JXB Advance Access originally published online on February 13, 2004
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Journal of Experimental Botany, Vol. 55, No. 397, pp. 695-709, March 1, 2004
© 2004 Oxford University Press
Regulation of Growth, Development and Whole Organism Physiology |
The effect of reduced glutathione on morphology and gene expression of white spruce (Picea glauca) somatic embryos
Received 17 July 2003; Accepted 25 November 2003
1 Department of Plant Science, University of Manitoba, Winnipeg, R3T 2N2 Manitoba, Canada
2 Department of Biological Sciences, University of Calgary, Calgary, T2N 1N4 Alberta, Canada
3 Forest Biotechnology Group, Department of Forestry, North Carolina State University, Raleigh, NC 27695-7247, USA
4 Baylor College of Medicine, 452A One Baylor Plaza, Houston, TX 77030, USA
* To whom correspondence should be addressed. Fax: +1 204 474 2578. E-mail: stasolla{at}ms.umanitoba.ca
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Abstract |
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Inclusions of reduced glutathione (GSH) in the maturation medium increased the conversion frequency of white spruce somatic embryos without the need of a partial drying treatment (PDT). This beneficial effect was the result of major alterations in morphology and gene expression during the maturation period. Compared with control embryos, GSH-treated embryos showed a differential accumulation of storage products, i.e. preferential deposition of starch, the reduced formation of protein bodies, and increased vacuolation of cells. These morphological changes correlated with extensive alterations of gene expression occurring throughout the maturation period. The transcript profiles of stage-specific embryos matured with or without GSH were analysed using a DNA microarray containing 2 178 cDNAs from loblolly pine (Pinus taeda). The efficiency of heterologous hybridization between spruce and pine species on microarrays has previously been documented. The results indicate that several genes involved in a variety of signal regulatory pathways were differentially expressed in developing GSH- treated embryos. The transcript levels of many genes involved in carbohydrate metabolism and protein synthesis were altered by the presence of GSH and denoted differences in physiology between treatments. Extensive changes in the expression of genes participating in hormone synthesis, nucleotide metabolism, and meristem formation were also observed and related to the post-embryonic performance of the embryos.
Key words: Embryogenesis, glutathione, microarray, transcript levels.
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Introduction |
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Desiccation is a natural event observed during the late phases of embryogensis. A severe decrease of moisture content, experienced by fully developed seed embryos, is a key event required for the termination of the developmental processes in preparation for germination (Kermode, 1990). The imposition of a water stress is also required in culture for increasing the conversion frequency of the embryos (Gray et al., 1987; Roberts et al., 1990; Attree et al., 1991). Methods for drying embryos, as summarized by Hay and Charest (1999), involve partial or full desiccation, depending on the rate and level of water loss. Both treatments enhance the conversion frequency of the embryos. The partial drying treatment (PDT), which involves a gradual and limited loss of moisture content was first reported by Roberts et al. (1990) and then utilized as a routine desiccation procedure for several coniferous species (reviewed by Stasolla and Yeung, 2003).
White spruce is one of the most widely distributed conifers in North America, where it is used for paper and pulp production. In recent years, several protocols for the propagation of spruce via somatic embryogenesis have been developed and they all require a desiccation treatment, including a PDT, at the end of the maturation period (Stasolla et al., 2002). The importance of the PDT in white spruce has been well documented. Compared with control embryos, white spruce somatic embryos subjected to a PDT were able to convert at high frequency and regenerated viable plants in culture (Kong and Yeung, 1992). Several studies have revealed that major physiological changes occur in the embryos during the PDT. These include alterations in storage product deposition (Joy et al., 1991), decreased synthesis of ethylene and abscisic acid (Kong, 1994), and variation in the pattern of nucleotide synthesis and utilization (Stasolla et al., 2001a). Responsiveness of conifer embryos to PDT appears to be species dependent. Optimal regeneration frequency was obtained after 10 d of PDT in white spruce somatic embryos, whereas more than 4 weeks of PDT were needed for the highest regeneration frequency in other coniferous species (Roberts et al., 1990). Furthermore, besides being relatively long, the PDT is also a tedious procedure, as individual embryos have to be selected and placed in multi-well tissue-culture plates. Therefore, optimization of the culture conditions, aimed at increasing the conversion frequency of the embryos without the imposition of a PDT, would be beneficial for a more efficient utilization of somatic embryogenesis.
Changes in the redox environment have pronounced effects on growth and development (Potters et al., 2002). Experimental manipulations of the ratio of several redox pairs, i.e. ascorbic acid/dehydroascorbic acid and reduced glutathione (GSH)/oxidized glutathione (GSSG), as well as alterations in the oxidize state effected by H2O2, have been shown to affect cell division and differentiation in several systems (Sanchez-Fernandez et al., 1997; Gardiner et al., 1998; de Pinto et al., 1999; Vernoux et al., 2000; Henmi et al., 2001; Ogawa and Iwabuchi, 2001). A reduced environment, effected by applications of ascorbic acid, improves white spruce embryo conversion by inducing cell proliferation in meristematic cells at the shoot apical pole (Stasolla and Yeung, 1999). Improve ments of the embryogenic process in spruce through manipulations of the GSH/GSSG ratio have also been documented (Belmonte and Yeung, 2004). A large fraction of GSH-treated embryos can be induced to convert into viable plants after direct transfer on the germination medium, without the imposition of a PDT. As an extension of this previous work and to contribute to the understanding of the molecular events associated with gymnosperm somatic embryogenesis, the steady-state transcript levels of 2 178 cDNAs from loblolly pine were compared in developing white spruce somatic embryos cultured in the absence (control) or presence (+GSH) of reduced glutathione. The utility of a pine cDNA array for studies on gene expression in spruce has been documented (van Zyl et al., 2002) and further utilized (Stasolla et al., 2003). The main objective of this work was to monitor the changes in transcript levels induced by GSH in relation to embryo morphology during development and post-embryonic growth. The results will be valuable for improving plant regeneration in conifers with modified media.
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Materials and methods |
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Plant material
White spruce (Picea glauca [Moench] Voss) embryogenic tissue was generated from zygotic embryos (Lu and Thorpe, 1987). Open pollinated seeds (lot numbers 7431580.1 and 7231587.2, respectively) were provided by the National Tree Seed Center (Fredericton, NB, Canada). Seeds were sterilized in 20% (v/v) commercial Javex bleach for 20 min and rinsed three times with sterile water. Dissected embryos were placed on induction (AE) medium (von Arnold and Eriksson, 1981) containing 10 µmol l–1 2,4 dichlorophenoxyacetic acid (2,4-D), 5 µmol l–1 N6-benzyladenine (BA), 5% (w/v) sucrose, and 0.8% (w/v) Purified agar (Becton-Dickinson), pH 5.8. The stock culture was maintained in the dark at 26 °C for 4–5 weeks. Embryogenic tissue was transferred onto a maintenance medium (1/2 strength LV medium containing 10 µmol l–1 2,4-D, 2 µmol l–1 BA, and 3% (w/v) sucrose) (Litvay et al., 1985) and was subcultured every 7 d. Somatic embryo development was initiated by transferring the embryogenic tissue onto solid maturation medium (1/2 strength LV medium containing 50 µmol l–1 abscisic acid [ABA], 5% (w/v) sucrose, and solidified with 0.4% (w/v) phytagel, pH 5.8) (Belmonte and Yeung, 2004). Reduced glutathione (GSH, 0.2 mM) was filter-sterilized and included in the autoclaved maturation medium, as reported by Belmonte and Yeung (2004). Although several levels of GSH were tested in the culture medium, this concentration (0.2 mM) resulted in optimal embryo conversion in several spruce cell lines. After 40 d, fully developed embryos were transferred onto the germination medium and the conversion of roots and shoots was recorded as a direct assessment of functionality (Stasolla and Yeung, 1999).
For studies on embryo morphology and post-embryonic growth, the effect of GSH was tested on three different cell lines: E(WS1), E(WS2), and E(WS3). Since very similar results were obtained for the three lines, only data obtained from the E(WS1) line were reported in this study. For micorarray analysis, tissue from the three lines was combined in equal amounts (1 g fresh weight) and utilized for the determination of transcript levels.
GSH and GSSG quantification
White spruce embryogenic tissue was collected on days 1, 4, and 7 of the 7 d maintenance period for glutathione analysis. Reduced and oxidized glutathione (GSH and GSSG) were determined by the 5,5'-dithio-bis-(2-nitrobenzoic acid)/GSSG reductase recycling assay according to the method of Zhang and Kirkham (1996).
Analysis of variance was used to compare the effect of exogenously supplied 0.2 mM GSH on endogenous levels of glutathione. Tukey’s Post-Hoc test for multiple variance was used to compare treatment effects within each group. All data were generated using the SPSS© version 10.0 statistical analysis software package.
Light microscopy
For light microscopic studies, control and GSH-treated embryos were fixed in 2.5% (v/v) glutaraldehyde and 1.6% (v/v) 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 2–3 µm sections were cut with glass knives on a Reichert-Jung 2040 Autocut rotary microtome. For general histological examination, sections were stained with the periodic acid–Schiff (PAS) reaction and counterstained with 0.05% (w/v) toluidine blue O in benzoate buffer, pH 4.4 (Yeung, 1999).
Microarray procedure
All microarray procedures were carried out as previously described (Stasolla et al., 2003). The 2 178 cDNAs were selected from 55 000 ESTs grouped in 9 000 contigs (Kirst et al., 2003). These ESTs were obtained from five different cDNA libraries: NXNV (Xylem Normal-wood Vertical), NXCI (Xylem Compression-wood Inclined), NXSI (Xylem Side-wood inclined), ST (Shoot Tip), and PC (Pollen Cone) (http://web.ahc.umn.edu/biodata/nsfpine/contig_ dir6). The cDNAs were selected closest to the 3'-end of the respective contig and were run on BLASTX against the A. thaliana database (www.arabi_all_proteins_v211200.tfa,MIPS). The best hit from the BLAST search was used for grouping the cDNAs into functional categories, as proposed for A. thaliana (http:// pedant.gsf.de). The selected cDNAs were transformed into E. coli XL-1 blue competent cells and the plasmids were isolated using Qiagen kits.
Probe preparation and printing
The cDNAs were PCR amplified in 50 µl reactions in 96-well reaction plates. Each 50 µl reaction contained 39.1 µl ddH2O, 0.5 µl of PCR reaction buffer containing 15 mM MgCl2 (Roche Molecular Biochemicals), 1 µl dNTPs, 1 µl of forward and reverse specific primers (10 µM), respectively, 0.4 µl Taq polymerase (5 U µl–1), and 2.54 µl of 100-fold diluted plasmid stock. Amplifications were carried out in MJ Research thermocyclers (Waltham, MA, USA) with the following conditions: denaturation at 94 °C for 30 s, annealing at 57 °C for 1 min, and elongation at 72 °C for 4 min. After 35 cycles, the final chain elongation was performed at 72 °C for 10 min. The PCR products were purified on Multiscreen filter plates (Millipore Corp. Bedford, MA) and analysed on ethidium bromide agarose gel. The purified DNA was denatured in 50% (v/v) dimethyl sulphoxide (DMSO), and spotted in four replicates onto CMT-GAPS aminosilane-coated glass microscope slides (Corning, Corning, NY), by using a 417 Arrayer (Affymetrix, Woburn, MA).
Target preparation
For each developmental stage of control and GSH-treated embryos, tissue (1 g fresh weight) was collected from three distinct cell lines, combined, and used for RNA extraction as described by Chang et al. (1993). cDNA probes were labelled using the aminoallyl procedure developed by De Risi (http://cmgm.stanford.edu/pbrown/protocols/index.html). RNA from each sample was labelled with Cy3 and Cy5 and used for reciprocal hybridizations. Hybridization and stringency washes were performed using the recent protocol from the Institute of Genomic Research (TIGR) (Hegde et al., 2000). The slides were scanned using a ScanArray 4000 Microarray Analysis System (GSI Lumonics, Ottawa, ON, Canada). Raw, non-normalized intensity values were collected with QUANTARRAY software (GSI Lumonics, Ottawa, ON, Canada). Using the quantification option, spots were visually inspected for spot morphology and background.
Experiment design and statistical analysis
A fully balanced, incomplete loop experimental design was used in this experiment, as proposed by Kerr and Churchill (2001). Gene significance was then estimated using the ‘mixed model system’ developed by Wolfinger et al. (2001) and Jin et al. (2001). This model, used in previous studies (Stasolla et al., 2003) is highly sensitive and shows that changes in gene expression less than 2-fold can be statistically significant (Jin et al., 2001). Briefly, the log2 transformed data (yijk) were subjected to a normalization model: yijk=µ+Ai+Dj+(AxD)ij+
ijk, where µ is the sample mean, Ai is the effect of the array, Dj is the effect of the dye, (AxD)ij is the effect of the array–dye interaction, and ij is the stochastic error. The residual values from this model were then fit into a gene-specific model in the form of rijk=µ+Ai+Tj+Nk+ijk, where Tj corresponds to the jth treatment (control and GSH), and Nk is the effect of the clone position on the array. Both models were implemented using PROC MIXED in SAS (SAS Institute Inc., SAS/STAT Software version 8, SAS Institute, Cary, NC, 1999). The least square means (probability value: P <0.01) and the differences in least square means between treatments were calculated from the gene-specific model and used for calculating fold changes (Stasolla et al., 2003). Fold changes were imported into GENESPRING, version 4.1 (Silicon Genetics, Redwood City, CA) and the ‘Make Tree’ function was used to perform hierarchical clustering of the genes.
Real time (RT)-PCR
The transcript levels of nine cDNAs (NXSI_044_F04, NXCI_053_ F03, NXSI_130_F05, NXNV_079_G01, NXSI_008_B02, NXSI_ 127_C02, NXNV_129_B12, ST_06_E01, NXNV_083_G07), which appeared differentially expressed during different stages of development between control and GSH-treated embryos in the microarray experiments were confirmed by RT-PCR. Total RNA was extracted as previously reported (Chang et al., 1993). First strand cDNA reverse transcription was performed as reported previously (Stasolla et al., 2003) The abundance of transcript levels was monitored on an ABI Prism 7900 Sequencer (Applied Biosystems) using SYBR Green PCR Master Mix (Applied Biosystems). The 18S amplicon was used as an internal control for normalization.
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Results |
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Development of white spruce embryos
White spruce somatic embryogenesis was divided into five distinct stages of development (Stasolla et al., 2003). Stage 1 was characterized by proliferating tissue maintained in the presence of 2,4-D and BA. At this stage, early filamentous embryos were observed. After 10 d on ABA-containing maturation medium (stage 2), the embryo proper increased in size. A well-developed shoot and root pole became visible after 20 d in culture (stage 3). After 30 d (stage 4), the embryos developed further, and a ring of cotyledons emerged from the shoot apical region. Fully mature embryos, characterized by well-developed cotyledons were observed at the end of the 40 d in culture (stage 5) (Fig. 1).
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Effects of GSH on embryo maturation and conversion
Inclusions of GSH increased the endogenous GSH level, but had no effects on the level of its oxidized form, i.e. GSSG. As a result, the GSH/GSSG ratio also increased (Fig. 2). After inclusion of GSH in the maturation medium, morphological differences between control and GSH-treated embryos started to appear early in culture.
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In control embryos, well-defined shoot and root apical meristems were visible at stage 3 of development (Fig. 3A, B). The shoot meristem was characterized by cytoplasmic apical cells located at the base of the forming cotyledons. Large root meristem initials were visible at the basal end of the embryos, between the procambial region and the root cap. A heavy accumulation of starch granules and storage protein bodies was observed at this stage (Fig. 3C). In GSH-treated embryos, many cortical and procambial cells appeared to be highly vacuolated (Fig. 3D, E). Mitotic figures were observed throughout the developing embryos, especially at the root pole (Fig. 3E). Compared with their control counterparts, GSH-treated embryos accumulated more starch, whereas protein bodies were absent at this stage of development (Fig. 3F).
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Completion of development occurred at stage 5 in both control and GSH-treated embryos. In control embryos, both root and shoot meristems were fully formed at this time and no mitotic figures could be detected (Fig. 4A, B). Storage protein bodies were abundant in the cytoplasm of many cells, and starch granules were still present (Fig. 4C). The cytological features of GSH-treated embryos showed a number of differences from the control. The most notable was that a majority of cells were vacuolated, especially in the procambial region (Fig. 4D). Judging from the protein stain, storage protein bodies were not as abundant, whereas a pronounced deposition of starch was observed in many cells (Fig. 4E, F).
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Marked differences in conversion frequencies, without the imposition of a PDT, were observed between control and GSH-treated embryos (Fig. 5). Embryos cultured with GSH during the maturation period showed improved shoot conversion (53% compared with 13% of control embryos), and overall embryo conversion (combined shoot and root conversion) (27% compared with 7% of control embryos) at germination. When the converted GSH-treated embryos were allowed to grow further, plantlets developed normally. Application of GSH in the maturation medium was also very effective in reducing the percentage of embryos unable to generate functional shoots and roots (12% compared with 75% of control embryos) (Fig. 5). Significant differences in the external morphology of germinating embryos matured with GSH were also observed (Fig. 6). Compared with germinating control embryos in which both shoot and root emergence was inhibited, GSH-treated embryos were able to convert into viable plantlets similar in morphology to those produced after the imposition of a PDT (Fig. 6).
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Global changes in transcript levels between control and GSH-treated embryos during development
Functional grouping of stage-specific genes differentially expressed between control and GSH-treated embryos during development is shown in Table 1. Compared with the early stages (stages 2 and 3), the total number of genes differentially expressed increased markedly during late phases of development (stages 4 and 5) (Table 1). An increasing percentage of differentially expressed genes, involved in metabolic processes and cell growth, was observed between control and GSH-treated embryos as maturation progressed. An opposite tendency was observed for genes participating in cell rescue and cell communication mechanisms (Table 1).
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Comparison of gene expression between neighbouring stages of embryo development in control and GSH-treated embryos revealed both quantitative and qualitative differences (Table 2). During the first three pair-wise comparisons (stages 1–2, 2–3, and 3–4) a lower number of genes were differentially expressed in GSH-treated embryos. The reverse was observed during late development (stage 4–5). Compared with control embryos, a higher percentage of genes involved in metabolic processes and cell growth were differentially expressed in developing GSH-treated embryos. Differences in expression between treatments were also observed for genes belonging to other functional categories (Table 2).
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Changes in transcript levels of representative genes belonging to the four largest categories on the array: metabolism, protein synthesis and destination, cell rescue, and transcription were analysed in detail.
Transcript levels of genes participating in metabolic processes
Representative genes involved in metabolic processes were grouped into five sub-categories: carbohydrate metabolism, nitrogen metabolism, cell wall modification and lignin formation, nucleotide and methionine metabolism, and hormonal control, and their transcript levels were compared between control and GSH-treated embryos during development (Fig. 7). Among genes participating in carbohydrate metabolism, two distinct groups were recognized. The transcript levels of the first group of genes, which included several glucanases, an acetyl-CoA synthase, and a phosphoenolpyruvate carboxylase, were higher in control embryos during the late phases of development. An opposite tendency was observed for genes of the second group, which were repressed in control embryos and included a transketolase, a fructokinase, and an aconitase hydratase (Fig. 7). Within genes involved in nitrogen metabolism, the transcript levels of a NADH-dependent glutamate synthase and an aspartate aminotransferase were higher in control embryos at stages 4 and 5, whereas those of genes encoding for glutamine synthase, asparagine synthase, and alanine aminotransferase were higher in GSH-treated embryos at stage 3. The transcript levels of almost all genes belonging to the sub-category ‘cell wall modification and lignin formation’ were induced in GSH-treated embryos during the late stages of maturation, with the exclusion of a chorismate mutase, a laccase precursor, and a laccase gene (Fig. 7). Among genes involved in nucleotide metabolism, the expression levels of an adenosine kinase and a uridinylate kinase-like protein were higher in developing embryos cultured with GSH. An opposite expression pattern was observed for a putative uracil phosphoribosyltransferase and a nucleoside diphosphate kinase. Differences in expression were also found for genes participating in methionine metabolism and processes related to hormonal regulation (Fig. 7).
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Transcript levels of genes involved in protein synthesis and destination and cell rescue mechanisms
Many genes involved in protein synthesis encoded ribosomal proteins and initiation and elongation factors. The transcript levels of several of these genes were higher in control embryos, especially at stages 4 and 5. A similar expression pattern was also observed for several late embryogenic abundant (LEA) proteins (Fig. 8). Within the sub-category ‘cell rescue mechanism’, transcripts for a heat shock protein 18, two hypothetical proteins, a thaumatin-like protein, and a glutathione S-transferase were increased in control embryos, whereas all the others were repressed, especially during the late stages of development. These included transcripts for an ascorbate peroxidase and two superoxide dismutases (Fig. 8).
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Transcript levels of genes involved in transcription mechanisms
Within the genes falling into the sub-category ‘transcription’, there were several involved in the control of the shoot apical meristem, i.e. CLAVATA1 (CLV1), NO APICAL MERISTEM (NAM), and ARGONAUTE (AGO). All these transcripts, with the exception of one of the two CLV 1 receptor kinase, were increased in GSH-treated embryos during the late phases of development (Fig. 9). A similar up-regulation was also observed for a scarecrow-like transcription factor. Differences in transcript levels between treatments were found for the remaining genes (Fig. 9).
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Validation of microarray data
Validation of the results of the microarray experiment was confirmed by RT-PCR studies of nine cDNAs representative of the different sub-categories discussed in this study: metabolism (NXCI_053_F03, NXSI_130_F05, NXSI_ 127_C02, and NXNV_079_G01), protein synthesis (NXSI_044_F04), cell rescue mechanisms (NXSI_008_ B02), and transcription (NXNV_129_B12, ST_06_E01, NXNV_083_G07). Changes in transcript levels were found between control and GSH-treated embryos at specific stages of development (Figs 7–9). Consistent results were obtained in the expression pattern (up- or down-regulation) of the selected clones, although variations in fold changes were observed between the two hybridization techniques (Table 3), as also reported previously (Stasolla et al., 2003).
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Discussion |
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Applications of GSH enhance spruce somatic embryo conversion without the imposition of a PDT and induce major changes in embryo morphology and gene expression. These changes are observed early during development (Fig. 3; Tables 1, 2), and persist throughout the embryogenic process (Fig. 4; Tables 1, 2). The beneficial effect of GSH on embryo conversion is probably due to the activation of a large number of regulatory pathways, as demonstrated by the different functions of the genes expressed during embryo development (Tables 1, 2).
Many of the genes affected by GSH treatment are involved in carbohydrate metabolism and protein synthesis. Within carbohydrate metabolism, two genes encoding for a triose phosphate isomerase and an aldolase were differentially expressed between control and GSH-treated embryos. The involvement of GSH in the regulation of these two enzymes was also shown in Arabidopsis, where their activities were dependent upon the formation of an intramolecular disulphite bridge with GSH (Ito et al., 2003). Therefore, it appears that GSH may influence the activity of important key enzymes by regulating their transcription and by participating in post-translational modifications. A correlation between changes in transcript accumulation and pattern of starch and protein accumulation is observed in developing embryos. The more pronounced accumulation of protein bodies in control embryos between stages 3 and 5 (Figs 3, 4) may be related to the increased transcript levels for several genes involved in protein synthesis, such as ribosomal proteins, initiation and translation factors, and late embryogenic abundant (LEA) proteins (Fig. 8). Accumulation of storage proteins allows embryos to withstand severe and prolonged water stress during the desiccation period, when they enter a quiescent phase (Kermode, 1990). The lower transcript levels of genes involved in protein synthesis, together with the reduced accumulation of protein bodies, indicate that GSH-treated embryos are not physiologically ready for desiccation. Rather, these embryos can switch to a germination mode without a PDT and without entering a quiescent phase. Contrary to previous studies, which have emphasized the requirement of a complete or ‘zygotic like’ protein set for successful germination of somatic embryos (Joy et al., 1991; Misra et al., 1993), the present work suggests that completion of storage product accumulation may not be critical for post-embryonic growth.
Several other differentially expressed genes are involved in nucleotide metabolism, including one adenosine kinase, which is induced in GSH-treated embryos (Fig. 7). This enzyme, which participates in the salvage of purine nucleosides, catalyses the conversion of adenosine to AMP (reviewed by Stasolla et al., 2004). In spruce, the activity of adenosine kinase is high during the PDT (Stasolla et al., 2001a), and increases at the onset of germination, in conjunction with the reactivation of the apical meristems (Stasolla et al., 2001b). In this latter study it was also demonstrated that conditions that enhance the conversion frequency of the shoot apical meristem in germinating embryos also increase the salvage of adenosine. Thus, it appears that higher transcript levels of adenosine kinase in GSH-treated embryos may contribute to the higher shoot conversion observed in the absence of PDT (Figs 5, 6). An active purine salvage pathway during the initial periods of growth and development contributes to the enlargement of the endogenous nucleotide pool, before the reactivation of the de novo synthesis of nucleotides, which appears to be a later event (Stasolla et al., 2003). Differences in transcript levels of genes encoding for uracil phosphotransferase, nucleoside diphosphate kinase, and uridinylate kinase (Fig. 7) further substantiate the involvement of GSH in nucleotide synthesis and utilization during embryonic growth. These results open exciting possibilities for improving somatic embryogenesis through experimental manipulations of the nucleotide biosynthetic pathways.
Genes belonging to the sub-category ‘hormonal control’ are also differentially expressed between treatments. Among them, there are several encoding for auxin-induced proteins, as well as one encoding for an anthranilate phosphoribosyltransferase, the enzyme which participates in the formation of the indole group during indole-3-acetic acid biosynthesis (reviewed by Bartel et al., 2001). Alterations in auxin synthesis and flow are crucial for the quality of the embryos produced in culture. In flowering plants, experimental inhibitions of auxin flow both in vivo (Steinmann et al., 1999) and in vitro (Ramesar-Fortner and Yeung, 2000) have deleterious effects on the quality of the embryos. Although no related information is currently available for gymnosperms, GSH-treated embryos may have different levels/distributions of endogenous auxin, which affect post-embryonic growth.
Another gene differentially expressed between treatments is aminocyclopropane-1-carboxylate (ACC), which is induced by GSH at any stage of development. Higher transcript levels of ACC oxidase have often been associated with increased ethylene accumulation (Hamilton et al., 1990), suggesting preferential production and, possibly, the accumulation of this hormone in GSH-treated embryos. The effect of GSH on ethylene production is likely to occur indirectly, through the salvage of ascorbic acid in the Halliwell–Asada cycle (Potters et al., 2002). The GSH-mediated recycling of ascorbic acid would promote ethylene synthesis through the activation of ACC oxidase, which uses ascorbic acid as a cofactor (reviewed by Gaspar et al., 1996). The involvement of ethylene during the initial phases of post-embryonic growth in culture is poorly defined. However, applications of ascorbic acid during germination of white spruce increase ethylene production (C Stasolla and EC Yeung, unpublished data), as well as shoot meristem conversion (Stasolla and Yeung, 1999). Thus, similar mechanisms related to ethylene synthesis and resulting in improved shoot conversion may be regulated by applications of GSH at maturation or ascorbic acid at germination.
The ability of the embryos to germinate and convert into viable plantlets is dependent upon the quality of the apical meristems produced during development (Kong and Yeung, 1992). In Arabidopsis, the acquisition of ‘meristematic identity’ of the cells at the apical poles of the embryos and the establishment of a functional meristem are regulated by a complex network of interactions among genes, which include ARGONAUTE (AGO), NO APICAL MERISTEM (NAM), and CLAVATA1 (CLV1) (Clark, 2001). The increased expression levels of several genes homologous to AGO, NAM, and CLV1 in embryos treated with GSH indicate that this compound may affect the quality of the shoot apical meristem, possibly by making it more responsive to germination conditions. Proper formation and maintenance of the shoot apical meristem rely on the precise regulation of cell division and differentiation, as an improper execution of these two events results in abnormal meristems and reduced growth (Fletcher and Meyerowitz, 2000). The improved shoot conversion observed in the presence of GSH indicates that this metabolite may affect cell division and differentiation in meristematic cells of spruce embryos. The involvement of GSH and its oxidized form, GSSG, in the control of cell division and differentiation has been reported in animal cultured cells (Smith et al., 2000).
In conclusion, this work demonstrates that applications of GSH increase the conversion frequency of white spruce somatic embryos, without an intervening desiccation period. Examination of transcript levels has revealed that the inductive effect of GSH on plant regeneration may be related to profound alterations in the expression of genes involved in important metabolic pathways and developmental processes. Altogether this information will be valuable for ongoing studies aimed at improving somatic embryogenesis in conifers through rational manipulations of culture conditions.
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Acknowledgements |
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This research was supported by Natural Sciences and Engineering Research Council of Canada Research Grants to CS and ECY, a National Science Foundation (USA) Grant to RRS, and by the NCSU Forest Biotechnology Industrial Research Consortium. The assistance of Mr Daniele Riscica is also acknowledged.
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References |
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