Cellular ascorbic acid regulates the activity of major peroxidases in the apical poles of germinating white spruce (Picea glauca) somatic embryos

Plant Physiology and Biochemistry 45 (2007) 188e198 www.elsevier.com/locate/plaphy

Research article

Cellular ascorbic acid regulates the activity of major peroxidases in the apical poles of germinating white spruce (Picea glauca) somatic embryos Claudio Stasolla a,*, Edward C. Yeung b a

Department of Plant Science, University of Manitoba, 222 Agriculture Building, Winnipeg, Manitoba, Canada 3RT 2N2 b Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4 Received 12 September 2006; accepted 19 February 2007 Available online 25 February 2007

Abstract In previous studies we have reported that applications of ascorbic acid (ACS) enhance the conversion frequency of white spruce somatic embryos by ‘‘rescuing’’ structurally disorganized meristems and inducing cell proliferation in the apical poles [C. Stasolla, E.C. Yeung, Ascorbic acid improves the conversion of white spruce somatic embryos, In Vitro Cell. Dev. Biol. Plant 35 (1999) 316e319]. In order to determine if the role played by this metabolite during embryo conversion is mediated by cellular peroxidases, the activity of guaiacol-, ferulic acid-, and ascorbic acid-dependent peroxidases were measured in the apical poles of germinating embryos with altered ASC levels. Changes in the endogenous ASC pool were achieved by treating the embryos with exogenously supplied ASC, L-galactono-g-lactone (GL) the last precursor of the de novo biosynthesis of ASC, and lycorine (L), an inhibitor of the last reaction leading to the synthesis of ASC. Our studies demonstrate the existence of a negative correlation between cellular ASC levels and activities of both guaiacol and ferulic acid peroxidases in root and shoot apices. A depletion of cellular ASC enhanced the rate of both guaiacol and ferulic acid oxidation at the apical poles of the embryos and resulted in meristem abortion. In contrast, the activity of guaiacol and ferulic acid peroxidases decreased below control levels if the endogenous ASC content of the embryos was experimentally increased. Fluctuations of total peroxidase activity following alterations in ASC pool were also confirmed by histochemical staining and in vitro studies. Overall our results suggest that a threshold of ASC level must be maintained in the apical poles of germinating embryos in order to inhibit peroxidase activities from cross-linking cell wall components and preventing post-embryonic growth. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Ascorbate; Conversion; Ferulic acid; Guaiacol; Meristems; Picea glauca

1. Introduction The ability of white spruce somatic embryos to convert into viable plantlets is strictly dependent upon the reactivation of the apical meristems at germination. Upon imbibition meristematic cells within the shoot and root apical poles start dividing, thus contributing cells to the surrounding tissues and allowing the growth of the embryos. Such a process, occurring normally in zygotic embryos, is often precluded in their

* Corresponding author. Tel.: þ1 204 474 6098; fax: þ1 204 474 7528. E-mail address: [email protected] (C. Stasolla). 0981-9428/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2007.02.007

somatic counterparts resulting in poor post-embryonic performance [14,24]. Failure of meristems to reactivate at germination has been ascribed to structural abnormalities in the apical poles, which harbor the meristematic cells [14]. In developing spruce somatic embryos such abnormalities include the formation of large intercellular spaces, improper accumulation of storage products, and differentiation of meristematic cells [14]. Reactivation of these ‘‘abnormal’’ meristems at germination, which is often precluded under standard culture conditions, can be induced upon applications of ASC [23]. Although the inductive effect of ASC on mitotic reactivation and cell growth is not well defined, our previous studies indicate that this metabolite may directly affect the synthesis of

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thymidine and uridine nucleotides, both of which are needed for nucleic acid synthesis [24]. Furthermore, histological studies conducted during tobacco shoot organogenesis have revealed that applications of exogenous ASC decrease the amount of phenolic compounds in developing shoots [12]. It is generally accepted that deposition and cross-linkage of phenolic compounds, driven by peroxidases, affect the architecture of the cell wall and ultimately cell elongation and cell division. As discussed in [8], increased peroxidase activity and cross-linkage of phenolic compounds often result in the stiffening of the cell wall and in the reduction of both cell elongation and cell division. Therefore, in order to understand the regulation of cell wall extensibility during growth, it is important to understand the biological factors regulating peroxidase activity. As reviewed in [3], these include the synthesis of peroxidases, their secretion at the wall site, and the availability of substrates, i.e. phenols and H2O2. Beyond their involvement in the cross-linkage of phenolics, peroxidases also participate in the formation of other bonds among cell wall structural components, such as glycoproteins and polysaccharides [3]. As such, measurements of major peroxidases are often indicative of the growth potential of the tissue. For example, total peroxidase activity is usually low in meristematic tissue, characterized by rapid cell division, and high in fully differentiated tissues, where both division and elongation have ceased [5]. Among the factors involved in the modulation of peroxidases activity, ASC seems to play a prominent role. A negative correlation between endogenous ASC levels and total peroxidase activity was observed in some systems [5]. High levels of ASC have demonstrated to inhibit peroxidase activity both in vitro [28,29] and in vivo [4]. Therefore, it is the objective of this study to determine whether experimental manipulations of the endogenous ASC level affect apical meristem growth in white spruce somatic embryos through regulation of cellular peroxidases. In order to test this hypothesis, total peroxidase activity, as well as peroxidases involved in the cross-linkage of cell wall components and removal of reactive oxygen species, were assayed by following the oxidation of guaiacol, ferulic acid, and ascorbic acid, respectively. These analyses were performed after altering the endogenous ASC content in germinating white spruce somatic embryos by using L-galactono-g-lactone (GL), the last precursor of the de novo biosynthesis of ASC, and lycorine (L), an inhibitor of the last reaction leading to the synthesis of ASC. 2. Materials and methods 2.1. Plant material White spruce (Picea glauca [Moench] Voss) embryogenic tissue was generated as previously reported [18]. Seeds collected from the Campus at the University of Calgary were surface sterilized with 20% Javex bleach for 20 min and rinsed three times in sterile water. Dissected zygotic embryos were cultured for 4e5 weeks on the induction medium AE [31], supplemented with 10 mM 2,4-dichlorophenoxyacetic acid (2,4-D), 5 mM N6-benzyladenine (BA), 5% (w/v) sucrose,

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and 0.8% Difco Bacto-agar, pH 5.8. The selected embryogenic tissue (E)WSC was transferred in a solid maintenance medium (AE medium containing 10 mM 2,4-D, 2 mM BA, 3% sucrose, 0.8% Difco Bacto-agar, pH 5.8) and subcultured every 7 days. Promotion of embryo development was achieved as previously described [14]. Briefly, 50 mg fresh weight of embryogenic tissue was spread on maturation medium (AE medium containing 50 mM abscisic acid [ABA], 5% sucrose, 0.8% Difco Bacto-agar, pH 5.8). After 40 days of incubation in the dark, mature somatic embryos, characterized by welldeveloped cotyledons were obtained. Embryo desiccation was carried out as reported by Roberts et al. [21]. Mature embryos (approx. 10), supported by a sterile filter paper disk (Whatman no. 1), were placed in the center wells of a 24well tissue culture plate (Falcon 3847, Franklin Lakes, NJ, USA). High relative humidity was generated by filling the outer wells of the plate with sterile water. The embryos were incubated for 10 days in the dark before germination. Germination was achieved by placing the partially dried embryos on germination medium (half strength AE medium supplemented with 1% sucrose and devoid of growth regulators), and incubated in a 16-h photoperiod (photon flux density of 90e 95 mmol2 s1, PAR; 380e800 nm). 2.2. Alterations of the endogenous ASC levels and tissue sampling Several metabolites were utilized in this experiment for altering the endogenous ASC level of the germinating embryos. Ten microliters of liquid germination medium (control), or 10 ml of liquid germination medium with 1 mM ascorbic acid (ASC), 2 mM L-galactono-g-lactone (GL), and/or 100 mM lycorine (L), were applied every 24 h on germinating embryos. Each solution, pH 5.8 was filter sterilized with a 0.2 mM Nalgene syringe filter. Prior to each application, the embryos were transferred onto fresh germination medium to avoid the accumulation of degradation products of the metabolites applied the previous day. Embryos were collected at days 0, 4, 2, 6, and 12, rinsed in distilled water and utilized for ASC measurements and enzyme assays. The concentrations of ASC, GL, and L used in this experiment have been found to be optimal for the embryos produced by the (E)WSC line in altering the endogenous ASC level without having toxic effects on the germinating embryos. Furthermore, the effects of these metabolites described for the (E)WSC line were also reproducible in other lines, i.e. (E)WS2 and (E)WS3, although with different optimal concentrations. Data from these other lines will be included in the text to validate the most important results. Unless specified, at least three independent biological replicates were used for each time point in the experiment. 2.3. ASC measurements Germinating embryos were ground in 5% metaphosphoric acid (1:5 w/v) at 4  C to prevent ASC oxidation and degradation.

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The homogenate was then centrifuged for 20 min at 16,000  g and the supernatant was collected for analysis of ASC. Ascorbate levels were measured as previously reported [26].

and counterstained with 0.05% (w/v) toluidine blue O in benzoate buffer, pH 4.4 [33]. The preparations were examined and photographed with a Leitz Aristoplan light microscope.

2.4. Enzyme extraction and assays of peroxidase activities

2.7. Cytochemical localization of total peroxidase activity in germinating embryos

Germinating embryos or shoot and root segments (1 mm from the apical tips) excised from the germinating embryos were homogenized at 4  C with a medium containing 50 mM potassium phosphate buffer, pH 6.0, 1 mM EDTA, 2 mM DTT, Triton (0.5% v/v), and PVPP (1.5% w/v). After centrifugation at 4  C for 20 min at 16,000  g, the supernatant was desalted on a pre-packed column of Sephadex G-25 (NAP-25, Pharmacia, Biotech Inc.) and utilized for the enzyme assays. Ascorbate peroxidase activity was measured by following the hydrogen peroxide-dependent oxidation of ASC by means of the decrease in absorbance at 265 nm. The reaction mixture contained 50 mM ASC, 90 mM H2O2, and 50 mM potassium phosphate buffer, pH 6.5 [1]. Guaiacol peroxidase (GP) activity was determined as described in previous work [4]. The reaction, carried out in a mixture containing 50 mM sodium phosphate, pH 6.0, H2O2 (0.03% v/v), and 3 mM guaiacol, was followed spectrophotometrically for 1 min at 470 nm. Ferulic acid peroxidase (FP) activity was measured in a reaction mixture similar to that utilized for GP determination, without guaiacol and with the addition of 0.2 mM ferulic acid. The reaction was followed for 1 min at 310 nm [4]. Protein was determined according to Lowry et al. [17] using BSA as a standard. For statistical analysis, the StudenteNewmaneKeuls test [36] was utilized.

Germinating embryos were fixed for 3 h in 4% paraformaldehyde buffered with 0.05 M phosphate buffer, pH 6.9. After washing, the tissue was dehydrated in a ethanol series and then infiltrated and embedded in Historesin (Leica, Markham, Canada) [34]. Sectioning was carried out with glass knives on a Reichert-Jung 2040 Autocut microtome. Serial longitudinal sections were cut at a thickness of 4 mm. For cytochemical localization of total peroxidase activity, the sections were stained for 15 min in 50 mM sodium phosphate buffer, pH 6.0 containing hydrogen peroxide (0.1%) and 3 mM 3,3-diaminobenzidine as a non-specific electron donor [10]. Control slides were incubated in a similar medium devoid of hydrogen peroxide. The preparations were examined and photographed with a Leitz Aristoplan light microscope. 2.8. Effects of ASC on ferulic acid and guaiacol oxidation In order to determine whether applications of ASC affected the oxidation of ferulic acid and guaiacol in vitro, 20 mg of protein extracted from 6-day-old germinating control embryos was utilized for assaying guaiacol and ferulic acid peroxidases, as described above. For this experiment, however, increasing concentrations of ASC (25, 50, or 100 mM) or 2 units of ASC oxidase (Sigma) were added to the spectrophotometric cuvette prior to reading. The delay in the oxidation of ferulic acid or guaiacol was monitored for more than 2 min at 310 nm for ferulic acid and 470 nm for guaiacol.

2.5. Native PAGE analysis of total peroxidase activity 3. Results Treated embryos were ground in the same extraction buffer utilized for enzyme assay (1:2 w/v). After centrifugation at 4  C for 20 min at 16,000  g, the supernatant was used for electrophoresis analysis. For staining, the gels were incubated at 4  C in 0.05 M sodium phosphate buffer pH 6.0, containing hydrogen peroxide (3%), 0.1 M CaCl2, and 3 mM 3-amino9-ethyl carbazole, a non-specific electron donor for total peroxidases [23]. 2.6. 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, Markham, Canada) [34]. Sectioning was carried out with glass knives on a Reichert-Jung 2040 Autocut microtome. Serial longitudinal sections were cut at a thickness of 3 mm. For general histological examinations, the sections were stained with the periodic acid-Schiff (PAS) procedure

3.1. Endogenous ASC content of treated embryos Modulations in the endogenous ASC levels were carried out by exogenous applications of ASC [2], GL, the last precursor in the de novo synthesis of ASC [11], and L which is a potent inhibitor of the conversion of GL to ASC [6]. The endogenous ASC content of control embryos produced by the (E)WSC line was close to 2 nmol embryo1 after 2 days in germination, and increased as germination progressed (Fig. 1). Applications of L and GLþL resulted in a decline of the endogenous ASC content. After 12 days of germination, in fact, the ascorbate levels of (L)- and (GLþL)-treated embryos were close to 8 nmol embryo1 respectively, compared to 16.3 nmol embryo1 of control embryos. Higher levels of endogenous ascorbate were measured in embryos germinated in the presence of exogenous ASC, GL, and ASCþL. As revealed by statistical analysis, significant differences were observed in the endogenous ASC level between control and all the other treatments after a few days in culture.

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to that of control (C) embryos. Applications of lycorine alone (L) or L-galactono-g-lactone plus lycorine (GLþL), resulted in a dramatic decrease in conversion frequency (21.1% and 26.9%, respectively), whereas only a slight decline in the percentage of embryos able to convert into viable plantlets was observed in the presence of ascorbic acid plus lycorine (ASCþL) (Fig. 2). A reduction in conversion frequency of more than 60% was also observed for embryos produced by the lines (E)WS2 and (E)WS3 when germinated in the presence of L or GLþL. In these lines the conversion frequency of untreated (control) embryos was more than 86% and it was not increased by applications of exogenous ASC (data not shown).

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Fig. 1. Effects of L-galactono-g-lactone (GL), lycorine (L), and ascorbic acid (ASC) treatments on endogenous ASC concentration of germination white spruce somatic embryos. Values are means  SE of three independent experiments. C, control. * indicates values that are significantly different from control (P < 0.01).

At day 12, the endogenous ASC level of embryos generated from the other two lines, i.e. (E)WS2 and (E)WS3 more than doubled in the presence of exogenous ASC, GL, and ASCþL. Applications of GLþL and L significantly lowered the level of endogenous ASC (data not shown).

After 2 days of germination the shoot pole of the control embryos had a flat-shaped appearance. Resumption of mitotic activity in the meristematic cells was observed around day 4 (Fig. 3A). During the following days in culture, continual cell division in the apical pole resulted in a dome-shaped shoot apical meristem (Fig. 3B). A similar growth pattern was also observed in ASC-treated embryos and in that percentage of ASCþL-treated embryos able to convert into viable plantlets (data not shown). Resumption of mitotic activity was a rare event in shoot apices of L-treated embryos. Accumulation of phenolic substances, especially in cell walls, and vacuolation of the meristematic cells resulted in growth cessation and meristem abortion (Fig. 3C and D). The root apical meristem of the somatic embryos was composed of large, densely cytoplasmic cells. At the beginning of the germination period, the root apical meristem of the somatic embryos consisted of large cells occupying the central region of the embryonic root (Fig. 3E). In control embryos and in embryos cultured with ASC and ASCþL reactivation of the root meristems, as denoted by the presence of mitotic figures, was observed around day 4 and continued during the following days in culture (Fig. 3F). In many L-treated embryos the root apical meristem failed to reactivate. Cells in the root pole 100

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Fig. 3. Micrographs showing the effects of the endogenous ASC levels on the structure of the apical meristems of germinating embryos. Mitotic activity (arrowheads) in the shoot apical meristems of control embryos was observed after 4 days in culture (A). Continuation of mitotic activity in control meristems resulted in the appearance of a dome-shaped shoot primordium (arrow), which became visible after 8 days in the germination medium (B). Shoot meristem reactivation was not observed in embryos cultured with lycorine (L). These meristems accumulated phenolic compounds (arrow), especially on the cell walls of apical cells after 4 days in culture (C). Continual deposition of phenolics (arrowheads) and vacuolization of the sub-apical cells (*) was observed during the following days in culture in the meristem of L-treated embryos (D). The root meristem of control embryos were characterized by the presence of a large cluster of cells located in the central region of the embryonic root (E). In these embryos, meristem reactivation was observed after day 2, as denoted by the presence of several mitotic figures (arrows) (F). Cells within the root meristems of L-treated embryos never divided. Accumulation of phenolic compounds (arrowhead) and vacuolation (*) started to appear after 2 days in culture (G). All scale bars ¼ 40 mm. The growth pattern of control embryos was similar to that of ASC, GL, and ASCþGL treated embryos; whereas the growth pattern of L-treated embryos was similar to that observed for embryos cultured in the presence of GLþL.

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started to elongate and accumulated large vacuoles and phenolic substances. After 6 days in culture, such meristems were poorly organized and the formation of intercellular air spaces resulted in cell separation and meristem abortion (Fig. 3G). Similar morphological changes to those described for the (E)WSC embryos were also observed for the embryos produced by the lines (E)WS2 and (E)WS3 when the endogenous ASC level was experimentally altered (data not shown).

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In intact control embryos produced by the line (E)WSC the activity of ferulic acid peroxidase increased as germination progressed. In embryos treated with ASC, ASCþL, or GL the activity of this enzyme was lower than that of their control counterparts after a few days in culture. An opposite tendency was observed in embryos cultured in the presence of L and GLþL (Fig. 4). A low ferulic acid peroxidase activity was also observed in shoot apexes excised from embryos treated with ASC, GL, or ASCþL. Increasing activity of this enzyme during the germination period was measured for control, as well as in shoot apexes of GLþL and L-treated embryos. At the end of the culture period the highest peroxidase activity was observed in shoot apexes of embryos cultured in the presence of GLþL and L (Fig. 4). In root segments the highest levels of ferulic acid oxidation were observed in the presence of L and GLþL at any day in culture (Fig. 4). Very similar changes in ferulic acid oxidation were also observed in (E)WS2 and (E)WS3 embryos. However, when these embryos were treated with either L or GLþL the activity of ferulic acid peroxidase increased of only 80% compared to control values (data not shown). For all treatments the activity of guaiacol peroxidase gradually increased in intact embryos during the germination period (Fig. 5). Higher levels of guaiacol oxidation were observed in embryos cultured with L and GLþL. A similar but more pronounced profile was observed in shoot segments. Compared to control values the activity of guaiacol peroxidase increased markedly after day 6 in shoots of L and GLþL treated embryos, whereas it remained low in shoots of embryos cultured in the presence of ASCþL. GL, and ASC (Fig. 5). In control root segments guaiacol oxidation was low during the first 6 days in culture and increased thereafter. A pronounced increase in guaiacol peroxidase activity was also measured after only 4 days in roots of embryos germinated in the presence of L and GL (Fig. 5). As observed for the shoot apices, guaiacol oxidation was lowest in roots excised from embryos treated with ASC, ASCþL, or GL at the end of the culture period. Similar changes in guaiacol peroxidase activity were also observed for the embryos produced by the lines (E)WS2 and (E)WS3 (data not shown). Regardless of treatment, the activity of ascorbic acid peroxidase increased in embryos and apical segments during the 12 days of germination (Fig. 6). No changes in ascorbic acid peroxidase among treatments were observed in embryos produced by the lines (E)WS2 and (E)WS3 (data not shown).

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3.5. Histochemical localization of peroxidases in shoot and root apices Histochemical localization of peroxidase activity was determined in shoot and root apices of control and treated embryos. Staining for total peroxidase activity, not observed in partially dried embryos (data not shown), was first detected after 2 days of germination. At day 6 peroxidase activity was localized mainly along the hypocotyl axis of control embryos (data not shown), whereas both apical meristems had low staining

C. Stasolla, E.C. Yeung / Plant Physiology and Biochemistry 45 (2007) 188e198

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Fig. 5. Effects of L-galactono-g-lactone (GL), lycorine (L), and ascorbic acid (ASC) applications on the in vivo activity of guaiacol-dependent peroxidase in germinating white spruce somatic embryos and in shoot and root segments. Values are means  SE. C, control. 1 unit ¼ 0.1 Abs. min1 mg protein1.

Fig. 6. Effects of L-galactono-g-lactone (GL), lycorine (L), and ascorbic acid (ASC) applications on the in vivo activity of ASC-dependent peroxidase in germinating white spruce somatic embryos and in shoot and root segments. Values are means  SE. C, control. 1 unit ¼ 1 nmol ASC oxidized min1 mg protein1.

intensity (Fig. 7A,B). Compared to control embryos, shoot and root meristems of (L)-treated embryos stained more intensely (Fig. 7C,D). In contrast, exogenous applications of ASC, ASCþL, or GL decreased the staining intensity in the apical poles of the embryos (Fig. 7E,F). However, upon further development as root and new leaf primordial emerged, a similar staining pattern was observed between control embryos and embryos with increased level of endogenous ASC (data not shown). A similar staining pattern to that observed for the (E)WSC embryos was also observed for the embryos produced by the other two lines, i.e. (E)WS2 and (E)WS3.

At this stage, three light bands were visible in control embryos (Fig. 8). Treatments that increased the endogenous ASC level, i.e. embryos cultured with ASC, ASCþL, or GL, only had one visible band. A similar staining profile to that observed in control embryos, but with bands more intensely stained, was observed in embryos in which the endogenous ASC level was reduced by applications of L or GLþL (Fig. 8).

3.6. Polyacrylamide gel electrophoresis (PAGE) As revealed by native PAGE analysis, total peroxidase activity appeared in germinating embryos after 2 days in culture.

3.7. ASC reduces the oxidation of both ferulic acid and guaiacol Both ferulic acid- and guaiacol-dependent peroxidases were inhibited in vitro by the presence of ASC (Fig. 9). The inhibition time of both peroxidases was proportional to the ASC concentrations added in the spectrophotometric cuvette, and the ASC inhibition ceased as soon as all ASC in the

Fig. 7. Histochemical localization of total peroxidase activity in shoot and root apices of control (C), ASC-treated embryos (ASC) and lycorine treated embryos (L). Reduced staining for total peroxidase activity was observed in the cells of both shoot apical meristem (arrowhead) (A) and root apical meristem (arrowheads) (B) of control embryos. Many cells in the apical meristems (C, D) of L-treated embryos stained for total peroxidases. Very fain staining was observed in shoots (E) and roots (F) of ASC-treated embryos. Peroxidase activity was detected in both cell wall and cytoplasm of these cells. Slides were incubated in the presence of 3-amino-9-ethyl carbazole, a non-specific electron donor for total peroxidases (see Materials and Methods). All scale bars ¼ 40 mm.

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reaction mixture was oxidized. Removal of ASC from the extraction buffer though applications of ASC oxidase did not affect the rate of ferulic acid and guaiacol oxidation (Fig. 9).

4. Discussion

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Fig. 8. Electrophoretic profiles of proteins extracted from 6-day-old control embryos and embryos treated with ascorbic acid (ASC) and lycorine (L). The gel was stained for total peroxidase activity. 150 mg protein were loaded per lane.

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Reactivation of the apical meristems at germination is a complex process, requiring a proper balance between cell proliferation and differentiation. Through a series of coordinated cell divisions, both shoot and root apical meristems contribute cells to the surrounding tissues allowing the embryo to grow. In white spruce somatic embryogenesis meristem reactivation is a critical event required for successful conversion of the embryos into viable plants. Failure to resume mitotic activity in the apical poles has been observed in those embryos which perform poorly at germination [14]. Ascorbic acid is a metabolite required for fundamental cellular processes, including cell division and differentiation. High levels of endogenous ASC are often measured in tissue undergoing intense growth [1], while exogenous applications of ASC promote cell division in several systems, including onion and corn roots, Lupinus albus seedlings, and tobacco cultured cells [2,7,13,15,16]. Two previous studies indicate that this metabolite may play an important role during spruce somatic embryogenesis. First, alterations in ASC metabolism occur during spruce somatic embryo development, where they delineate important morphogenic events [26]. Second, exogenous applications of ASC have been found to enhance the conversion frequency of white spruce somatic embryos in poor embryogenic lines, possibly by promoting cell division in structurally disorganized meristems [24]. Data from the present work suggest that apical meristem reactivation at germination is strictly dependent upon the availability of a critical level of endogenous ASC. Depletion of the cellular ASC pool results in meristem abortion, and correlates to the activation of guaiacol and ferulic acid peroxidases which have been shown to participate in the cross linkage of cell wall components.

Fig. 9. Effects of different concentrations of ascorbic acid (ASC) and 2 units of ASC oxidase on the oxidation rate of ferulic acid and guaiacol dependent peroxidases. For each experiment 20 ml of protein extracted from control embryos cultured for 6 days on germination medium were utilized.

Experimental manipulations of the endogenous ASC level through exogenous applications of ASC, GL, or L have a profound effect on white spruce embryo conversion. Specifically, inhibition of the de novo synthesis of ASC, effected either by L which inhibits the conversion of GL to ASC [11], or by a combination of GLþL (Fig. 1), results in meristem abortion at germination, thus reducing the ability of the somatic embryos to convert into viable plants (Fig. 2). This poor post-embryonic performance of the embryos is due solely to the depletion of the cellular ASC, and not to a possible toxic effect of L, since it is almost completely reverted if L is applied in conjunction with ASC (Figs. 1 and 2). Conversely, high conversion frequencies, similar to the already high values of control embryos, were observed in embryos with increased levels of endogenous ASC, effected by applications of either ASC or GL (Fig. 2). From these results it is evident that the amount of cellular ASC is an important prerogative for successful embryo regeneration. Supporting this notion is the observation that the endogenous ASC level of the good quality somatic embryos utilized in this study (88% conversion frequency) is more than double that of poor quality embryos (33% conversion frequency), produced by other lines [26]. Furthermore, conversion frequency of poor quality embryos can be enhanced by increasing the endogenous ASC level [24]. The deleterious effects of low levels of endogenous ASC on meristem reactivation observed in spruce embryos may be the

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result of increased peroxidase activity, which through the cross-linkage of cell wall components, including phenolic compounds, prevents cell proliferation and meristem conversion. Our data show that with reduced ASC levels, effected by applications of L or GLþL, the abortion of both shoot and root apical meristems is accompanied by an increased activity of both ferulic acid and guaiacol peroxidase (Figs. 3e5). In situ studies also confirm in that the apical poles of embryos with decreased L level stain more intensely for total peroxidase activity than their control counterparts (Fig. 7). Conversely, treatments that increase the endogenous ASC level, i.e. ASC, ASCþL, and GL, inhibited both ferulic acid- and guaiacol-dependent peroxidases after a few days on germination medium, and reduced the staining against total peroxidases in the apical poles of the embryos. Thus a negative correlation between activities of both ferulic acid and guaiacol peroxidases in the apical poles and successful conversion of the embryos exists in white spruce. A similar correlation was also documented in other systems [9,30], including gymnosperms. Sanchez et al. [22] suggested that polymerization of ferulic acid and phenolics, catalyzed by peroxidases, enhances cell wall rigidity and strength, thus leading to growth cessation in pine tissue. The accumulation of phenolic compounds observed in the wall of meristematic cells of embryos with reduced levels of ASC (Fig. 3), together with the increased peroxidase activities (Figs. 4 and 5) support the notion that similar growth inhibitory processes operate in spruce. The key role played by ascorbic acid in the regulation of peroxidase dependent polymerization of phenolic compounds has been emphasized in many studies. De Gara and Tommasi [5] proposed that ASC might facilitate the removal of H2O2 by acting as a substrate for ASC peroxidase. Specifically, increasing activities of this enzyme would reduce the availability of hydrogen peroxide utilized by secretory peroxidases for cell wall cross-linking. Besides its higher affinity for H2O2 [5], a negative correlation between ASC peroxidase activity and major peroxidases activities was observed in several systems, including white spruce zygotic embryogenesis (Stasolla and Yeung, unpublished data). In the present study, however, no correlation was observed between the patterns of guaiacol and ferulic acid oxidation and ASC peroxidase activity, which shows no major differences among treatments (Fig. 6). A high ASC content in germinating white spruce somatic embryos might affect peroxidases directly, possibly by altering their synthesis and/or secretion. As shown by the electrophoretic studies different levels of endogenous ASC seem to changes the peroxidase isoenzyme profile (Fig. 8). Several investigations have indicated that ASC may be utilized as a non-specific electron donor by several classes of peroxidases [19,32]. In vitro studies conducted in this experiment confirm this observation. Peroxidase activities measured with ferulic acid and guaiacol were completely abolished by applications of ASC, and this inhibition was released after the ASC that was present in the assay medium was oxidized (Fig. 9). Ascorbic acid (ASC) was responsible for this control, as neither GL nor L affected peroxidase activities in vitro (data not shown). In agreement with our findings, oxidation of

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ferulic acid and guaiacol [4], as well as oxidation of hydroxycinnamic acids and hydroxycinnamyl alcohols [2,29] by peroxidase/hydrogen peroxide systems, were almost completely inhibited in the presence of ASC. These results were explained by the ability of ASC to re-reduce the intermediate phenoxyl radical compounds, through its oxidation to ascorbate free radicals [28,29]. It must be mentioned, however, that the ASCinhibition of wall associated peroxidases, including ferulic acid peroxidase, only occurs if sufficient ASC is present in the apoplast. Although the apoplastic ASC level was not measured in this experiment, Rautenkranz et al. [20] demonstrated that movement of ASC from symplastic compartment to the apoplast is possible in plant cells. In conclusion, the results of this investigation clearly indicate that endogenous ASC levels play a fundamental role during meristem reactivation at germination. With the resumption of mitotic activity in the meristematic cells, a large availability of ASC may be needed for inhibiting the activity of major cellular peroxidases, including those responsible for the polymerization of cell wall components, such as ferulic acid. This would result in cell wall relaxation which is necessary to allow cell division in meristematic cells. As such, manipulations of the culture medium which increase the endogenous ASC content would be beneficial to enhance embryo conversion, especially for those lines characterized by low ASC content. Acknowledgments The research reported in this paper was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) scholarship to C.Stasolla and by NSERC Research Grants to E.C.Yeung Professor Laura de Gara, University of Bari, Italy, is gratefully acknowledged for providing lycorine. References [1] O. Arrigoni, L. De Gara, F. Tommasi, R. Liso, Changes in the ascorbate system during seed development of Vicia faba L, Plant Physiol. 99 (1992) 235e238. [2] O. Arrigoni, G. Calabrese, L. De Gara, M.B. Bitonti, R. Liso, Correlation between changes in cell ascorbate and growth of Lupinus albus seedlings, J. Plant Physiol. 150 (1997) 302e308. [3] K.J. Biggs, S.C. Fry, Phenolic cross-linking at the cell wall, in: D.J. Cosgrove, D.P. Knievel (Eds.), Physiology of Cell Expansion during Plant Growth, American Society of Plant Physiologists, Rockville, MD, 1997, pp. 46e57. [4] M.C. Cordoba-Pedregosa, J.A. Gonzales-Reyes, M.S. Canadillas, P. Navas, F. Cordoba, Role of apoplastic and cell wall peroxidases on the stimulation of root elongation by ascorbate, Plant Physiol. 112 (1996) 1119e1125. [5] L. De Gara, F. Tommasi, Ascorbate redox enzymes: a network of reactions involved in plant development, in: Recent Research and Development in Phytochemistry, vol. III, Research Signpost, 1999, pp. 1e15. [6] L. De Gara, C. Paciolla, F. Tommasi, R. Liso, O. Arrigoni, In vivo inhibition of galactono-g-lactone conversion to ascorbate by lycorine, J. Plant Physiol. 144 (1994) 649e653. [7] T. de Pinto, M.C.D. Francis, L. De Gara, The redox state of the ascorbatedehydroascorbate pair as a specific sensor of cell division in tobacco BY-2 cells, Protoplasma 209 (1999) 90e97. [8] S.C. Fry, Cross-linking of matrix polymers in the growing cell wall of angiosperms, Annu. Rev. Plant Physiol. 37 (1986) 165e186.

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