Journal of Experimental Botany, Vol. 52, No. 357, pp. 761-769,
April 15, 2001
© 2001 Oxford University Press
Original Papers |
Involvement of ethylene in the maturation of black spruce embryogenic cell lines with different maturation capacities
Centre de Recherche en Biologie Forestière, Pavillon C.-E. Marchand, Université Laval, Québec, Qc G1K 7P4, Canada
Received 23 October 2000; Accepted 7 November 2000
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Abstract |
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To examine the possible relationship between ethylene and the capacity of embryogenic cell lines to produce mature somatic embryos of black spruce (Picea mariana (Mill.) B.S.P.), two embryogenic cell lines which exhibit different maturation capacities were used to analyse ethylene biosynthesis and that of its immediate precursor, 1-aminocyclopropane-1- carboxylic acid (ACC). Several compounds known to alter ethylene metabolism were also evaluated for their effect on the number of mature somatic embryos produced. The results showed that in the high capacity cell line, ethylene production and endogenous ACC pools were less than in the low capacity cell line. It was also demonstrated that limiting ethylene biosynthesis by adding inhibitors of ethylene biosynthesis or its physiological action to the maturation medium promoted somatic embryo production for the low capacity cell line. Conversely, lowering ethylene biosynthesis reduced the number of somatic embryos in the high capacity cell line. These results were further substantiated by the finding that the effects of amino-oxyacetic acid (AOA), an inhibitor of ethylene biosynthesis, were partially reversed by adding ethylene to both embryogenic cell lines. It is concluded that ethylene is implicated in somatic embryogenesis of black spruce and that the low capacity cell line had excess, i.e. supraoptimal, ethylene production, whereas the high capacity cell line had nearly optimal ethylene production. The relationship between ethylene and other phytohormones, and the possible effects of the interaction between ethylene and polyamines on the maturation of the somatic embryos are discussed.
Key words: Embryo maturation, ethylene, Picea mariana, somatic embryogenesis.
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Introduction |
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In higher organisms, growth and development of somatic cells are regulated by multiple controls. It has become increasingly evident that these controls are based on various regulatory compounds which can stimulate, inhibit or in some way condition cell growth and development. In this context, hormones have received attention in physiological studies of both plants and animals.
Ethylene (C2H4) is a phytohormone that plays an important role in every phase of plant growth and development (Abeles et al., 1992
). Its biosynthetic pathway has been well established (reviewed by Yang and Hoffman, 1984; Kende, 1993). In higher plants, ethylene is synthesized from methionine (Met) through S-adenosylmethionine (SAM) and 1-aminocyclopropane-1-carboxylic acid (ACC). Considerable progress has been made in the genetic and molecular dissection of the ethylene-response pathway (Kieber, 1997; Johnson and Ecker, 1998).
In plant cell, tissue and organ culture, the influence of ethylene on the regulation of different physiological processes occurring in in vitro culture, particularly during somatic embryogenesis, is not fully understood. This has led to studies on the effects of ethylene on different steps of somatic embryogenesis and have yielded conflicting results and conclusions both in various angiosperm species (for review see Biddington, 1992) and conifers (Kumar et al., 1989; Kvaalen, 1994; Kong and Yeung, 1994; Li and Huang, 1996; Selby et al., 1996; Afele and Preveen, 1995; El Meskaoui and Tremblay, 1999; El Meskaoui et al., 2000). In the present study, our objective was to determine if the ability of an embryogenic cell line to produce mature embryos, i.e. maturation capacity, could be associated with its patterns of ethylene production.
Thus, two embryogenic cell lines having different maturation capacities were used to investigate ethylene metabolism during maturation of black spruce somatic embryos. First, ethylene metabolism during somatic embryo maturation was analysed by quantifying ethylene biosynthesis and levels of endogenous 1-aminocyclopropane-1-carboxylic acid. Second, the effect of ethylene on the maturation of black spruce somatic embryos was studied by adding modulators of ethylene metabolism. This was done by (1) using pure ethylene enrichment, (2) modifying tissue sensitivity to ethylene with silver nitrate, an ethylene antagonist, and (3) modifying the biosynthetic pathway of ethylene by addition of ACC or amino-oxyacetic acid, an inhibitor of ACC synthase. It was hypothesized that the high capacity cell line had an optimal ethylene production capacity, i.e. that embryo production would decrease for any treatment which resulted in either higher or lower rates of ethylene biosynthesis. On the other hand, we hypothesized that the low capacity cell line had a supraoptimal ethylene production capacity and would respond accordingly to treatments which modified its capacity to produce ethylene.
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Materials and methods |
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Plant material
Two embryogenic cell lines (M-282 and M-393.1) of black spruce (Picea mariana (Mill.) B.S.P.) were induced from mature zygotic embryos and had been maintained for 2 and 3 years, respectively, through 14 d subcultures on half Litvay's medium (HLM) (Tremblay, 1990
). To allow maturation of somatic embryos, embryogenic tissues were transferred after 7 d on the fresh maintenance medium onto HLM medium supplemented with 45 µM (±)-cis, trans-abscisic acid (ABA), 6% sucrose and 0.4% (w/v) GelriteTM gellan gum (Schweizerhall Inc., NJ) as previously described (El Meskaoui and Tremblay, 1999).
Effects of ACC, AOA, and AgNO3
For both embryogenic cell lines, the effects of 1-aminocyclopropane-1-carboxylic (ACC), amino-oxyacetic acid (AOA) and silver nitrate (AgNO3) on maturation of somatic embryos were investigated by comparing, through separate experiments (1 experiment/compound), the effect of different levels of ACC (0, 5, 10, and 100 µM), AgNO3 (0, 0.5, 1, and 2 mM) and AOA (0, 5, 10, and 100 µM). These compounds were filter-sterilized before being added to 90x15 mm Petri dishes containing 35 ml of maturation medium. For each embryogenic cell line, each compound was tested in an independent experiment. Each experiment followed a completely randomized design with five replicates (1 replicate=1 Petri dish) per treatment and five pieces of 80 mg (±5 mg) of embryogenic tissues per Petri dish.
Ethylene production measurement
In parallel with the treatment in the Petri dishes described above, measurement of ethylene biosynthesis was conducted with 80 mg of black spruce embryogenic tissues cultured in a 15 ml VacutainerTM tube containing 7 ml of maturation medium solidified at 45° slope. These tubes were exposed to the same treatments and environmental conditions as descibed above. For experiments which describe the profile of ethylene production for each embryogenic cell line, two batches of 42 tubes each were used. For the experiments intended to determine the effects of ACC, AOA and AgNO3 on ethylene production for each embryogenic cell line, two batches of 20 tubes per treatment were used in the experiments. Within each experiment, each batch was alternatively hermetically capped to allow ethylene accumulation for 24 h. At specific times, 1 ml of gas was removed from the head space of the culture tube and injected into a gas chromatograph (GC) equipped with a 6'x0.125'' OD Porapak R column (100/120 mesh) and a flame ionization detector for ethylene analysis. Oven temperature was set at 80 °C with nitrogen as a carrier gas (flow rate of 30 ml min-1). The amount of ethylene was quantified using a Hewlett-Packard 3390A integrator. Ethylene in nitrogen (10 µl l-1) (Scott Specialty Gases, South Plainfield, NJ, USA) was used to calibrate the GC before analysis. After each measurement the tubes were capped with a sponge stopper for incubation. Tubes containing only medium were used as controls.
Assay of endogenous ACC
Five pieces of embryogenic tissue (80 mg each) were cultured in Petri dishes containing 35 ml maturation medium. The volume of gas space and medium available per embryogenic tissue was 8 ml and 7 ml, respectively. At specific times, three replicates (Petri dishs) were used for endogenous ACC analysis. Two hundred mg fresh weight of tissue per replicate per treatment was used for ACC extraction in 6 ml 85% ethanol for 40 min, followed by a 20 min centrifugation at 13 000 g at 4 °C. The supernatant was concentrated under vacuum at 35 °C and the residue was dissolved in 4 ml distilled water. ACC content in the extracts was assayed by its conversion to ethylene (Lizada and Yang, 1979). Ethylene was determined by gas chromatography as described above. Experiments were set up as completely randomized designs with three replicates (1 replicate=1 Petri dish) per treatment and and five pieces of 80 mg (±5 mg) of embryogenic tissue per Petri dish.
Ethylene enrichment system
To evaluate the impact of the ethylene enrichment on the process of somatic embryo maturation, a system was designed to keep cultures under four different microenvironmental conditions: (a) 5 µl l-1 C2H4; (b) 10 µl l-1 C2H4; (c) 30 µl l-1 C2H4; all balanced with 0.034% CO2 and 21% O2, and (d) a control containing no ethylene but 0.034% CO2 with 21% O2 (v/v). The system was constructed using four small growth chambers. The sealing of each Plexiglas chamber made it possible to circulate the modified gas mixture. The samples were placed on a false floor to allow for the circulation of gases. To avoid culture dehydration, the gases were hydrated by bubbling through distilled water. The relative humidity inside the chambers was about 90±5%. Sterility was achieved by passing the air through a 0.22 µM air filter before injection into the growth chambers. The flux was kept constant day and night. Ethylene enrichment was provided by cylinders containing a mixed calibrated gas. The experiment was a randomized complete block design with three blocks, and 10 Petri dishes per treatment per block with five pieces of 80 mg (±5 mg) embryogenic tissues per Petri dish. Embryogenic tissues were placed on maturation medium in unsealed Petri dishes.
In addition, to detect a possible interaction between ethylene and its biosynthetic inhibitors, embryogenic tissues were cultured on a maturation medium supplemented with 10 µM AOA and placed in the ethylene enrichment system (30 µl l-1). This experiment was a completely randomized design with two replicates (growth chamber) per treatment and 10 Petri dishes per replicate with five pieces of 80 mg (±5 mg) of embryogenic tissue per Petri dish.
Data collection
After 5 weeks on maturation medium, the cotyledonary somatic embryos were determined as to be normal or abnormal as described previously (El Meskaoui and Tremblay, 1999).
Tissue processing for microscopy
The effects of ethylene on the amount of intercellular space in the somatic embryo shoot meristems were studied using light microscopy. At the end of the maturation period, somatic embryos which matured either under ethylene enrichment or without ethylene enrichment were harvested. At least 10 embryos were collected for each treatment and were fixed overnight at 4 °C in 4% formaldehyde in 25 mM phosphate buffer (pH 6.8). Samples were washed three times in buffer, dehydrated in an ethanol series and then embedded in LR White resin (Marivae, Halifax, Canada) according to current procedures (Newman, 1989). Semi-thin (1.5 µm) sections were incubated first with periodic acid for 8 min, rinsed in distilled water and then treated in the dark with Schiff reagent for 15 min to reveal carbohydrates. After rinsing with distilled water, the sections were then stained with the naphthol blue black solution for 12 min at 37 °C to reveal proteins, washed with distilled water, treated with 7% acetic acid for 30 s, rinsed with water and dried. They were mounted in Eukitt (EMS, Fort Washington, PA, USA) and examined with a polyvar Reichert-Jung microscope.
Environmental conditions
Maintenance and maturation of embryos for all of the experiments described above were performed under the conditions described previously (El Meskaoui et al., 2000).
Statistical analyses
All experiments were repeated at least twice and representative data are presented. For each experiment, homogeneity of variance was verified by Bartlett's test (Sokal and Rohlf, 1995). Data from maturation were analysed using the SAS GLM procedure (SAS Institute Inc., Cary, NC, USA) and means were compared using Bonferroni's multiple range test (Sokal and Rohlf, 1995) at P=0.05.
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Results |
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Effect of ethylene enrichment and ACC supply on black spruce somatic embryo maturation
Addition of ACC to the maturation medium resulted in a decrease in the total number of mature somatic embryos produced in both embryogenic cell lines compared to controls. Furthermore, the total number of embryos decreased with increasing concentration of ACC (Table 1
). Increasing the level of ethylene by ethylene enrichment (pure ethylene) in the culture gas phase did not alter the production of mature somatic embryos in the high capacity cell line (M-282), but significantly reduced it in the low capacity cell line (M-393.1) at the highest ethylene level, 30 µl l-1 (Table 1), indicating a possible negative feedback of ethylene. Furthermore, both embryogenic cell lines growing on maturation medium with either added ACC or ethylene exhibited a brown colour in contrast to those growing on the control medium.
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Effect of AgNO3 and AOA on maturation of black spruce somatic embryos
Compared to controls, addition of the ethylene inhibitors, AOA (10 or 100 µM) or AgNO3 (1 or 2 mM) to the maturation medium significantly decreased the number of mature somatic embryos produced for the high capacity cell line. In contrast, addition of AOA (10 µM) or AgNO3 (1 mM) to the maturation medium significantly promoted the production of mature somatic embryos in the low capacity cell line except for an inhibitory effect at high concentrations of AOA (100 µM) (Table 2). Furthermore, under 30 µl l-1 ethylene enrichment, the stimulatory effect of AOA was reversed in the low capacity cell line (Fig. 1A) while the inhibitory effect of AOA was partially reversed in the high capacity line (Fig. 1B). AOA limited the browning of both embryogenic cell lines, while AgNO3 was ineffective in reducing this phenomenon.
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Time-course of ethylene production in black spruce embryogenic tissues during maturation
The control tube without embryogenic tissue never showed any detectable ethylene production. Preliminary experiments had revealed that the rate of ethylene production was constant during 24 h for both cell lines (data not shown). Therefore, the rate of ethylene production was determined by assaying its accumulation during 24 h. The time-course of ethylene evolution during the maturation period varied for the different embryogenic cell lines (Fig. 2A, B). The low capacity cell line produced considerably more ethylene, with the maximum rate exceeding that of the high capacity cell line by more than 4-fold during the first half of the culture period and by much more than this after this period. In the low capacity cell line, ethylene production was about 250 pmol g-1 fresh weight h-1 at the beginning of the culture (first 24 h). During the following 4 d, it declined steadily to reach a minimum value of approximately 60 pmol g-1 fresh weight h-1. After this, the rate of ethylene production increased gradually to reach a maximum level of approximately 400 pmol g-1 fresh weight h-1 at the end of the maturation period (Fig. 2A). However, in the high capacity cell line, ethylene production was about 65 pmol g-1 fresh weight h-1 at the beginning of the culture (first 24 h). During the following 8 d, it declined steadily to reach a value of approximately 25 pmol g-1 fresh weight h-1. After this, the rate of ethylene production slightly increased to reach a maximum level of approximately 45 pmol g-1 fresh weight h-1 and then declined steadily to reach a minimum value of approximately 10 pmol g-1 fresh weight h-1 at the end of the maturation period (Fig. 2B).
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Time-course of the intracellular levels of ACC in black spruce embryogenic tissues during maturation
In tissues grown on maintenance medium before transfer to maturation, intracellular ACC levels were higher in both lines than during maturation (about 110 and 90 nmol g-1 fresh weight in the high and low capacity cell lines, respectively) (Fig. 3A, B). After transfer to maturation medium, intracellular ACC levels declined rapidly after 5 d to reach a value of approximately 25 and 40 nmol g-1 fresh weight in higher and less responsive lines, respectively. Beyond this period, the level of intracellular ACC increased slightly followed by a decrease that stabilized at approximately 10 nmol g-1 fresh weight at the end of the culture period in the high capacity cell line (Fig. 3B). In the low capacity cell line, however, the intracellular ACC level continued to decline to a minimum value of approximately 25 nmol g-1 fresh weight at day 12, then stabilized at approximately 30 nmol g-1 fresh weight for the remainder of the culture period (Fig. 3A).
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Effect of AgNO3, ACC, and AOA on ethylene production
Compared to controls, addition of ACC and AOA to the culture medium increased and decreased, respectively, the ethylene production of both cell lines (Fig. 4A, B). When 10 µM ACC was added, ethylene levels were significantly higher than in the control without ACC during the entire culture period. On the other hand, the addition of 10 µM of AOA to the maturation medium significantly decreased ethylene production in both cell lines. Addition of 1 mM silver nitrate affected ethylene production only slightly.
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Effect of AOA and exogenous ACC on intracellular levels of ACC
The effects of addition of AOA or exogenous ACC to the maturation medium on intracellular levels of ACC in embryogenic tissue during the maturation period were determined for both cell lines (Fig. 5A, B). Addition of exogenous ACC to the maturation medium on cellular levels of ACC showed that the level of endogenous ACC in the ACC-treated tissue was significantly higher in the presence of 10 µM of exogenous ACC than in the control throughout the maturation period for a low capacity cell line (Fig. 5A). However, the presence of ACC to the maturation medium significantly increased but only after the first 5 d of culture (Fig. 5B). Furthermore, addition of AOA to the maturation medium reduced cellular levels of ACC during the culture period of both cell lines.
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Effect of ethylene on the shoot apical meristem of black spruce somatic embryos
No difference was observed in the organization of the shoot apical meristem of mature cotyledonary somatic embryos developed in the absence (control) or presence of ethylene (30 µl l-1) (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In the present study, the possible role of ethylene in maturation of conifer somatic embryo was investigated on two selected embryogenic cell lines of black spruce. Genotype is one of the most important factors affecting maturation. However, it is possible to modulate somatic embryo production with different treatments. The embryogenic cell lines used in this study were selected because they responded differently during the maturation stage of somatic embryogenesis. Somatic embryo production of the line M-282 was much higher than that of M-393.1 (Tables 1
, 2). Based on their mature somatic embryo production capacity, M-282 and M-393.1 can be considered as high and low capacity cell lines, respectively. The comparison of their ethylene production during maturation revealed that the low capacity cell line exhibited higher rates of production than the high capacity cell line. Similarly, endogenous ACC levels in the low capacity cell line were higher compared to those in the high capacity cell line. This suggests that embryo production capacity might be inversely related to ethylene production rate during maturation.
In order to investigate how ethylene metabolism might be associated with this difference in maturation capacity, ethylene metabolism was altered using several modulators molecule. First, an ethylene enrichment approach was taken by increasing the ethylene level in the vicinity of cultures. Ethylene enrichment resulted in a decrease in the number of mature somatic embryos produced in the low capacity cell line whereas it had no effect in the high capacity cell line, indicating a possible feedback of ethylene (Table 1).
Second, modification of tissue sensitivity to ethylene was performed by using AgNO3, an inhibitor of ethylene action which acts by combining with the ethylene receptor and preventing the cell from responding to ethylene. Addition of AgNO3 in the culture medium reduced somatic embryo production in the high capacity cell line while it promoted embryo production in the low capacity cell line (Table 2). As well, AgNO3 slightly increased ethylene production in both embryogenic cell lines (Fig. 4A, B). This increase, however, was not statistically significant. An increase in ethylene production by AgNO3 has also been observed in other studies (Roustan et al., 1990; Chraibi et al., 1991; Kong and Yeung, 1994; Nissen, 1994; Santos et al., 1997). The mechanism for the increase in ethylene production in response to AgNO3 is not well understood, but it is speculated that the absence of ethylene activity following treatment with AgNO3 may influence the negative feedback regulation of ethylene biosynthesis (Kende, 1993).
The effect of influencing the metabolic pathway of ethylene was also examined. This was done by incorporating ACC, a precursor of ethylene biosynthesis, into the culture medium or AOA, an inhibitor of ACC synthase. In both embryogenic cell lines, the addition of AOA markedly decreased both ethylene production (Fig. 4A, B) and the endogenous ACC levels (Fig. 4A, B), while the addition of ACC markedly increased it (Fig. 4A, B). Furthermore, AOA promoted somatic embryo production in the low capacity cell line and reduced it in the high capacity cell line (Table 2). The addition of ACC reduced somatic embryo production in both cell lines (Table 1). At the same time the addition of ethylene to the high capacity cell line did not inhibit somatic embryo production, thus confirming earlier results (El Meskaoui and Tremblay, 1999). Therefore, the effect of ACC on embryo maturation appears to be independent of the ethylene. Similarly, growth inhibition of Norway spruce embryogenic tissue (Kvaalen, 1994), and inhibition of black spruce (El Meskaoui and Tremblay, 1999), and carrot somatic embryos by ACC (Nissen, 1994) were not mediated by ethylene.
On another hand, combining the results of this work, these results also indicate that limiting ethylene biosynthesis was beneficial to somatic embryo maturation in the low capacity cell line. Conversely, lowering ethylene production was not beneficial to maturation in the high capacity cell line. This is further substantiated by the finding that the stimulatory and inhibitory effects of AOA were reversed by adding ethylene to the low and high embryogenic cell lines (Fig. 1A, B), respectively. These results suggest that the low capacity cell line was sensitive to decreases in its ethylene biosynthetic capacity and thus appears to have a supraoptimal ethylene production capacity. In the high capacity cell line, on the other hand, ethylene biosynthetic capacity could be considered optimal since any treatment that either increased or decreased ethylene biosynthesis resulted in lower mature somatic embryo production. Consistent with these results, ethylene enhanced embryogenesis in anthers that produce ethylene slowly whereas it reduced embryogenesis in anthers that produce ethylene rapidly (Cho and Kasha, 1989). Similarly, the effect of ethylene has also been found to be positive (Nissen, 1994) or negative (Roustan et al., 1989, 1990) for carrot somatic embryogenesis in different cell lines which exhibited different capacities to produce ethylene. In conifer species, ethylene effects on different stages of somatic embryogenesis can not be generalized (Kumar et al., 1989; Kvaalen, 1994; Kong and Yeung, 1994; Li and Huang, 1996; Selby et al., 1996; Afele and Preveen, 1995; El Meskaoui and Tremblay, 1999), but might be attributed to the sensitivity of the embryogenic cell line to ethylene. The results of the present study clearly demonstrate that ethylene plays a crucial role in the regulation of somatic embryo maturation in black spruce. Although the precise mechanism by which ethylene regulates somatic embryogenesis is not known, it is believed that a sensitivity of the tissue to ethylene can be regulated by endogenous growth factors as well as by their balance. It is well documented that plant hormones are capable of regulating ethylene biosynthesis (Yang and Hoffman, 1984; Abeles et al., 1992). Among the plant hormones, the effect of auxin (IAA) on ethylene biosynthesis and its mechanism of action have been thoroughly investigated (Yang and Hoffman, 1984). ABA has also been shown to affect ethylene biosynthesis in different plant species (Abeles et al., 1992).
The maturation medium of conifers commonly contains ABA which is necessary for embryo maturation in these species (Jain et al., 1995). Thus, it may be that somatic embryogenesis is regulated by the interaction between endogenous ABA and ethylene metabolism. A link between ethylene and ABA has been reported in oat leaves and in apple slices, where ABA decreases ethylene production through a decrease in ACC synthesis (Tan and Thimann, 1989). In the present study, before transfer to the maturation medium, the endogenous ACC level was very high and then decreased through the maturation stage in both embryogenic cell lines (Fig. 2A, B). Moreover, the endogenous ACC level in embryogenic tissues of black spruce and white spruce growing on maintenance medium, without ABA but with auxin (2,4-D) and cytokinin (BA), was very high compared to those in the embryogenic tissues cultivated on maturation medium (data not shown). In the present study, ABA might act on ACC synthesis as shown by the decrease of endogenous ACC during maturation of both embryogenic lines. In white spruce, ABA reduced ethylene production and promoted maturation (Kong and Yeung, 1994). It is important to note that ABA had both promotive and inhibitory effects on somatic embryos maturation depending on its concentration. Thus, the optimal maturation capacity depends mainly on the sensitivity of the embryogenic cell line to ABA. On another hand, the absence of auxin and cytokinin in the maturation medium might also have caused the observed decline in ACC. Removal of 2,4-D and/or BA from the maintenance medium of embryogenic tissue of Norway spruce caused a rapid fall in the endogenous ACC level (Kvaalen, 1994). Thus, both presence of ABA or absence of auxin/cytokinin may be responsible for reduced ACC synthesis in cultures during maturation. In fact Yoon et al., using the promotor region of an auxin-inducible ACC synthase gene (VR-ACS6), found that both auxin and cytokinin stimulated expression of this gene whereas ABA and ethylene supressed its expression in transgenic tobacco (Yoon et al., 1999). Analyses of endogenous ABA and other hormones such as auxin and cytokinin, together with alteration of ethylene metabolism, will help to clarify their interaction in conifer somatic embryogenesis.
The polyamines and ethylene biosynthetic pathways share a common precursor, S-adenosylmethionine (Yang and Hoffman, 1984). It has been postulated that ethylene and polyamines may regulate each other's biosynthesis (Minocha and Minocha, 1995). In the present study, AOA reduced ACC synthase activity as shown by the decrease in endogenous ACC pools and in ethylene production. Therefore, it is believed that this inhibition resulted in an accumulation of S-adenosylmethionine (SAM), which participates in the methylation of nucleic acids and serves as a donor of aminopropyl moieties for polyamine synthesis. Furthermore, it has been shown that SAM levels may be involved in the control of somatic embryogenesis and in the regulation of development of somatic embryos (Munksgaard et al., 1995).
Thus, it is possible that somatic embryogenesis was regulated by the interaction between ethylene and polyamine metabolism. In carrot, it has been hypothesized that the competitive interaction between ethylene and polyamines may be an important regulatory factor controlling somatic embryogenesis (Galston and Kaur-Sawhney, 1995). Somatic embryogenesis in conifers is known to be quite different from that in angiosperms. Despite this, evidence for the participation of polyamines in the control of conifer zygotic and somatic embryogenesis continues to accumulate (Santanen and Simola, 1992, 1994; Minocha and Minocha, 1995; Sarjala et al., 1997; Kong et al., 1998; Minocha et al., 1999). These studies indicate that the cellular levels of polyamines are associated with the differentiation and development of conifer somatic embryos.
It is well known that polyamines promote growth and delay senescence whereas ethylene may accelerate senescence in tissue culture. According to this study's observations, ethylene appears to be involved in the browning and senescing of the tissues since the application of either ethylene or ACC increased these phenomena. On the other hand, application of AOA led to reduced browning in both cell lines. The mechanism by which ethylene causes browning is not clear, but it is thought that ethylene could activate the synthesis of oxidative enzymes or could inhibit the synthesis of protective enzymes (Abeles et al., 1992). It has been reported that ethylene can inhibit polyamines biosynthesis which are recognized as antisenescence agents (Galston and Kaur-Sawhney, 1995; Egea-Cortines and Mizhari, 1991).
It has also been indicated that ethylene can negatively affect the quality of white spruce somatic embryos by forming large intercellular spaces in the shoot apex that can be partly responsible for the low conversion rate of the somatic embryos into plants (Kong and Yeung, 1994). However, in the present study as in previous study (El Meskaoui et al., 2000) ethylene did not affect the quality of somatic embryos. Indeed, somatic embryos produced under a constant supply of ethylene during the maturation show no abnormality in cell structural organization of the caulinary pole.
In conclusion, these results show the importance of endogenous ethylene production during maturation of black spruce somatic embryos. Ethylene may play a role in the variability of mature somatic embryo production of different cell lines. Depending on the embryogenic cell line, a reduction in ethylene production may be beneficial to embryo development in embryogenic cell lines which have a supraoptimal level of ethylene production, whereas it may not be beneficial in embryogenic lines which have an optimal level of ethylene production. Moreover, the fact that ethylene production is modulated by other hormones, which are also necessary for in vitro development (Abeles et al., 1992), makes the interactions between ethylene and other endogenous growth factors crucial in the regulation of mature somatic embryo production. Such studies are now in progress.
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Acknowledgments |
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We thank Dr H Margolis for critical reading of the manuscript, Dr H Chamberland for technical assistance with tissue processing for microscopy, and the Ministry of Natural Resources of Quebec (Ste-Foy, Que, Canada) for providing control pollinated seeds. This research program was financially supported by a grant from the Ministry of Industry, Trade, Science and Technology (Synergie program) in partnership with BECHEDOR Inc., PAMPEV Inc., and CPPFQ Enr. to FM Tremblay.
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Notes |
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1 To whom correspondence should be addressed. Fax: +1 418 656 7493. E-mail: aelmeskaoui{at}caramail.com
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