Journal of Experimental Botany, Vol. 52, No. 365, pp. 2301-2311,
December 1, 2001
© 2001 Oxford University Press
Original Papers |
Analysis of carbohydrate metabolism enzymes and cellular contents of sugars and proteins during spruce somatic embryogenesis suggests a regulatory role of exogenous sucrose in embryo development
Centre de Recherche en Biologie Forestière, Pavillon Charles-Eugène Marchand, Université Laval, Sainte-Foy, Québec G1K 7P4, Canada
Received 19 April 2001; Accepted 12 July 2001
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Abstract |
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Carbohydrate metabolism was investigated during spruce somatic embryogenesis. During the period of maintenance corresponding to the active phase of embryogenic tissue growth, activities of soluble acid invertase and alkaline invertase increased together with cellular glucose and fructose levels. During the same time, sucrose phosphate synthase (SPS) activity increased while sucrose synthase (SuSy) activity stayed constant together with the cellular sucrose level. Therefore, during maintenance, invertases were thought to generate the hexoses necessary for embryogenic tissue growth while SuSy and SPS would allow cellular sucrose to be kept at a constant level. During maturation on sucrose-containing medium, SuSy and SPS activities stayed constant whereas invertase activities were high during the early stage of maturation before declining markedly from the second to the fifth week. This decrease of invertase activities resulted in a decreased hexose:sucrose ratio accompanied by starch and protein deposition. Additionally, carbohydrate metabolism was strongly modified when sucrose in the maturation medium was replaced by equimolar concentrations of glucose and fructose. Essentially, during the first 2 weeks, invertase activities were low in tissues growing on hexose-containing medium while cellular glucose and fructose levels increased. During the same period, SuSy activity increased while the SPS activity stayed constant together with the cellular sucrose level. This metabolism reorganization on hexose-containing medium affected cellular protein and starch levels resulting in a decrease of embryo number and quality. These results provide new knowledge on carbohydrate metabolism during spruce somatic embryogenesis and suggest a regulatory role of exogenous sucrose in embryo development.
Key words: Picea, carbohydrate metabolism, invertase, somatic embryogenesis, sucrose phosphate synthase, sucrose synthase.
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Introduction |
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In plant cell culture, the addition of exogenous sugar to the culture medium is an absolute necessity for nearly all tissues. Among carbohydrates, sucrose has generally been used as the most suitable source of carbon and energy for growth of plant tissues (George, 1993
). Sucrose degradation is the first step for carbon utilization by plant cells. The breakdown of sucrose is mediated by two enzymes, invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13), allowing the subsequent utilization of the hexoses produced. On the basis of pH optima, plant tissues contain two types of invertase: an acid invertase, located in vacuoles and cell walls, and an alkaline invertase, located in the cytoplasm (Hauch and Magel, 1998). The reactions catalysed by invertases are irreversible while those catalysed by sucrose synthase, a cytoplasmic enzyme, are reversible. On the other hand, sucrose biosynthesis occurs in the cytoplasm and involves the activity of sucrose phosphate synthase (EC 2.4.1.14) (Huber and Huber, 1992; Frommer and Sonnewald, 1995; Peace et al., 1995). In plant tissues, soluble invertases play an important role in the regulation of sugar composition (Yelle et al., 1988; Obenland et al., 1993; Scholes et al., 1996; Kingston-Smith et al., 1999) and sucrose partitioning (Tang et al., 1999) whereas SuSy is correlated with starch and cell wall synthesis (King et al., 1997; Winter and Huber, 2000).
Somatic embryogenesis offers an excellent experimental system to study the physiological and biochemical aspects of embryo development. Among the medium components reported to play a dominant role in the development of somatic embryos (Jain et al., 1995), one of the most important is the exogenous source of sugar. Sucrose being the most commonly used carbon source in the medium, its effect on induction, maintenance and maturation of somatic embryos has been investigated (Finer et al., 1989; Tremblay and Tremblay, 1991b, 1995; Iraqi and Tremblay, 2001). Even if sucrose in the maturation medium is hydrolysed into its monomers, equimolar concentrations of glucose and fructose in the medium did not replace the positive effects of sucrose on embryo maturation (Tremblay and Tremblay, 1995; Taber et al., 1998; Iraqi and Tremblay, 2001). In addition, hydrolysis of sucrose in the maturation medium results in an increased osmotic pressure of the medium. However, simulation of this increasing osmotic pressure in the maturation medium did not improve somatic embryo production (Iraqi and Tremblay, 2001). These authors suggested that sucrose might act as a factor regulating the maturation of spruce somatic embryos through its action as a signal for the synthesis of storage proteins, especially the 42, 35 and 22 kDa polypeptides. Although an understanding of carbohydrate metabolism is important to optimize growth and embryo yield in several species, information on the activity of the main enzymes involved in carbohydrate metabolism during the conifer somatic embryogenesis process is lacking. Since carbohydrate metabolism can markedly differ from one species to another, the questions addressed in the present paper were extended to black spruce and white spruce. These two species vary in terms of embryo production and plantlet development (Iraqi and Tremblay, 2001), white spruce being generally superior to black spruce with regard to plantlet conversion rate.
The first objective of the current study was to describe carbohydrate metabolism during the maintenance stage. To do so, soluble acid invertase (Ac Inv), alkaline invertase (Alk Inv), sucrose synthase (SuSy), and sucrose phosphate synthase (SPS) were studied in relation to growth and levels of endogenous soluble sugars (sucrose, glucose and fructose). The second objective was to elucidate the relationship between exogenous sugar supply, carbohydrate metabolism and the resulting embryo development during maturation. This study represents the first report attributing respective roles to the enzymes involved in carbohydrate metabolism in terms of growth and embryo development during spruce somatic embryogenesis.
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Materials and methods |
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Plant material
Embryogenic tissues were induced from mature zygotic embryos of black spruce (Picea mariana [Mill.] BSP) (line M-286) and white spruce (Picea glauca [Moench.] Voss) (line G-316) as previously described (Tremblay, 1990
). They had been maintained by 14 d subcultures on HLM-1 medium (Tremblay, 1990) before use.
Maintenance experiment
Seven portions (30±5 mg each) of embryogenic tissue were placed in Petri dishes (100x15 mm) containing 24 ml of HLM-1 maintenance medium (Tremblay, 1990). For each species, dry weight, soluble sugar level, and enzyme activity were monitored throughout the maintenance period at days 0, 5, 7, 10, and 15. This experiment was conducted as a completely randomized design with seven tissues per Petri dish (replicate). For each sampling date, five Petri dishes were randomly selected for dry weight (DW) and sugar analysis, and another five Petri dishes for enzyme assays. DW was determined after lyophilization. The relative growth rate (RGR) was determined by fitting a polynomial function of the form Ln(DW)=a0+a1D+
+anDn, where D=sampling date, and by differentiating the resulting function. The degree of the polynomial function was derived from the significance of the cubic terms (Poorter and Lewis, 1986; Lamhamedi et al., 1997). The RGR was used to eliminate the effect of embryogenic tissue sizes. Soluble sugar contents and enzyme activities were determined as described below.
Maturation experiment
Seven days after transfer onto fresh maintenance medium, portions (90±5 mg) of embryogenic tissue were placed in Petri dishes (100x15 mm) containing 24 ml of maturation medium. This medium consisted of Litvay's salts (Litvay et al., 1985) used at half-strength, supplemented with 1 g l-1glutamine, 1 g l-1 casein hydrolysate (Tremblay and Tremblay, 1991a), and 45 µM (±) cis,trans-abscisic acid (ABA). It was solidified with 0.4% (w/v) GelriteTM gellan gum and the pH was adjusted to 5.7 before autoclaving at 121 °C. Glutamine, ABA, and carbohydrates were filter-sterilized and added to the cooled medium.
To determine the effect of the medium sugar composition on carbohydrate metabolism during maturation of black spruce and white spruce somatic embryos, two treatments were compared: (1) 6% (w/v) of sucrose (anhydrous, BDH Inc., Toronto, Canada), and (2) 3.16% (w/v) each of glucose (anhydrous, BDH Inc.) and fructose (anhydrous, Fisher, Fair Lawn, NJ, USA). For each treatment and species, DW, sugar content, total protein content, and enzyme activities were monitored after 0, 1, 2, 3, 4, and 5 weeks of maturation. This experiment was conducted as a completely randomized design with three tissues per Petri dish (replicate) per treatment. At each sampling time, five Petri dishes were removed for DW and sugar analysis and another five Petri dishes were used for protein analysis and enzyme assays.
Sugar extraction and analysis
Embryogenic tissues were homogenized to a very fine powder in liquid nitrogen and then sugar extraction was carried out (Yelle et al., 1991). Briefly, ethanol at 80% (v/v) containing mannitol as internal standard, was added to each sample which was then kept at 80 °C in a water bath for 20 min before centrifugation for 15 min at 13000 g. After supernatant removal, the pellet was resuspended and re-extracted twice in 80% ethanol to recover the residual soluble sugars. The combined supernatants constituted the soluble sugar fraction. The extracts were evaporated in a SpeedVac and resuspended in water before injection into an HPLC system (712 WISP, Waters, Milford, MA, USA). An aqueous solution of 0.005% (w/v) Ca-EDTA (Sigma, St Louis, MO, USA) flowing at 0.5 ml min-1 was used as the mobile phase. The sugar column (Sugar-Pak 1, 6.5x300 mm, Waters) was used at 75 °C and eluted sugars were detected with a differential refractometer (Waters). Peaks were quantified using anhydrous sucrose, glucose (BDH Inc.), fructose (Fisher, Fair Lawn, NJ, USA), and mannitol (Sigma).
Starch was analysed by an enzymatic digestion method (Robinson et al., 1988). Briefly, the pellet left after soluble sugar extraction was washed three times in 80% (v/v) ethanol and incubated for 30 min at 55 °C to evaporate all remaining ethanol. The dry pellet was solubilized in 0.2 N NaOH and boiled for 30 min at 100 °C. After cooling at room temperature, citrate buffer (pH 4.5) containing 100 units of amyloglucosidase and 70 units of 
-amylase (Sigma) was added to each sample before incubation at 55 °C for 3 h. Glucose in the extract was determined by using the glucose kit (HK) (Sigma) according to manufacter's recommendations.
Enzyme extraction and determination of total protein content
All the steps for enzyme extraction and desalting were performed at 4 °C. Embryogenic tissues were homogenized to a fine powder in liquid nitrogen. Enzymes were extracted (Déjardin et al., 1997) by resuspending the powdered tissues in 50 mM HEPES buffer, pH 7.5, containing 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 10% (v/v) glycerol, 1 mM PMSF, and 1% (w/v) insoluble PVP. The homogenates were centrifuged at 13000 g for 15 min, and the supernatants were desalted by passage through a 4 ml Sephadex G-25 column pre-equilibrated with 50 mM HEPES buffer, pH 7.5, containing 10 mM MgCl2, 1 mM EDTA, and 1% (w/v) BSA. Insoluble proteins were extracted as described previously (Xu et al., 1996). Both soluble and insoluble protein contents were determined according to Bradford's method (Bradford, 1976). Total protein content represents the sum of the two protein fractions.
Enzyme assay
Soluble acid invertase and alkaline invertase were assayed as described previously (Déjardin et al., 1997) with some modifications. Briefly, desalted enzyme extract was incubated with 100 mM sucrose in either sodium acetate buffer (pH 4.5) for acid invertase or HEPES buffer (pH 7.5) for alkaline invertase. Reactions were allowed to progress for 15 min at 30 °C and then stopped by boiling. Control reactions contained boiled extract. The reaction buffer was centrifuged at 13000 g for 3 min, then hexoses were measured spectrophotometrically using coupling enzymes (King et al., 1997). The reaction of coupling enzymes was started with 5 µl ATP (100 mM), 5 µl NAD (40 mM), 2 units of Glc-6-P dehydrogenase, 2 units of hexokinase, and 3 units of glucoisomerase.
SuSy activity was assayed in the direction of sucrose breakdown (Déjardin et al., 1997) with some modifications. Briefly, desalted enzyme extract was incubated in HEPES buffer (pH 6.5) containing 100 mM sucrose and 2 mM UDP. Reactions, started by the addition of desalted extract, were incubated at 30 °C for 15 min and stopped by boiling. A control reaction without UDP estimated invertase activity. After centrifugation at 13000 g for 3 min, the production of hexoses was quantified as described above.
SPS activity was assayed in a HEPES buffer at pH 7.5 (Klann et al., 1993) containing 10 mM MgCl2, 10 mM Fru-6-P, 40 mM Glc-6-P, and 10 mM UDP-Glc. Reactions, started by the addition of desalted extracts, were incubated at 28 °C for 10 min. They were stopped by the addition of 100 µl of 30% KOH (w/v) and boiling for 10 min. Sucrose in the supernatant was determined by the anthrone method (Weber et al., 1996b). In all cases it was ensured that activity was linearly related to time and the amount of extract.
Embryo number and germination
After 5 weeks in maturation, the numbers of normal (morphology similar to zygotic embryos) and abnormal (abnormality in shape or in the cotyledon position) embryos were determined from five replicates per treatment of each species. For each maturation treatment, normal somatic embryos (10 embryos/Petri dish, n=5) were transferred onto a germination medium (Khlifi and Tremblay, 1995) solidified with 1% (w/v) GelriteTM gellan gum. The number of embryos developing an epicotyl was determined after 9 weeks in germination.
Environmental conditions
Maintenance, maturation and germination phases occurred under a 16 h photoperiod at 23 °C. An irradiance of 5–10 µmol m-2 s-1 given by Gro-Lux WS (Sylvania, Ontario, Canada) fluorescent lamps was used for the maintenance, 10–15 µmol m-2 s-1 given by Vita-Lite Plus (Duro-test, Houston, TX, USA) fluorescent lamps for maturation, and 70–85 µmol m-2 s-1 given by Vita-Lite fluorescent lamps for germination.
Statistical analysis
The number of embryos and the frequencies of embryos developing an epicotyl (calculated as the number of embryos with an epicotyl/total number of embryos transferred to the germination medium) were analysed using the SAS GLM analysis of variance procedure (SAS Institute Inc., Cary, NC, USA) followed by a Student test (Sokal and Rohlf, 1995). Homogeneity of variances was verified by Bartlett's test. A transformation of the epicotyl frequency data by arcsine (sqr (p/100)) was needed before statistical analysis to make treatment variances homogeneous.
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Results |
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Dry weight and relative growth rate of embryogenic tissues during maintenance
During the maintenance period, time-courses of dry weight accumulation were similar for black spruce and white spruce embryogenic tissues (Fig. 1
). For both species, relative growth rate was characterized by an exponential phase until the tenth day followed by a slight decrease until the end of the maintenance period (Fig. 1).
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Sucrose, glucose and fructose levels in the tissues during maintenance
During maintenance, the changes in the levels of sucrose, glucose and fructose in the embryogenic tissues followed similar patterns for both black spruce and white spruce (Fig. 2). For both species, endogenous sucrose levels stayed constant at about 15 mg g-1 DW throughout the 15 d of maintenance. Endogenous glucose and fructose increased to reach their maximum levels between days 5 and 10 before a decrease to their initial levels by the end of the maintenance period.
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Ac Inv, Alk Inv, SuSy, and SPS activities during maintenance
Both species showed similar activity profiles but at different levels for Ac Inv, Alk Inv, SuSy, and SPS during the maintenance period. For both species, Ac Inv, Alk Inv and SPS showed a peak of activity between days 5 and 7 followed by a decrease to their initial levels by the end of the maintenance period (Fig. 3). By contrast, SuSy activity remained constant throughout the maintenance period at about 45 nmol mg-1 protein min-1 for black spruce and 35 nmol mg-1 protein min-1 for white spruce. The two species differed in their Ac Inv, Alk Inv and SPS activity levels. For example in black spruce, Ac Inv activity increased from 150 at day 0 to 320 nmol mg-1 protein min-1 at day 7 before a decrease to about 120 nmol mg-1 protein min-1 on day 15, whereas white spruce Ac Inv activity increased from 50 at day 0 to 165 nmol mg-1 protein min-1 at day 7 before a decrease at 40 nmol mg-1 protein min-1 at day 15.
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Embryo production and plantlet development
For both species, the best embryo production was observed for tissues matured on 6% sucrose (Table 1). Replacing sucrose in the medium by glucose and fructose significantly decreased the number of normal embryos from 48 to 18 for black spruce and 36 to 12 for white spruce while it did not affect the number of abnormal embryos. This resulted in a normal to abnormal ratio significantly higher for the sucrose treatment than for the glucose and fructose treatment. The carbohydrate treatments during the maturation period also affected the ability of the embryos to develop into plants. Embryos matured on 6% sucrose developed their epicotyls at frequencies of 57% and 83% for black spruce and white spruce respectively (Table 1). However, when embryos were produced on a medium containing glucose and fructose, their ability to develop an epicotyl was significantly lowered to 30% and 47% for black spruce and white spruce, respectively.
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0.01 (**), at P
Sucrose, glucose, fructose, and starch levels in the tissues during maturation
Cellular levels of soluble sugars (sucrose, glucose and fructose) and starch were determined each week throughout the maturation period (Figs 4, 5). Within the first week of maturation on a medium containing 6% sucrose, the cellular levels of soluble carbohydrates had increased to their maximum values (Fig. 4). Thereafter, and until the end of the maturation period, cellular sucrose levels remained constant at about 75 and 45 mg g-1 DW for black spruce and white spruce, respectively, while glucose and fructose levels decreased to reach a plateau in the last week. For example, glucose content fell between weeks 1 and 5 from 150 to 45 mg g-1 DW in white spruce tissues and from 120 to 30 mg g-1 DW in black spruce tissues. For both species, replacing sucrose in the medium by glucose and fructose did not affect the endogenous level of sucrose while it increased significantly the cellular levels of glucose and fructose throughout the maturation period (Fig. 4).
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) 6% sucrose; (
) 3.16% of both glucose and fructose. Bars represent standard deviation with n=5.When considering the starch content of tissues placed on a medium containing 6% sucrose, the results showed that the major cellular starch accumulation happened during the first 2 weeks of the maturation period. During this time, the starch content increased from about 50 mg of glucose g-1 DW for both species to 150 mg of glucose g-1 DW for black spruce and 90 mg glucose g-1 DW for white spruce (Fig. 5
). For both species, when sucrose was substituted in the medium by glucose and fructose, the cellular starch level was significantly lowered throughout the maturation period (Fig. 5).
Total protein contents in the tissues during maturation
Each week of the maturation period, total proteins were analysed in black spruce and white spruce embryogenic tissues (Fig. 6). For both species, the total protein content in tissues cultured on 6% sucrose gradually increased from 20–25 initially to about 80–100 mg g-1 DW at the end of the maturation period. However, for both species, when sucrose in the medium was substituted by glucose and fructose, the total protein content in the tissues remained constant at about 30 mg g-1 DW throughout the maturation period.
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Ac Inv and Alk Inv activities during maturation
Measurements of Ac Inv and Alk Inv activities in black spruce and white spruce embryogenic tissues during maturation showed that the transfer from a maintenance medium containing 1% sucrose to a maturation medium containing 6% sucrose resulted in a peak of invertase activities after 1 week (Fig. 7). After this time, invertase activities decreased rapidly to reach final levels significantly lower than initial levels. For black spruce, Ac Inv activity dropped from 790 at week 1 to about 300 at week 2 to finally reach 50 nmol mg-1 protein min-1 at week 4. For white spruce, Ac Inv activity decreased linearly from 790 to about 130 nmol mg-1 protein min-1 at week 5. Similarly, Alk Inv activity dropped from 660 at week 1 to 20 nmol mg-1 protein min-1 at week 3 for black spruce while for white spruce it dropped from 370 to 26 nmol mg-1 protein min-1 at week 5. Substitution of sucrose by glucose and fructose in the medium significantly lowered the Ac Inv and Alk Inv activities in black spruce tissues during the first 2 weeks. For white spruce, replacement of sucrose by glucose and fructose in the medium significantly lowered Ac Inv activity during the entire maturation period, and Alk Inv activity during the first four weeks.
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SuSy and SPS activities during maturation
SuSy and SPS activities for both species are shown in Fig. 8. Throughout the whole maturation period and independently of the carbohydrate treatment, SPS activity stayed constant at about 100 and 80 nmol mg-1 protein min-1 for black spruce and white spruce, respectively (Fig. 8). The SuSy activity of tissues incubated on the sucrose medium stayed constant at about 35 nmol mg-1 protein min-1 for both species during the whole maturation period. For both species, replacing sucrose in the medium by glucose and fructose significantly increased the SuSy activity in tissues particularly during the first weeks of the maturation period while it did not affect the SPS activity.
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Discussion |
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As far as is known, carbohydrate metabolism has not been investigated during spruce somatic embryogenesis. Here, the implication of sucrose metabolic enzymes on tissue growth and somatic embryo development for black spruce and white spruce is reported. Furthermore, direct evidence is provided that carbohydrate metabolism can be reoriented by varying the carbohydrate source in the maturation medium.
During the first 10 d of maintenance, black spruce and white spruce embryogenic tissues are characterized by a high growth rate (Fig. 1), which is usually attributed to the hexoses produced from the cleavage of sucrose by invertases (reviewed by Weber et al., 1997). These data confirm for spruce that the high growth rate observed during the first 10 d was accompanied by an accumulation of glucose and fructose in the tissues (Fig. 2) together with increased activities of Ac Inv and Alk Inv (Fig. 3). SuSy, another enzyme able to hydrolyse sucrose, appears to play a less important role than invertases in this process since its activity in spruce stayed constant throughout the maintenance period.
Contrary to hexoses, the sucrose level in embryogenic tissues stayed constant throughout the maintenance period (Fig. 2). Over the same time, SPS activity increased following a pattern similar to that of the invertases while the SuSy activity stayed constant (Fig. 3). It therefore appears that the main role of SPS and SuSy during spruce maintenance would be to keep a constant cellular sucrose level, which is necessary to maintain a gradient between the medium ‘source’ and the tissue ‘sink’ (reviewed by Winter and Huber, 2000). Therefore, during maintenance of spruce embryogenic tissues, carbohydrate metabolism is characterized by high invertase activities leading to a nutritive role of sucrose, but also by SPS and SuSy activities to preserve a sucrose gradient between the medium and the tissue.
Carbohydrate metabolism was also studied during the maturation of black spruce and white spruce somatic embryos. In spite of the differences observed in terms of embryo production and plantlet development (Table 1), the two species present qualitatively similar results with regard to cellular sugar status, enzyme activities and reserve accumulation but at different levels. Therefore, in this study, it was not possible to explain the different performances of these two species through carbohydrate metabolism. Somatic embryo maturation involved a transfer of embryogenic tissues from a maintenance medium containing 1% sucrose to a maturation medium containing 6% sucrose. As a consequence of this sucrose increment in the medium, enzyme assays showed that Ac Inv and Alk Inv reached their maximum activities within the first week of maturation (Fig. 7). This confirms for the spruce that sucrose abundance stimulates invertase activities through the induction of their corresponding genes (Koch, 1996; Gibson, 2000). As a consequence and during the same period of time, glucose and fructose levels increased to reach their maximum levels (Fig. 4), confirming for spruce embryogenic tissues the relationship between invertase activity and hexose contents as reported for sink organs such as seeds (Weber et al., 1997) and fruits (Sturm, 1999). During the first week of maturation, the cellular sucrose level increased by 3 to 4 times compared to maintenance while SuSy and SPS activities stayed constant at levels similar to the highest values measured during maintenance. These results suggest that, for spruce, both sucrose resynthesis in the cells via SuSy and/or SPS (Geigenberger and Stitt, 1993; Déjardin et al., 1997; Winter and Huber, 2000) and direct uptake from the medium (Thom and Maretzki, 1992) might be involved during maturation, but further investigation is still necessary before to conclude.
From the 2nd to the 5th week of maturation, Ac Inv and Alk Inv activities decreased rapidly resulting in decreased cellular glucose and fructose levels while the cellular sucrose level remained constant (Figs 4, 7). The consequence is a low hexose:sucrose ratio which is known to favour the storage functions (Weber et al., 1996a; Wobus and Weber, 1999). This is in opposition to the first week of maturation, which is characterized by a high cellular hexose:sucrose ratio, known to favour growth. Therefore, it is concluded that for both black spruce and white spruce, the first week of maturation corresponds to a period of growth characterized by a high glucose and fructose demand, whereas the remainder of maturation constitutes a period for development and storage, as supported by the accumulation of protein and starch (Figs 5, 6).
The relationship between carbohydrate metabolism and somatic embryo development was studied further by modifying the carbohydrate source in the maturation medium. As previously observed (Tremblay and Tremblay, 1995; Iraqi and Tremblay, 2001), replacing sucrose in the maturation medium by equimolar concentrations of glucose and fructose decreased the embryo number and their germination capacity (Table 1), but also severely modified the carbohydrate metabolism. Among the modifications observed on hexose-containing medium were reduced invertase activities (Fig. 7) and increased levels of cellular glucose and fructose (Fig. 4). Since the invertase activities were reduced, the only way to explain the observed hexose increment is through a direct uptake from the medium. Therefore, the pool of hexoses resulting from direct uptake could not support the same embryo development as the pool of hexoses normally produced by sucrose cleavage via invertases. In general, during maturation, the cellular sucrose level was not affected by varying the carbohydrate source in the maturation medium (Fig. 4). In the absence of exogenous sucrose, the pool of sucrose in the tissues growing on hexose-containing medium should result from sucrose resynthesis in the cells via SuSy and/or SPS (Geigenberger and Stitt, 1993; Déjardin et al., 1997; Winter and Huber, 2000). In spruce tissues, SuSy activity increased on hexose-containing medium compared to sucrose-containing medium but, unexpectedly, SPS activity did not change (Fig. 8). Therefore, in the absence of sucrose in the medium, it is suggested that the main role of SuSy is to maintain constant cellular sucrose levels throughout the maturation period. Such a situation appeared detrimental to the maturation of black spruce and white spruce somatic embryos since it resulted in a reduction of embryo production (Table 1) and storage deposition (Figs 5, 6). It has been proposed that different sugars and intermediates in sugar metabolism may have a signalling function, especially hexoses and sucrose (Sturm and Tang, 1999; Smeekens, 2000). In particular, the induction of gene expression has been related to the level of phosphorylation of the hexoses by hexokinase (Kingston-Smith et al., 1999). Here, on hexose-containing medium, high cellular levels of free hexoses due to the direct uptake of glucose and fructose by the tissues did not promote the accumulation of storage products, but rather led to a reorientation of the carbohydrate metabolism towards cellular sucrose synthesis. Conversely, on sucrose-containing medium, hexoses produced by the cleavage of sucrose via invertases could be a signal to induce glucose and fructose phosphorylation which is necessary to enter other metabolic pathways (Halford et al., 1999; Farrar et al., 2000). Consequently, these results are consistent with the hypothesis that glucose and fructose generated by the cleavage of sucrose via invertases could initiate changes in gene expression (Halford et al., 1999; Farrar et al., 2000). Therefore, in the presence of sucrose in the medium, sucrose hydrolysis via invertases may play an important role in spruce embryo development, confirming the findings on carrot (Tang et al., 1999), but more specifically for spruce, invertases may be linked to the control of protein and starch synthesis.
In conclusion, the implication of carbohydrate metabolism during spruce somatic embryogenesis is reported for the first time here. During the first 10 d of maintenance and during the first week of maturation, carbohydrate metabolism was oriented to sustain growth and was characterized by high invertase activities leading to high cellular glucose and fructose levels. During the remaining maturation period, carbohydrate metabolism was essentially oriented towards storage deposition and embryo development. Replacing sucrose by glucose and fructose in the maturation medium was accompanied by an imbalance in invertase and SuSy activities, resulting in an alteration of storage reserve accumulation and, consequently, in a depression of somatic embryo production and germination capacity. Thus, these results suggest a regulating role of exogenous sucrose during spruce embryo development via the regulation of the carbohydrate metabolism, essentially through the activation of invertases.
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Acknowledgments |
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This work was financially supported in part by the Ministry of Industry, Trading, Science and Technology in association with Pampev Inc., Bechedor Inc., and CPPFQ Enr. (Synergie program) (grant to FMT) and by the Ministry of Natural Resources of Quebec (scholarship to DI). We thank Dr VQ Le, Dr M Lamhamedi, Dr A El Meskaoui, DC Stowe, and Dr C Bomal for critical reading of the manuscript.
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Notes |
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1 To whom correspondence should be addressed. Fax: +14186567493. E-mail: francine.tremblay{at}sbf.ulaval.ca
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