP through an active purine salvage pathway, increased the number of early filamentous embryos protruding from the embryogenic tissue (Belmonte et al., 2003). However, applications of GSH during the later phases of embryogenesis were not beneficial and resulted in the production of a high percentage of abnormal embryos unable to regenerate viable plants. Contrary to GSH, applications of GSSG did not have a profound effect on tissue proliferation during the early phases of embryogenesis, but improved the quality of the embryos during the later stages of development. Compared with controls, GSSG-treated embryos had more cotyledons, thus denoting improved functionality of SAM, and a better ability to convert into viable plantlets (Belmonte and Yeung, 2004).
Based on the evidence above, it appears that culture conditions during spruce somatic embryogenesis can be optimized through experimental modulations of the glutathione redox state. An increase in embryo number and quality can be obtained through an initial imposition of a reduced state (GSH), which promotes cell proliferation and the formation of immature embryos, followed by a switch towards an oxidative state (GSSG) which promotes proper embryonic development. To test this hypothesis, and to investigate the role of the glutathione redox pair during embryogenesis, experiments were performed in which spruce embryos, initially cultured in the presence of GSH, were transferred onto GSSG-containing media at different stages of development. This made it possible to discover optimal treatments that increase the number of embryos able to regenerate viable plants. Differences in quality between control and treated embryos were correlated to the storage product deposition pattern, the cellular organization of SAMs, and to the accumulation of ethylene, which has been shown to affect meristem architecture and function (Kong and Yeung, 1994). Finally, the localization pattern of HBK1, a SAM ‘molecular marker’ gene with preferential expression to the meristematic cells of the shoot pole (Sundas-Larsson et al., 1998), was followed in both control and treated embryos in order to estimate meristem integrity.
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Materials and methods |
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Plant material
White spruce, Picea glauca (Moench) Voss, embryogenic tissue derived from immature zygotic embryos was isolated according to the methods of Lu and Thorpe (1987)
. Immature seeds were collected from the campus at the University of Calgary, Calgary, Alberta, Canada, sterilized in 20% commercial Javex bleach for 20 min, and then rinsed three times in sterile distilled water. Dissected embryos were placed on half-strength Litvay (
LV) (Litvay et al., 1985) induction medium containing 10 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 5 µM N6-benzyladenine (BA), and 5% sucrose, and solidified with 0.8% Becton Dickinson purified agar, pH 5.8. The initiated stock cultures were kept in the dark at 26 °C for 4–6 weeks. Embryogenic tissue was transferred to a solid maintenance medium ( LV medium containing 10 µM 2,4-D, 2 µM BA, and 3% sucrose) and subcultured every 7 d to fresh medium. Maturation of somatic embryos was established by spreading 100 mg fresh weight of embryogenic tissue directly onto solid maturation medium ( LV supplemented with 50 µM filter-sterilized abscisic acid (ABA), 5% sucrose, and solidified with 0.4% phytagel, pH 5.8) (Belmonte and Yeung, 2004).
To test the effect of exogenously supplied GSH and GSSG on the redox status of glutathione during the maturation period, 0.1 mM of GSH or 1.0 mM GSSG was filter-sterilized and included into the cool autoclaved maturation medium before being poured. These concentration were used as they have been optimized in previous studies (Belmonte and Yeung, 2004; Stasolla et al., 2004). For transfer experiments, 100 mg of tissue was spread onto 0.1 mM GSH containing media for 3, 7, 14, or 30 d before being directly transferred onto GSSG-containing medium for the remainder of the maturation process. Embryos were transferred at 20 d regardless of treatment to new medium. Embryo number was scored at the completion of the 40 d maturation period. The ability of the embryogenic tissue to produce mature cotyledonary embryos was scored according to the methods detailed in Belmonte and Yeung (2004). Embryos were divided into two groups (A and B), based on their ability to convert. Those embryos that formed four or more cotyledons, and possessed a greater ability to convert were classified as Group A. Conversely, embryos that formed three or fewer cotyledons and did not convert readily were classified as Group B.
The partial drying treatment (PDT) of mature somatic embryos was followed according to the method developed by Roberts et al. (1990). At the completion of the 10 d PDT, white spruce somatic embryos were allowed to develop on hormone-free germination medium ( LV containing 1% sucrose, solidified with 0.8% agar, pH 5.8) for 8 weeks. Plates were placed under light (photon flux density of 90–95 µE m–2 s–1, PAR; 380–800 nm) with a 16 h photoperiod and maintained at 25 °C. Conversion, defined as the emergence and development of both root and shoot, was scored at the end of the 8-week germination period (Stasolla and Yeung, 1999).
The effects of GSH and GSSG applications were replicated in three different cell lines with similar results. Data presented in this paper were generated using the (E)WS1 line, which was characterized in previous work (Belmonte and Yeung, 2004).
Glutathione measurements and glutathione reductase activity
Tissue was collected on days 10, 20, 30, and 40 for glutathione analysis. Glutathione measurements and activity of glutathione reductase (GR) were performed exactly as reported by Zhang and Kirkham (1996).
Light microscopy
For light microscopy, samples were fixed in 2.5% glutaraldehyde and 1.6% paraformaldehyde buffered with 0.05 M phosphate buffer, pH 6.9, dehydrated with methyl cellosolve followed by two changes of absolute ethanol, and then infiltrated and embedded in Historesin (Leica Canada, Toronto) (Yeung, 1999). Serial sectioning (3 µm) was carried out on a Reichert-Jung 2040 Autocut rotary microtome. These sections were stained with periodic acid–Schiff (PAS) reaction for total carbohydrates, and counterstained with amido black 10B for protein or toluidine blue O (TBO) for general histological organization (Yeung, 1984). At least 50 embryos per treatment, per developmental stage, per replicate were fixed and processed as outlined above according to the methods of Yeung (1999).
RNA in situ hybridization
Chemical fixation and tissue processing:
Developing embryos were fixed in 4% (w/v) freshly prepared paraformaldehyde in PBS pH. 7.4, vacuum infiltrated for 15 min, and incubated on a rotator for 3 h at room temperature. The samples were dehydrated in an ethanol series (30%, 50%, 70%, 95%, 100%, 100%) for 45 min at 4 °C and left overnight in 100% ethanol. The samples were treated with increasing concentrations of xylene at room temperature and incubated overnight at 42 °C in xylene and a few pellets of paraffin. The tissue was then incubated at 60 °C and the xylene was slowly replaced with molten paraffin. After eight changes with paraffin, blocks were made and the paraffin-embedded embryos were sectioned at a thickness of 7 µm using disposable blades in a Leica (RM 2145) microtome. Prior to hybridization, paraffin was removed with two changes of xylene for 15 min, and the sections were rehydrated.
Probe preparation and hybridization:
A 345 bp fragment containing the 3'-untranslated region of HBK1 was subcloned in Bluescript SK+ plasmid (Stratagene) and used for in vitro transcription using digoxigenin-11-UTP, as described in the DIG RNA labelling kit (Roche Molecular Biochemicals). Sense and antisense probes were stored at –80 °C prior to hybridization.
Tissue treatments and prehybridization washes were conducted exactly as described in Cantón et al. (1999). Sections were hybridized with equal concentrations (25 µg ml–1) of sense or antisense probe in 1x Denhardt's, 1 mg ml–1 tRNA, 10% dextran sulphate, 50% formamide, and 1x salts (Regan et al., 1999). Probes were denatured at 65 °C for 5 min and hybridization was carried out at 50 °C for 16 h. Post-hybridization washes and antibody treatment were performed as described by Regan et al. (1999). Detection of DIG-labelled probes was carried out using a Wester Blue solution (Promega) containing 1 mM Levamisol. Depending on the developmental stages of the embryos, color development occurred between 3–12 h.
Ethylene measurements:
Ethylene production was measured using headspace gas analysis. Six pieces of 0.1 g of embryogenic tissue were placed on 30 ml media in a plastic Petri plate. A set of Petri plate lids were modified to accommodate ethylene measurements. A 6 mm hole was drilled in the lid and a 6 mm diameter rubber septum was inserted into the hole. Ethylene was measured on days 10, 20, 30, and 40. On the days of ethylene analysis, the Petri plates were placed in a laminar flow hood and the lids were removed for 5 min to flush out any residual ethylene. The lids were replaced with sterilized lids containing the rubber septa. Each plate was double wrapped with commercial plastic wrap (Saran Wrap®) to prevent gas leakage. The plates were incubated for 6 h. At the end of the incubation period, a 1 ml sample was withdrawn from the Petri plate headspace via the rubber septum using a 1 ml syringe. The total headspace volume was 60 ml.
The 1 ml gas sample was injected into a Photovac 10S plus gas chromatograph (Photovac, Inc., Waltham, MA, USA) equipped with a photo-ionization detector and a 3.2 mx2.45 mm 60/80 mesh Carbopack B column (1.5%xE-60/1% H3PO4; Supelco, Oakville, ON, Canada). Four plates for each day of growth, i.e. day 10, 20, 30, and 40, were used for the measurements.
Statistical analysis
For maturation frequency and conversion, and endogenous glutathione levels, Tukey's Post-Hoc test for multiple variance (Zar, 1999) was used to compare differences between treatments and control. For total fresh weight and ethylene measurements, Tukey's Post-Hoc test for multiple variance was used to compare treatment effects at each stage of development. All data in these studies were generated using the SPSS© v 10.0 statistical software package analysed at the 5% level.
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Results |
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Effects of glutathione on embryo maturation
Pronounced differences in embryo number and quality were observed under various glutathione redox treatments (Fig. 1). Of the total population of mature control embryos at the cotyledonary stage, about 40% showed four or more cotyledons (Group A). Inclusion of 0.1 mM GSH into the maturation medium was effective at producing a larger total population of somatic embryos (Fig. 1). The mean total number of embryos generated from 100 mg of tissue increased from about 50 in control tissue to about 70 when embryos were treated with 0.1 mM GSH throughout the 40-d maturation period. This increased number was, however, mostly due to the higher production of poor quality embryos with fewer than four cotyledons (Group B). Inclusion of 1.0 mM GSSG significantly increased the percentage of better quality embryos, with a marginal increase in total embryo population.
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0.05. C, control; DnT, embryos treated with GSH for n days, followed by GSSG for the remaining culture period (see Materials and methods for details).Treatments in which embryos were transferred from a GSH-containing medium to a GSSG-containing medium improved the number of good quality embryos (Group A). When the transfer from GSH to GSSG was performed after 7 d (D7T), the number of Group A embryos produced from 100 mg of embryogenic tissue increased from 20.7 (control) to about 70 (Fig. 1). This increase was also responsible for a higher total population of embryos (A+B) observed after a day 7 transfer. Prolonged treatments with 0.1 mM GSH, before transfer to GSSG-containing media, decreased the percentage of Group A embryos (Fig. 1). Embryo number and quality were also very low when embryos were initially cultured in the presence of GSSG and then transferred onto GSH-containing medium (data not shown).
Embryo maturation was accompanied by an increase in fresh weight of the tissue. Compared with controls, the increased in fresh weight of treated tissue producing the highest number of embryos (D7T) was reduced over the maturation period (Fig. 2A). To determine whether this difference was due to the developing embryos or to the subtended non-embryo-forming tissue, different measurements were carried out. The low embryo/non-embryo-forming tissue ratio indicates that the increase in fresh weight observed in control tissue is mainly due to the proliferation of the non-embryo-forming tissue (Fig. 2B). Conversely, in tissue treated with GSH for 7 d followed by a transfer onto GSSG-containing medium for the remaining culture period (D7T), the fresh weight increase was due to the growth of developing embryos (Fig. 2B).
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Effect of glutathione on conversion frequency
Following the 8-week germination period, embryo conversion (i.e. root and shoot emergence) was examined in both Group A and B embryos (Fig. 3). About 50% of control A embryos were able to generate a functional root and shoot (R+S). More than 30% of these embryos did not show any apical growth during germination (NR+NS, Fig. 3A). Shoot and root conversion of Group A embryos was significantly increased in transfer experiments. Transfer from GSH to GSSG after 7 d (D7T) resulted in the production of embryos with the highest conversion frequency (Fig. 3A). More than 80% of these embryos were able to generate a functional shoot and root during germination. Prolonged culture in the presence of GSH (D14T and D30T) was not beneficial for embryo regeneration.
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Overall, the conversion frequency of Group B embryos was low for all treatments. A large fraction of these embryos aborted on the germination medium and were not able to regenerate plantlets. The transfer experiments (D3T, D7T, D14T, and D30T) increased root conversion, but did not have any beneficial effects on shoot emergence (Fig. 3B). This supported the notion that the number of cotyledons is a reliable characteristic for screening ‘good’ from ‘poor’ quality embryos.
Changes in the glutathione redox state during maturation
Changes in reduced (GSH) or oxidized glutathione (GSSG) during embryo maturation were compared between control tissue and tissue producing the largest number of good quality embryos (D7T). Compared with control tissue, the endogenous levels of both forms of glutathione were higher in treated tissue (Fig. 4A). The GSH level of treated tissue decreased during maturation, resulting in a shift in the GSH/GSSG ratio towards a more oxidized state (i.e. a low ratio) (Fig. 4B). This flux of the glutathione pool toward its oxidized state was not observed in control tissue, where the GSH/GSSG ratio increased over time (Fig. 4B).
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The activity of glutathione reductase (GR), the enzyme responsible for the conversion of GSSG to GSH was higher in control embryos at all stages of development. A sharp increase in activity was observed at day 40 (Fig. 5). A steady, but less pronounced increase in the activity of this enzyme was also observed in D7T embryos.
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Embryo morphology
Irrespective of the treatment, the early pattern of embryo development, i.e. histodifferentiation, appeared to be similar (data not shown). Differences in structure between treatments were observed on day 20 (Fig. 6A–G). Compared with control embryos, increased mitotic activity was detected in developing cotyledons and of embryos treated with GSH for 7 d, followed by GSSG (D7T) (Fig. 6B, C). In these embryos, the subapical cells of the shoot pole and the cortical cells accumulated a larger amount of storage products, mainly starch (Fig. 6D–G). At the end of the maturation period the SAM of control embryos was generally poorly organized and was characterized by the presence of intercellular spaces, which often separated the subapical cells of the meristem (Fig. 6I). The subapical cells (Fig. 6I) and the subtended procambial cells (Fig. 6J) were vacuolated and had few storage products in their cytoplasm. In D7T embryos, the structure of the SAM was much more organized. Large intercellular spaces were absent in the shoot pole (Fig. 6K). The subapical (Fig. 6K) and procambial cells (Fig. 6L) did not have large vacuoles and accumulated many storage products. Judging from the vesicular nature of the cytoplasm, a large amount of storage lipid bodies were also present (photo not shown). Differences in storage product deposition were observed in the cortical cells. Compared with control embryos, where starch was the predominant storage product in the cortex (Fig. 6M), the cortical cells of D7T embryos preferentially accumulated proteins and lipid bodies (Fig. 6N).
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Glutathione modulation of ethylene production
Significant differences in ethylene production between control and D7T embryos were visible after day 10 in culture (Fig. 7). Compared with control embryos, where the endogenous level of ethylene increased markedly after the first 10 d in culture, ethylene production did not increase in D7T embryos (Fig. 7).
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Localization of HBK1, a SAM-‘molecular marker’ gene
Shoot meristem integrity was further investigated by following the localization pattern of HBK1, a gene with preferential expression in the shoot apical meristem of spruce (Sundas-Larsson et al., 1998
). Probes were generated from the 3'-untranslated sequence to avoid possible cross-hybridization with other HBK genes (Hjortswang et al., 2002). RNA hybridization studies indicate that the localization pattern of HBK1 was similar in both control and D7T embryos during the first 10 d in culture (Fig. 8A–C). The localization of HBK1 transcripts was mainly restricted to the apical pole of the developing embryos. Differences in expression patterns were visible at day 20, when the localization of HBK1 was restricted to the apical cells of the control embryos (Fig. 8H), but was extended to the subapical cells in treated embryos (Fig. 8I). At day 40 HBK1 mRNAs still accumulated in the apical cells of control SAMs. However, the expression of this gene was reduced in poorly organized meristems which were disrupted by intercellular air spaces (Fig. 8K–M). In D7T embryos the localization of HBK1 mRNAs was retained in both apical and subapical cells (Fig. 8N).
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Discussion |
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Simple manipulations of the tissue-culture environment, through inclusions of glutathione in the medium, have been shown to improve the quality of embryos in different somatic embryogenic systems (Earnshaw and Johnson, 1987
; Jain et al., 1988). The present study indicates that an imposed switch of the glutathione pool from a reduced to an oxidized state increases the number of spruce somatic embryos (Figs 1, 2) and their ability to convert into viable plants (Fig. 3). The inclusions of GSH for 7 d in the maturation medium seems to favour cleavage polyembryony, the splitting of the immature somatic embryos, responsible for the proliferation of the embryogenic tissue. The initial GSH treatment increases the number of early-stage embryos which are then able to develop and mature properly in response to GSSG. The imposition of an oxidative environment effected by GSSG later in culture may be important for blocking cell expansion (de Pinto et al., 1999) and cell cycle progression (Reichheld et al., 1999), thus reducing proliferation of the embryogenic tissue and promoting proper embryo growth and development (Fig. 1). A high percentage of poor quality embryos is always observed in the absence of GSSG (Fig. 1). It is important to note that the beneficial switch towards a more oxidized environment, as revealed by the decreasing GSH/GSSG ratio over the maturation period of treated embryos, is not due to an increase in the endogenous GSSG level, since its level remains constant during the 40 d, but to a decrease of cellular GSH (Fig. 4). The endogenous level of this metabolite increased in control embryos, whereas it decreased steadily in D7T embryos (Fig. 4A). During the first 30 d in culture these opposite trends cannot be ascribed to the activity of glutathione reductase (GR), which catalyses the conversion of GSSG to GSH. The activity of this enzyme is higher in control embryos at day 10 and similar to that of treated embryos at days 20 and 30 (Fig. 5). However, during the last 10 d in culture the lower activity of GR in treated embryos may be responsible for the switch of the glutathione pool towards the oxidized state (i.e. low GSH/GSSG ratio) (Figs 4B, 5).
Alterations in glutathione metabolism have profound effects on embryo development. The higher numbers of mitotic figures observed in the forming cotyledons of developing (day 20) treated embryos, as well as the pronounced deposition of storage product (Fig. 6), are indications of improved development. In particular, the increased mitotic activity observed in treated embryos may be due to the high level of endogenous GSH still present in these embryos at day 20 (Fig. 4). The effect of this metabolite in promoting cell proliferation is well established in the literature (de Pinto et al., 1999; Smith et al., 2000; Belmonte et al., 2003, 2005). Accelerated cell division in the apical pole of developing treated embryos may be required for the formation of better quality cotyledonary embryos, having more cotyledons (Fig. 1) and organized shoot apical meristems (Fig. 6).
Compared with their control counterparts, the subapical and procambial cells of D7T embryos had small vacuoles and accumulated a larger amount of storage products. The accumulation of storage products observed in treated cells may be required for the acquisition of desiccation tolerance (Misra et al., 1993; Stasolla et al., 2004). The ability to withstand the imposed drying treatment and to restore metabolic processes promptly at germination is especially important for meristematic cells, which are responsible for the formation of functional organs. A reactivation failure of the meristematic cells at germination, common among control embryos (Fig. 3), results in poor post-embryonic performance and low conversion frequency. The differential pattern of storage product deposition observed between embryo types also suggests the presence of distinct physiological and metabolic processes. In treated embryos the initial deposition of starch granules, followed by lipids and protein bodies during the later stages of development, is reminiscent of the accumulation pattern observed in zygotic embryos (Yeung et al., 1998). The switch in storage product composition is precluded in control embryos, where starch is always the preferential product throughout the maturation period. A proper accumulation and subsequent mobilization of storage products is critical for successful regeneration. As also reported in this study, the regulation of storage product distribution and deposition has been shown to be under the control of the glutathione redox state (Rhazi et al., 2002; De Gara et al., 2003).
The improved architecture of the shoot apical meristems in treated embryos may be due to a reduction of ethylene production (Fig. 7). Accumulation of this hormone in the culture vessel has been directly implicated with the formation of intercellular spaces leading to meristem abortion at germination (Kong and Yeung, 1992). It appears that in spruce cells ethylene production is under the control of the glutathione redox state. A switch of the glutathione pool towards the reduced form, i.e. GSH, increases ethylene accumulation (Belmonte, 2003), possibly through the activation of ACC oxidase (Stasolla et al., 2004). An opposite trend is observed with the imposition of an oxidized environment, i.e. a low GSH/GSSG ratio (Fig. 8). Therefore, it appears that the oxidized environment which was imposed during the last weeks of embryo development, reduces ethylene accumulation, thereby preventing the formation of intercellular spaces and improving the quality of the shoot apical meristems. In line with this observation an experimental decrease in ethylene biosynthesis, effected by AVG, prevents cell–cell separation of the meristematic cells and results in an improvement of meristem architecture (Kong and Yeung, 1994).
Shoot apical meristem integrity was also examined by following the expression pattern of HBK1, a spruce gene which is preferentially localized in the meristematic cells of the apical pole (Sundas-Larsson et al., 1998). Based on its localization pattern during spruce embryogenesis (Fig. 8) and vegetative bud formation (Sundas-Larsson et al., 1998), it is speculated that HBK1 may have a primary role in the formation and maintenance of the shoot apical meristem, similar to that described for SHOOT MERISTEMLESS in Arabidopsis (Long et al., 1996; Sundas-Larsson et al., 1998). Both genes, in fact, encode homeodomain proteins of the KNOTTED class (Long et al., 1996; Hjortswang et al., 2002). In Arabidopsis, SHOOT MERISTEMLESS defines stem cell identity by repressing cell differentiation, thus maintaining the cells in a continual meristematic state (Barton and Poethig, 1993; Endrizzi et al., 1996). During spruce somatic embryo development, expression of HBK1 is strictly correlated to meristem quality, since its signal is reduced in those meristems disrupted by the presence of intercellular spaces (Fig. 8L, M). Furthermore, the enlarged expression pattern observed in meristematic cells of treated embryos (Fig. 8I, N) indicates that a switch of the glutathione pool towards the oxidized state may also be required for the formation of proper meristems, by conferring stem fate identity to a larger population of cells. The presence of a larger group of meristematic cells would then guarantee a higher conversion rate and better regeneration frequency.
In conclusion, this study clearly demonstrates that experimental manipulations of the glutathione redox state during spruce embryogenesis have profound effects on the embryogenic output and the quality of the embryos produced in culture. The initial applications of GSH would favour proliferation of the immature embryos, whereas a switch towards the oxidized state, through inclusions of GSSG, would promote proper development. Besides enhancing the structure of the shoot apical meristems, through inhibition of ethylene production, the oxidative environment (low GSH/GSSG ratio) may be required for the acquisition of cell stem identity of the meristematic cells. Overall, the glutathione-treated embryos have a zygotic-like appearance and are able to regenerate viable plants at high frequency.
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Acknowledgements |
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This research was supported by Natural Sciences and Engineering Research Council of Canada Grants to CS, ECY, and DMR. The generous financial contribution of CELLFOR and the technical help of Mr Luit are also appreciated.
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References |
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