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Polyamine Content and Somatic Embryogenesis in Papaver somniferum Cells Transformed with sam-1 Gene
f. Plant Physiol. Vol. 154. pp. 729-734 (1999) http://www. urbanfischer.de/journals/j pp
JOUR.ALOF
Plani Physjoloay © 1999 URBAN & FISCHER
Polyamine Content and Somatic Embryogenesis in Papaver somniferum Cells Transformed with sam-1 Gene SANAA NABHA 1 *,FREDERIC LAMBLIN 2 , FRAN<;:OISE GILLET\ DoMINIQUE LAURAIN\ MARC FLINIAux\ ALAIN DAvm 2 , and ANNIE ]ACQUIN 1 1
Laboratoire de Pharmacognosie et Phytotechnologie, Universite de Picardie Jules Verne, Faculte de Pharmacie, 1 rue des Louvels, 80037 Amiens Cedex, France
2
Laboratoire de Biotechnologie et Physiologie V egetales, Universite de Picardie Jules Verne, Faculte de Sciences, 33 rue Saint Leu, 80039 Amiens Cedex, France
Received July 13, 1998 ·Accepted October 25, 1998
Summary
Papaver somniferum calluses transformed with the sam-1 gene from Arabidopsis thaliana, which encodes a SAM-synthetase, were subcultured over a 4-year period. The stability of the expression as well as the level of SAM synthetase activity were evaluated in 5 transgenic cell lines (STSI, STSII, STSIII, STSIV, STSV) and in the control. All transgenic cell lines exhibited a level of SAM-synthetase activity higher than that of the control. The enhancement of the enzyme activity has been confirmed by Northern blot analyses. The level of polyamines (putrescine, spermidine, spermine and 1,3-diaminopropane) was also evaluated in cell lines cultured either in Linsmaier and Skoog medium containing growth regulators (LS) or on a hormone free medium (LSHF). Cell lines cultured in LS medium showed a variable yield of the polyamine content. Putrescine was the major polyamine. After transfer to a hormone free medium, all of the cell lines, except STSIV, were able to form embryo-like structures. In this condition, the polyamine level decreases about 4-fold, spermidine being the most abundant. Key words: Papaver somniferum, transgenic cells, sam-1 gene, SAM synthetase activity, polyamines, somatic embryogenesis. Abbreviations: DTT = dithiothreitol; EDTA = ethylene diamine tetraacetic acid; dCTP = deoxycytidine triphosphate; LS = Linsmaier and Skoog medium; LSHF = Linsmaier and Skoog hormone free medium; PA =Polyamine; DAP = 1,3 diaminopropane; SAM-S = S-Adenosyl Methionine Synthetase; Tris = trishydroxymethylaminomethane. Introduction
SAM-S catalyzes the conversion of L-methionine to Sadenosyl-L-methionine (SAM) (Tabor and Tabor, 1984), which is the major methyl group donor for numerous methylation reactions. It plays a key role in the biosynthesis of polyamines and ethylene. Polyamines are polybasic aliphatic amines. Putrescine is synthesized from ornithine and arginine by ornithine decarboxylase and arginine decarboxylase (Slocum et a!., 1984; Smith, 1990). Spermidine and spermine are synthesized by the addition of an aminopropyl group to putrescine. This * Correspondence.
aminopropyl group is provided by S-adenosylmethionine via S-adenosylmethionine decarboxylase (Smith, 1990). 1,3Diaminopropane (DAP) is synthesized from the oxidation of spermidine and spermine by polyamine oxidase. Polyamines play a key role in numerous processes of plant development including growth, senescence and response to stress (Galston, 1983), as well as floral initiation and differentiation (Flores and Martin-Tanguy, 1991; Martin-Tanguy, 1997). They seem to be also involved in organogenesis and somatic embryogenesis in a number of plant cell cultures (Montague et a!., 1978; Robie and Minocha, 1989; Minocha eta!., 1991; Amarasinghe eta!., 1996). The sam-1 gene from Arabidopsis thaliana encoding the S-adenosylmethionine synthetase (SAM-S) has been success0176-1617/99/154/729 $ 12.00/0
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S. NABHA, F. LAMauN, F. GILLET, D. LAuRAIN, M. FLINIAUX, A. DAVID, and A. }ACQUIN
fully transferred into Papaver somniferum cells (Belny et al., 1997). Five transgenic cell lines have been obtained. In this paper, we report on the evolution of sam-1 gene expression over a 4-year period taking as reference the results published by Belny et al. (1997). We also evaluate at the end of this period, the level of endogenous PA as well as the capacity of the cells to develop somatic embryogenesis.
Materials and Methods
Culture conditions Seeds of Papaver somniferum C. V. exotica were obtained from Clause Co., Paris, France. Calluses were initiated using hypocotyl explants. Untransformed calluses used as control were subcultured on a solid Linsmaier and Skoog (1965) medium (LS) supplemented with 2,4-dichlorophenoxyacetic acid (0.1 mg/L), 6-benzylaminopurine (0.1mg/L), sucrose (30g/L) and 0.8o/o (w/v) agar (Difco). Five transformed cell lines (Belny et a!., 1997) expressing the foreign sam-] gene from Arabidopsis thaliana were cultured on an LS solid medium supplemented with the antibiotic paromomycin sulfate (lOOmg/L) (Sigma). Control and transgenic calluses were maintained at 25 ± 2 ·c in the dark and were subcultured on a fresh medium every 3 weeks. Although the cultures did not necessarily have a unicellular origin, they were called cell lines for convenience.
Growth measurement For estimation of the growth rate between two subcultures, calluses were carefully collected, freeze-dried and weighed (SD ± 5 o/o). Growth rate was the ratio between the dry weight after a 21-day culture period and the dry weight of the inoculum.
SAM-synthetase assays The SAM-S activity was determined on calluses that had been maintained and subcultivated during 4 years. Five hundred mg of calluses (fresh weight) were collected 13 days after the last subculture (exponential growth phase). Proteins were extracted using a method described in the literature (Boerjan et a!., 1994). The cells were ground in 0.5 mL of extraction buffer (0.1 mol/L Tris, pH 7.5, 2 mmol/L EDTA, 20 o/o glycerol, 20 mmol/L ~-mercaptoethanol, 1 mmol/L DTT). After centrifugation, the protein concentration was determined according to Bradford (1976). The SAM-S activity was determined according to Peleman et a!. (1989) and Mathur and Sachar (1991) in two protein extracts and expressed in nKatg-1 protein.
Northern blot analysis Total RNA was extracted from calluses collected 13 days a&er subculture using the RNeasy plant mini kit (Qiagen). Samples (10 !lg total RNA) were denaturated, run on a MOPS-formaldehyde 1.2 o/o agarose gel and then blotted by capillary transfer to HybondN nylon membranes (Amersham). Blotted RNA was fixed by baking at 80 ·c for 2 h. Prehybridizations and hybridizations were performed according to Sambrook et a!. (1989), at 42 ·c in 50 o/o formamide, 5 x SSC, 0.5 o/o SDS and 100 11g/mL salmon sperm DNA. A 974 bp 2urified fragment of the sam- I coding sequence was labelled with (a-33 P) dCTP (Redeprime DNA labelling system, Amersham) and used as probe. Membranes were washed once in 2xSSC, 0.1 o/o SDS at 65 ·c. Hybridization was detected by exposure to X-Omat films (Kodak) at -80 ·c. To compare the relative amounts of RNA,
the membranes were rehybridized with a 25S rRNA eDNA probe. Hybridization signal intensities were estimated using a phosphor Imager (Storm 840, Molecular Dynamics) and for each RNA sample the quantification index (I) was calculated as the ratio of the intensity of the sam-] signal to the 25 rRNA signal.
Induction ofembryogenesis For the expression of embryogenesis, control and transgenic calluses were transferred to an LS hormone-free medium (LSHF) in Petri dishes placed at 25 ± 2 ·c in darkness, with three Petri dishes for each cell line. The number of embryogenic structures was estimated by stereomicroscopic observations on three samples of 100 mg fresh weight of each Petri dish. Data presented correspond to the mean value of the 9 samples.
Analysis ofpolyamine content For the polyamine (PA) determination, cells were collected 3 weeks after subculture, freeze-dried and then homogenized in 1 N perchloric acid (5 mL/100 mg dty weight). The homogenates were centrifuged for 45 min at 32,000 gn at 4 ·c. An aliquot of the supernatant was used for analysis of free PAs, another aliquot from the supernatant and the pellet were hydrolysed with 6 N HCl for 12 h at 110 ·c for analysis of both conjugated and bound PA, respectively. After adding diaminooctane (internal standard), 200 IlL of the extracts were dansylated following the procedure described by Flores and Galston (1982). The PAs were determined by HPLC by comparison with authentic standards (Sigma), using a Thermo Separation Products model1500 pump fitted with a 20 IlL injection loop, a C 18-reversed phase column (Kromasil, 4.6 X 25 mm, 5 11m particles), a fluorescence detector (Waters) equipped with an excitation filter (333 at 365 nm) and an emission filter (485 at 522 nm) coupled with an integrator (Chromfet). The PAs were eluted using a linear gradient of methanol (from 60 o/o to 100 o/o) and water at a flow rate of 1 mL/min (Smith and Davies, 1985). The elution times were the following: 13.2 min for DAP, 14.9 min for putrescine, 19.2min for spermidine and 22.6min for spermine.
Results and Discussion
SAM synthetase activity in control and transgenic calluses SAM-S activity was analyzed in 4-year-old Papaver somniferum cultures {one control and five transgenic cell lines) (Fig.
1). These results were compared with those obtained 3 years earlier by Belny et al. (1997) with the same cell lines. In young cultures, only one transgenic cell line (STSV) showed a SAM-S activity about 1.8-fold higher than that of the control (47 nkat/g protein). All of the others showed a lower level of SAM-S activity. This was probably due to a partial cosuppression of both the resident and the foreign sam genes (Belny et al., 1997). This phenomenon can affect many endogenous genes after introduction of homologous transgenes {Meyer, 1996), which has already been observed in tobacco plants transformed with the Arabidopsis thaliana sam-] gene (Boerjan et al., 1994). In 4-year-old cell lines, we first noticed that the SAM-Sactivity of the control dramatically decreased {about two-fold) from values of initial cultures. This could be partially explained by somaclonal variations that may occur during 4 years of in vitro culture in the presence of growth regulators.
Somatic Embryogenesis in Papaver Cells
1
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0.02 0.13 0.56 0.38 0.48 1.0
PS
STSI
STSII STSIII STSIV STSV
Cell lines Fig. 1: SAM-S activity in control (PS) and transgenic (STSI, STSII, STSIII, STSIV, STSV) cell lines of Papaver somniferum cultured in LS medium for 4 years. Total protein was extracted 13 days after subculture. Each column represents the mean of the enzyme activity determined for two successive subcultures. Asterisk (*) indicates a significant difference from the control (p <0.05) according to the bilateral Dunnett's test.
On the contrary, the enzyme activity increased significantly in all of the transgenic cell lines, as confirmed by statistical analysis (bilateral Dunnett's test). The STSIV cell line exhibited the highest level of activity (77 nkat/g protein), about 4-fold higher than the control (18 nkat/g protein). The cell lines STSI, STSII, STSIII and STSV had a SAM-S activity 3-fold higher than the control. This could result from the selection conditions under which the transgenic cells were maintained during 4 years. The calluses that could be still chimerical at the time of the first analysis, now contained more and more transgenic cells.
Fig. 2: Northern blot analysis of total RNAs isolated from control and transgenic cell lines 13 days after subculture. Ten micrograms of RNA samples from control, STSI, STSII, STSIII, STSIV and STSV were loaded in lanes 1 to 6, respectively. The blot was hybridized using a 33 P-labelled sam-] probe. Quantification index (I) was calculated using a Phosphor Imager and taking into account the relative amount of RNA in each lane determined after rehybridization of the blot with a 25S ribosomal RNA eDNA probe.
Polyamine content in calluses cultured in a LS medium Figure 3 presents the polyamine content (putrescine, spermidine, spermine, DAP) of the transgenic cell lines (STSI, STSII, STSIII, STSIV, STSV) and the control cultured in a LS medium. The total polyamine content of the transgenic cell lines was lower than the control (PS) except for STSIV (52!lmol/g dry weight and 34!lmollg dry weight, respectively). Polyamine content was the lowest in the STSV cell line. (17!lmol/g dry weight). Among the four analyzed poly60r-----~~====~------~
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To confirm that the increase of the SAM-S activity resulted from the transgene expression, Northern blot analysis was performed on total RNA extracted from cell lines 13 days after a subculture (Fig. 2). All transgenic cell lines showed a stronger hybridization signal compared with the control. A very weak signal could be observed for the control, probably due to a non-specific hybridization with mRNAs of sam endogenous genes. The determination of the quantification index revealed higher levels of sam-] transcripts in STSV (I= 1.0) than in STSII and STSIV (0.56 and 0.48, respectively). Cell line STSI, which had the lowest SAM-S activity, showed the lowest level of sam-1 mRNA. These results indicated that all transgenic cell lines expressed the sam-] gene and that there is a positive correlation between transgene expression and enzyme activity.
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Cell lines Fig. 3: Polyamine content (Jlmol/g dry weight) of Papaver somniferum calluses cultured in an LS medium; PS: untransformed calluses; STSI, STSII, STSIII, STSIV, STSV: transgenic cell lines.
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S. NABHA, F. LAMBLIN, F. GILLET, D. LAURAIN, M. FuNIAUX, A. DAVID, and A. ]ACQUIN
Table 1: Free, conjugated and bound polyamine content (f.Lmollg dry weight) of Papaver somniferum calluses cultured in an LS medium; PS: untransformed calluses; STSI, STSII, STSIII, STSIV, STSV: transgenic cell lines (mean values of2 replicates).
Table 2: Growth rate of Papaver somniferum cell lines; estimation by the ratio between the dry weight of the biomass 3 weeks after subculture and the dry weight of the inoculum (PS: untransformed cal!uses; STSI, STSII, STSIII, STSIV, STSV: transgenic cell lines).
PS
STSI
STSII
STSIII
STSIV
STSV
Cell lines
Growth rate
Putrescine Free Conjugated Bound Total
6.10 14.65 1.80 22.55
1.87 6.25 1.72 9.84
4.17 8.25 0.70 13.32
5.44 2.97 5.22 13.63
24.49 10.82 7.63 42.94
4.44 2.09 2.41 8.94
Spermidine Free Conjugated Bound Total
PS STSI STSII STSIII STSIV STSV
4.0 4.7 5.2 6.7 3.3 4.5
2.47 4.83 1.03 8.33
1.20 4.25 1.70 7.15
2.66 4.50 1.03 8.19
2.41 3.57 3.73 9.71
3.29 0.95 1.82 6.06
2.83 1.25 1.76 5.84
Spermine Free Conjugated Bound Total
0.30 1.03 0.15 1.48
0.46 1.20 0.31 1.97
0.80 1.42 0.46 2.68
3.13 1.38 0.91 5.42
0.69 0.11 0.40 1.20
0.95 0.50 0.34 1.79
DAP Free Conjugates Bound Total
0.45 0.84 0.08 1.37
0.15 0.27 0.07 0.49
0.36 0.40 0.06 0.82
0.34 0.15 0.16 0.65
0.69 0.90 0.40 1.38
0.30 0.10 0.11 0.51
amines, putrescine was the most abundantly accumulated, especially in STSIV. All of the cell lines contained more spermidine than spermine. DAP was the least abundant compound. The levels of the three different forms of PA (free, conjugated and bound) are presented in Table 1. The conjugated form was predominant in the control (PS), STSI and STSII cell lines, approximatively 60 o/o of total PA. The free PA form was most dominant in STSIV (about 60 o/o of total PA), in STSV (about 50 o/o of total PA), and in STSIII (about 40 o/o of total PA), except for spermidine. In STSIV, the high level of PA (52!-lmol/g dry weight including about 43!-lmol/g dry weight of putrescine) is due to all of the forms of putrescine (about 25!-lmol/g dry weight free putrescine, 11!-lmol/g dry weight for the conjugated form and 8!-lmol/g dry weight for the bound form). It has been suggested in the literature (Aribaud et al., 1994; Burtin et al., 1989) that such high levels of free and conjugated PAs (especially putrescine and spermidine) could inhibit cell multiplication and supress differentiation in leaf explants of tobacco and Chrysanthemum morifolium cultured in vitro. In spindle trees, it has been shown that a high intracellular level of putrescine supresses somatic embryogenesis (Bonneau et al., 1995). Moreover, the formation of somatic embryo is associated with a significant decrease in putrescine level. From these results, we decided to evaluate the growth and the embryogenic ability of the studied cell lines.
Growth ofPapaver somniferum calluses In a preliminary work (data not shown), we observed that the growth of the culture during a 21-day period was linear
for all cell lines. Growth weight was estimated by the ratio of the dry weight after 21 days of culture and dry weight of the inoculum, for each cell line (Table2). It is remarkable to note that STSIV, the cell line that accumulated the highest level of PA, presented the lowest growth rate.
Somatic embryogenesis ofPapaver somniferum calluses Some of the cell lines displayed an irregular and spontaneous somatic embryogenesis. In order to know whether this
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Fig. 4: Somatic embryogenesis in Papaver somniferum calluses. Cell line STSV was cultured for 21 days in LSHF medium; (a): Globular embryos (G), Torpedo embryo (T) (x 8), (b): Torpedo stage (x 20), (c): Cotyledonary stage (x20).
Somatic Embryogenesis in Papaver Cells
Table 3: Somatic embryogenesis of Papaver somniferum cell lines:
20~-----------------------.
lliiil Spermine
the embryogenic ability is expressed by the number of embryogemc structures per g of fresh weight. PS: untransformed calluses; STSI, STSII, STSIII, STSIV, STSV: transgenic cell lines. Cell lines
Embryo somatic number/g fresh weight
PS STSI STSII STSIII STSIV STSV
64± 2 90±14 169±42 16± 6 0 300±67
capacity was linked to the transgene expression and/or to the intracellular PA levels, the cell lines were transferred to a hormone-free medium. This condition is known to facilitate somatic embryogenesis (Ribnicky eta!., 1996). Within a cell line, the morphology of the embryo-like structures varied greatly; globular, torpedo and heart cotyledonary stage embryos (Fig. 4). In all cases, except for the STSIV, embryo-like structures appeared about 10 days after subculture. The number of embryo-like structures regularly increased during 3 weeks of culture, but with large variations in the values depending on the cell line. After 3 weeks (Table 3), 3 cell lines (STSI, STSII, STSV) were more embryogenic than the control (PS) and the other two (STSIII, STSIV) were less embryogenic. STSV appeared to be the most embryogenic. Surprisingly, STSIV, which exhibited the highest level of SAM-S activity and the highest putrescine level, was the least embryogenic cell line. This result is in agreement with those obtained by Martin-Tanguy (1997) relating an inhibitory effect of free and conjugated putrescine on embryogenic capacity.
Polyamine content in calluses cultured in LSHF medium In order to correlate PA and somatic embryogenesis, PA content was measured after 21 days of culture in a hormonefree medium (Fig. 5). Similarly to cells cultivated in LS medium, we noted a cell line-dependent variability of the PA content. Some of the cell lines (STSIII, STSIV) contained more PA than the control, while the other ones, such as STSI, STSII, STSV, contained less than the control. However, in this condition, only the control accumulated putrescine, which was the major compound detected in all cell lines cultured in LS. After transfer of the cells to LSHF medium, spermidine was the major compound; spermine could also be detected. Only traces of DAP could be detected in all cell lines. A decrease in the total polyamine level (about 4-fold) could be noted, independently of the cell line (transformed or not). Among polyamines, putrescine was the most affected. These results are similar to the results reported by Tiburcio et a!. (1985), Santanen and Simola (1992), and Bonneau et a!. (1995). Indeed, they observed a significant decrease in putrescine levels associated with the formation of somatic embryos in tobacco, Picea abies and Euonymus europeaus L., respectively. This decrease could reflect a conversion of putrescine to spermidine (Men-
733
•
Spermidine
ILl Putrescine
PS
STSI
STSII STSIII STSIV STSV Cell lines
Fig. 5: Polyamine content (~mol/g dry weight) of Papaver somniferum calluses cultured in a hormone free-medium (LSHF); PS: untransformed calluses; STSI, STSII, STSIII, STSIV, STSV: transgenic cell lines.
goli et al., 1989). Spermidine and spermine did not decrease so dramatically as putrescine. The spermidine content decreased slightly in PS, STSI and STSIII, which were less embryogenic and decreased 2-fold in STSII and STSV, which were the most embryogenic cell lines. It can even be noted that the spermidine content of STSIV was higher than that of STSIV cultured in an LS medium (about lliJ.mol/g dry weight versus 61J.mol/g dry weight). Our results, except for STSIV, confirm those of Amarasinghe eta!. (1996) who observed a decrease in spermidine level in interior spruce calluses cultured on a medium for differentiation (without hormones). The free, conjugated and bound forms of spermidine, the most dominant PAin all of the cell lines cultured in LSHF medium, are given in Table 4. In all of the transformed cell lines, the main part of spermidine was accumulated in bound form (Table 4). This form represents about 60% (STSIII) to 93% (STSIV, STSV) of the total spermidine. It is worth noting that spermidine was accumulated mainly in free and conjugated forms when the cells were cultured in LS medium. Table 4: Free, conjugated and bound spermidine content (~mollg dry weight) of Papaver somniferum calluses cultured in a LSHF medium; PS: untransformed calluses; STSI, STSII, STSIII, STSHT, STSV: transgenic cell lines (mean values of2 replicates). PS
STSI
STSII
STSIII
STSIV
STSV
0.33 2.88 2.30 5.51
0.11 0.86 3.94 4.91
0.28 1.24 2.29 3.81
0.55 2.98 5.10 8.63
0 0.75 10.18 10.93
0 0.16 2.28 2.44
Spermidine Free Conjugated Bound Total
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S. NABHA, F. lAMBLIN, F. GILLET, D. LAuRAIN, M. FuNIAUX, A. DAVID, and A. ]ACQUIN
Concerning the embryogenic ability of the cell lines, a negative correlation was observed with the polyamine content. The most embryogenic cell line (STSV) had the lowest PA level and the non-embryogenic (STSIV) had the highest PA level. These results differ from those reported by numerous authors (Montague et al., 1978; Robie and Minocha, 1989; El Hadrami et al., 1989).
Conclusion
We noted an increase in the transgene expression (enzyme assays and Northern blots), as the cell lines were ageing. Morevee, the polyamine level and particularly that of putrescine was very heterogenous from one cell line to another one. This PA level, which decreased when calluses were transferred to a differentiation medium, was negatively correlated to embryogenesis in the various cell lines. Acknowledgements
We are grateful to Pro£ J. Martin-Tanguy for useful suggestions and critical reading of the manuscript. We thank «Le Biopole Vegetal>> Amiens-France for its financial support.
References
AMARASINGHE, V., R. DHAMI, and J. E. CARLSON: Polyamine biosynthesis during somatic embryogenesis in interior spruce (Picea glauca X Picea engelmannii complex). Plant Cell Rep. 15, 495499 (1996). AruBAUD, M., M. CARRE, and J. MARTIN-TANGUY: Polyamine metabolism and in vitro cell multiplication and differentiation in leaf explants of Chrysanthemum morifolium Ramat. Plant Growth Regul. 15, 143-155 (1994). BELNY, M., D. HEROUARD, B. THOMASSET, H. DAVID, A. JACQUINDUBREUIL, and A. DAVID: Transformation of Papaver somniferum cell suspension cultures with sam-] from A. thaliana results in cell lines of different S-adenosylmethionine synthetase activiry. Physiol. Plantarum 99, 233-240 (1997). BoERJAN, W., G. BAuw, M. VAN MoNTAGU, and D. INZE: Distinct phenotypes generated by overexpression and suppression of S-adenosyl-L-methionine synthetase reveal developmental patterns of gene silencing in tobacco. Plant Cell 6, 1401-1414 (1994). BoNNEAU, L., N. BERANGER-NOVAT, J. MoNIN, and J. MARTINTANGUY: Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis. Plant Growth Regul. 16, 5-10 (1995). BRADFORD, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 (1976). BuRTIN, D., ]. MARTIN-TANGUY, M. PAYNOT, and N. RossiN: Effects of the suicide inhibitors of arginine and ornithine decarboxylase activities on organogenesis, growth, free polyamine and hydroxycinnamoylputrescine levels in leaf explants of Nicotiana Xanthi n.c. cultivated in vitro in a medium producting callus formation. Plant Physiol. 89, 104-110 (1989). EL HAnRAMI, I., N. MicHAux-FERRIERE, M. P. CARRON, and J. D'AuZAc: Variabilite donale du potentiel embryogene chez Hevea brasiliemis; relations avec les Polyamines (PA) et les Peroxydases (PO) des cals. C. R. Acad. Sc. Paris, t. 308, serie III, p. 205-211 (1989).
FLORES, H. E. and A. W. GALSTON: Analysis of polyamines in higher plants by high performance liquid chromatography. Plant Physiol. 69,701-706 (1982). FLORES, H. E. and J. MARTIN-TANGUY: Polyamines and plant secondary metabolites. In: SLOCUM, R. D. and H. E. FLORES (eds.): Biochemistry and Physiology of Polyamines in Plants. CRC Press, Boca Raton Florida, pp. 57-76 (1991). GALSTON, A. W.: Polyamines as modulators of plant development. Biosc. 33, 382-388 (1983). LINSMAIER, E. M. and F. SKOOG: Organic growth factor requirements of tobacco tissue cultures. Physiol. Plantarum 18, 100-127 (1965). MARTIN-TANGUY, J.: Conjugated polyamines and reproductive development: Biochemical, molecular and physiological approaches. Physiol. Plantarum 100, 675-688 (1997). MATHUR, M. and R. C. SACHAR: Phytohormonal regulation of S-adenosylmethionine synthetase and S-adenosylmethionine levels in dwarf pea epicotyls. FEBS Lett. 287, 113-117 (1991). MENGOLI, M., N. BAGNI, G. LucCARINI, V. N. RoNCHI, and D. SERAFINI-FRACASSINI: Daucus carota cell cultures: Polyamines and effect of polyamine biosynthesis inhibitors in the preembryogenic phase and different embryo stages. J. Plant Physiol. 134, 389394 (1989). MEYER, P.: Homology-dependent gene silencing in plants. Annu. Rev. Plant Physiol. Plant Mol. Bioi. 47, 23-48 (1996). MINOCHA, S. C., R. MINOCHA, and A. KoMAMINE: Effects of polyamine biosynthesis inhibitors on S-adenosylmethionine synthetase and S-adenosylmethionine decarboxylase activities in carrot cell cultures. Plant Physiol. Biochem. 29, 231-237 (1991). MoNTAGUE, M. ]., J. W. KoPPENBRINK, and E. G. JAWORSKI: Polyamine metabolism in embryogenic cells of Daucus carota. Plant Physiol. 62, 430-433 (1978). PELEMAN, J., W. BoERJAN, G. ENGLER, J. SEURINK, J. BorrERMAN, T. ALuorrE, M. VAN MoNTAGU, and D. INZE: Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetase. Plant Cell 1, 81-93 (1989). RrBNICKY, D. M., N. Iuc, J.D. CoHEN, and T. ]. CooKE: The effects of exogenous auxins on endogenous indole-3-acetic acid metabolism. Plant Physiol. 112, 549-558 (1996). RoBIE, C. A. and S. C. MINOCHA: Polyamines and somatic embryogenesis in carrot. I. The effects of difluoromethylornithine and difluoromethylarginine. Plant Sci. 65, 45-54 (1989). SAMBROOK, J., E. F. FRITSCH, and T. MANIATIS: Molecular cloning. A Laboratory Manual, Vol. 2, PP. 9.34-9.46. Cold Spring Harbor Laboratory Press, New York, NY. ISBN 0-87969-309-6 (1989). SANTANEN, A. and L. K. SIMOLA: Changes in polyamine metabolism during somatic embryogenesis in Picea abies. ]. Plant Physiol. 140, 475-480 (1992). SLOCUM, R. D., R. KAuR-SAWHNEY, and A. W. GALSTON: The physiology and biochemistry of polyamines in plants. Arch. Biochem. Biophys. 235, 283-303 (1984). SMITH, T. A. and P. J. DAVIES: Separation and quantification of polyamines in plant tissue by high performance liquid chromatography of their dansyl derivatives. Plant Physiol. 78, 89-91 (1985). SMITH, T. A.: Plant polyamines - metabolism and function. In: FLORES, H. E., R. N. ARTECA, and J. E. SHANNON (eds.): Polyamines and ethylene: Biochemistry, Physiology, and interactions. Amer. Soc. Plant Physiol., 1-23 (1990). TABOR, C. W. and H. TABoR: Polyamines. Annu. Rev. Bio. Chern. 53, 749-790 (1984). TIBURCIO, A. F., R. KAuR-SAWHNEY, R. B. INGERSOLL, and A. W. GALSTON: Correlation between polyamines and pyrrolidine alkaloids in developing tobacco callus. Plant Physiol. 78, 323-326 (1985).
JOUR.ALOF
Plani Physjoloay © 1999 URBAN & FISCHER
Polyamine Content and Somatic Embryogenesis in Papaver somniferum Cells Transformed with sam-1 Gene SANAA NABHA 1 *,FREDERIC LAMBLIN 2 , FRAN<;:OISE GILLET\ DoMINIQUE LAURAIN\ MARC FLINIAux\ ALAIN DAvm 2 , and ANNIE ]ACQUIN 1 1
Laboratoire de Pharmacognosie et Phytotechnologie, Universite de Picardie Jules Verne, Faculte de Pharmacie, 1 rue des Louvels, 80037 Amiens Cedex, France
2
Laboratoire de Biotechnologie et Physiologie V egetales, Universite de Picardie Jules Verne, Faculte de Sciences, 33 rue Saint Leu, 80039 Amiens Cedex, France
Received July 13, 1998 ·Accepted October 25, 1998
Summary
Papaver somniferum calluses transformed with the sam-1 gene from Arabidopsis thaliana, which encodes a SAM-synthetase, were subcultured over a 4-year period. The stability of the expression as well as the level of SAM synthetase activity were evaluated in 5 transgenic cell lines (STSI, STSII, STSIII, STSIV, STSV) and in the control. All transgenic cell lines exhibited a level of SAM-synthetase activity higher than that of the control. The enhancement of the enzyme activity has been confirmed by Northern blot analyses. The level of polyamines (putrescine, spermidine, spermine and 1,3-diaminopropane) was also evaluated in cell lines cultured either in Linsmaier and Skoog medium containing growth regulators (LS) or on a hormone free medium (LSHF). Cell lines cultured in LS medium showed a variable yield of the polyamine content. Putrescine was the major polyamine. After transfer to a hormone free medium, all of the cell lines, except STSIV, were able to form embryo-like structures. In this condition, the polyamine level decreases about 4-fold, spermidine being the most abundant. Key words: Papaver somniferum, transgenic cells, sam-1 gene, SAM synthetase activity, polyamines, somatic embryogenesis. Abbreviations: DTT = dithiothreitol; EDTA = ethylene diamine tetraacetic acid; dCTP = deoxycytidine triphosphate; LS = Linsmaier and Skoog medium; LSHF = Linsmaier and Skoog hormone free medium; PA =Polyamine; DAP = 1,3 diaminopropane; SAM-S = S-Adenosyl Methionine Synthetase; Tris = trishydroxymethylaminomethane. Introduction
SAM-S catalyzes the conversion of L-methionine to Sadenosyl-L-methionine (SAM) (Tabor and Tabor, 1984), which is the major methyl group donor for numerous methylation reactions. It plays a key role in the biosynthesis of polyamines and ethylene. Polyamines are polybasic aliphatic amines. Putrescine is synthesized from ornithine and arginine by ornithine decarboxylase and arginine decarboxylase (Slocum et a!., 1984; Smith, 1990). Spermidine and spermine are synthesized by the addition of an aminopropyl group to putrescine. This * Correspondence.
aminopropyl group is provided by S-adenosylmethionine via S-adenosylmethionine decarboxylase (Smith, 1990). 1,3Diaminopropane (DAP) is synthesized from the oxidation of spermidine and spermine by polyamine oxidase. Polyamines play a key role in numerous processes of plant development including growth, senescence and response to stress (Galston, 1983), as well as floral initiation and differentiation (Flores and Martin-Tanguy, 1991; Martin-Tanguy, 1997). They seem to be also involved in organogenesis and somatic embryogenesis in a number of plant cell cultures (Montague et a!., 1978; Robie and Minocha, 1989; Minocha eta!., 1991; Amarasinghe eta!., 1996). The sam-1 gene from Arabidopsis thaliana encoding the S-adenosylmethionine synthetase (SAM-S) has been success0176-1617/99/154/729 $ 12.00/0
730
S. NABHA, F. LAMauN, F. GILLET, D. LAuRAIN, M. FLINIAUX, A. DAVID, and A. }ACQUIN
fully transferred into Papaver somniferum cells (Belny et al., 1997). Five transgenic cell lines have been obtained. In this paper, we report on the evolution of sam-1 gene expression over a 4-year period taking as reference the results published by Belny et al. (1997). We also evaluate at the end of this period, the level of endogenous PA as well as the capacity of the cells to develop somatic embryogenesis.
Materials and Methods
Culture conditions Seeds of Papaver somniferum C. V. exotica were obtained from Clause Co., Paris, France. Calluses were initiated using hypocotyl explants. Untransformed calluses used as control were subcultured on a solid Linsmaier and Skoog (1965) medium (LS) supplemented with 2,4-dichlorophenoxyacetic acid (0.1 mg/L), 6-benzylaminopurine (0.1mg/L), sucrose (30g/L) and 0.8o/o (w/v) agar (Difco). Five transformed cell lines (Belny et a!., 1997) expressing the foreign sam-] gene from Arabidopsis thaliana were cultured on an LS solid medium supplemented with the antibiotic paromomycin sulfate (lOOmg/L) (Sigma). Control and transgenic calluses were maintained at 25 ± 2 ·c in the dark and were subcultured on a fresh medium every 3 weeks. Although the cultures did not necessarily have a unicellular origin, they were called cell lines for convenience.
Growth measurement For estimation of the growth rate between two subcultures, calluses were carefully collected, freeze-dried and weighed (SD ± 5 o/o). Growth rate was the ratio between the dry weight after a 21-day culture period and the dry weight of the inoculum.
SAM-synthetase assays The SAM-S activity was determined on calluses that had been maintained and subcultivated during 4 years. Five hundred mg of calluses (fresh weight) were collected 13 days after the last subculture (exponential growth phase). Proteins were extracted using a method described in the literature (Boerjan et a!., 1994). The cells were ground in 0.5 mL of extraction buffer (0.1 mol/L Tris, pH 7.5, 2 mmol/L EDTA, 20 o/o glycerol, 20 mmol/L ~-mercaptoethanol, 1 mmol/L DTT). After centrifugation, the protein concentration was determined according to Bradford (1976). The SAM-S activity was determined according to Peleman et a!. (1989) and Mathur and Sachar (1991) in two protein extracts and expressed in nKatg-1 protein.
Northern blot analysis Total RNA was extracted from calluses collected 13 days a&er subculture using the RNeasy plant mini kit (Qiagen). Samples (10 !lg total RNA) were denaturated, run on a MOPS-formaldehyde 1.2 o/o agarose gel and then blotted by capillary transfer to HybondN nylon membranes (Amersham). Blotted RNA was fixed by baking at 80 ·c for 2 h. Prehybridizations and hybridizations were performed according to Sambrook et a!. (1989), at 42 ·c in 50 o/o formamide, 5 x SSC, 0.5 o/o SDS and 100 11g/mL salmon sperm DNA. A 974 bp 2urified fragment of the sam- I coding sequence was labelled with (a-33 P) dCTP (Redeprime DNA labelling system, Amersham) and used as probe. Membranes were washed once in 2xSSC, 0.1 o/o SDS at 65 ·c. Hybridization was detected by exposure to X-Omat films (Kodak) at -80 ·c. To compare the relative amounts of RNA,
the membranes were rehybridized with a 25S rRNA eDNA probe. Hybridization signal intensities were estimated using a phosphor Imager (Storm 840, Molecular Dynamics) and for each RNA sample the quantification index (I) was calculated as the ratio of the intensity of the sam-] signal to the 25 rRNA signal.
Induction ofembryogenesis For the expression of embryogenesis, control and transgenic calluses were transferred to an LS hormone-free medium (LSHF) in Petri dishes placed at 25 ± 2 ·c in darkness, with three Petri dishes for each cell line. The number of embryogenic structures was estimated by stereomicroscopic observations on three samples of 100 mg fresh weight of each Petri dish. Data presented correspond to the mean value of the 9 samples.
Analysis ofpolyamine content For the polyamine (PA) determination, cells were collected 3 weeks after subculture, freeze-dried and then homogenized in 1 N perchloric acid (5 mL/100 mg dty weight). The homogenates were centrifuged for 45 min at 32,000 gn at 4 ·c. An aliquot of the supernatant was used for analysis of free PAs, another aliquot from the supernatant and the pellet were hydrolysed with 6 N HCl for 12 h at 110 ·c for analysis of both conjugated and bound PA, respectively. After adding diaminooctane (internal standard), 200 IlL of the extracts were dansylated following the procedure described by Flores and Galston (1982). The PAs were determined by HPLC by comparison with authentic standards (Sigma), using a Thermo Separation Products model1500 pump fitted with a 20 IlL injection loop, a C 18-reversed phase column (Kromasil, 4.6 X 25 mm, 5 11m particles), a fluorescence detector (Waters) equipped with an excitation filter (333 at 365 nm) and an emission filter (485 at 522 nm) coupled with an integrator (Chromfet). The PAs were eluted using a linear gradient of methanol (from 60 o/o to 100 o/o) and water at a flow rate of 1 mL/min (Smith and Davies, 1985). The elution times were the following: 13.2 min for DAP, 14.9 min for putrescine, 19.2min for spermidine and 22.6min for spermine.
Results and Discussion
SAM synthetase activity in control and transgenic calluses SAM-S activity was analyzed in 4-year-old Papaver somniferum cultures {one control and five transgenic cell lines) (Fig.
1). These results were compared with those obtained 3 years earlier by Belny et al. (1997) with the same cell lines. In young cultures, only one transgenic cell line (STSV) showed a SAM-S activity about 1.8-fold higher than that of the control (47 nkat/g protein). All of the others showed a lower level of SAM-S activity. This was probably due to a partial cosuppression of both the resident and the foreign sam genes (Belny et al., 1997). This phenomenon can affect many endogenous genes after introduction of homologous transgenes {Meyer, 1996), which has already been observed in tobacco plants transformed with the Arabidopsis thaliana sam-] gene (Boerjan et al., 1994). In 4-year-old cell lines, we first noticed that the SAM-Sactivity of the control dramatically decreased {about two-fold) from values of initial cultures. This could be partially explained by somaclonal variations that may occur during 4 years of in vitro culture in the presence of growth regulators.
Somatic Embryogenesis in Papaver Cells
1
*
..
2 3 4
731
5 6
ssm1 25SrRNA
20
0
0.02 0.13 0.56 0.38 0.48 1.0
PS
STSI
STSII STSIII STSIV STSV
Cell lines Fig. 1: SAM-S activity in control (PS) and transgenic (STSI, STSII, STSIII, STSIV, STSV) cell lines of Papaver somniferum cultured in LS medium for 4 years. Total protein was extracted 13 days after subculture. Each column represents the mean of the enzyme activity determined for two successive subcultures. Asterisk (*) indicates a significant difference from the control (p <0.05) according to the bilateral Dunnett's test.
On the contrary, the enzyme activity increased significantly in all of the transgenic cell lines, as confirmed by statistical analysis (bilateral Dunnett's test). The STSIV cell line exhibited the highest level of activity (77 nkat/g protein), about 4-fold higher than the control (18 nkat/g protein). The cell lines STSI, STSII, STSIII and STSV had a SAM-S activity 3-fold higher than the control. This could result from the selection conditions under which the transgenic cells were maintained during 4 years. The calluses that could be still chimerical at the time of the first analysis, now contained more and more transgenic cells.
Fig. 2: Northern blot analysis of total RNAs isolated from control and transgenic cell lines 13 days after subculture. Ten micrograms of RNA samples from control, STSI, STSII, STSIII, STSIV and STSV were loaded in lanes 1 to 6, respectively. The blot was hybridized using a 33 P-labelled sam-] probe. Quantification index (I) was calculated using a Phosphor Imager and taking into account the relative amount of RNA in each lane determined after rehybridization of the blot with a 25S ribosomal RNA eDNA probe.
Polyamine content in calluses cultured in a LS medium Figure 3 presents the polyamine content (putrescine, spermidine, spermine, DAP) of the transgenic cell lines (STSI, STSII, STSIII, STSIV, STSV) and the control cultured in a LS medium. The total polyamine content of the transgenic cell lines was lower than the control (PS) except for STSIV (52!lmol/g dry weight and 34!lmollg dry weight, respectively). Polyamine content was the lowest in the STSV cell line. (17!lmol/g dry weight). Among the four analyzed poly60r-----~~====~------~
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To confirm that the increase of the SAM-S activity resulted from the transgene expression, Northern blot analysis was performed on total RNA extracted from cell lines 13 days after a subculture (Fig. 2). All transgenic cell lines showed a stronger hybridization signal compared with the control. A very weak signal could be observed for the control, probably due to a non-specific hybridization with mRNAs of sam endogenous genes. The determination of the quantification index revealed higher levels of sam-] transcripts in STSV (I= 1.0) than in STSII and STSIV (0.56 and 0.48, respectively). Cell line STSI, which had the lowest SAM-S activity, showed the lowest level of sam-1 mRNA. These results indicated that all transgenic cell lines expressed the sam-] gene and that there is a positive correlation between transgene expression and enzyme activity.
D DAP
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Spermidine
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PS
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STSII STSID STSIV STSV
Cell lines Fig. 3: Polyamine content (Jlmol/g dry weight) of Papaver somniferum calluses cultured in an LS medium; PS: untransformed calluses; STSI, STSII, STSIII, STSIV, STSV: transgenic cell lines.
732
S. NABHA, F. LAMBLIN, F. GILLET, D. LAURAIN, M. FuNIAUX, A. DAVID, and A. ]ACQUIN
Table 1: Free, conjugated and bound polyamine content (f.Lmollg dry weight) of Papaver somniferum calluses cultured in an LS medium; PS: untransformed calluses; STSI, STSII, STSIII, STSIV, STSV: transgenic cell lines (mean values of2 replicates).
Table 2: Growth rate of Papaver somniferum cell lines; estimation by the ratio between the dry weight of the biomass 3 weeks after subculture and the dry weight of the inoculum (PS: untransformed cal!uses; STSI, STSII, STSIII, STSIV, STSV: transgenic cell lines).
PS
STSI
STSII
STSIII
STSIV
STSV
Cell lines
Growth rate
Putrescine Free Conjugated Bound Total
6.10 14.65 1.80 22.55
1.87 6.25 1.72 9.84
4.17 8.25 0.70 13.32
5.44 2.97 5.22 13.63
24.49 10.82 7.63 42.94
4.44 2.09 2.41 8.94
Spermidine Free Conjugated Bound Total
PS STSI STSII STSIII STSIV STSV
4.0 4.7 5.2 6.7 3.3 4.5
2.47 4.83 1.03 8.33
1.20 4.25 1.70 7.15
2.66 4.50 1.03 8.19
2.41 3.57 3.73 9.71
3.29 0.95 1.82 6.06
2.83 1.25 1.76 5.84
Spermine Free Conjugated Bound Total
0.30 1.03 0.15 1.48
0.46 1.20 0.31 1.97
0.80 1.42 0.46 2.68
3.13 1.38 0.91 5.42
0.69 0.11 0.40 1.20
0.95 0.50 0.34 1.79
DAP Free Conjugates Bound Total
0.45 0.84 0.08 1.37
0.15 0.27 0.07 0.49
0.36 0.40 0.06 0.82
0.34 0.15 0.16 0.65
0.69 0.90 0.40 1.38
0.30 0.10 0.11 0.51
amines, putrescine was the most abundantly accumulated, especially in STSIV. All of the cell lines contained more spermidine than spermine. DAP was the least abundant compound. The levels of the three different forms of PA (free, conjugated and bound) are presented in Table 1. The conjugated form was predominant in the control (PS), STSI and STSII cell lines, approximatively 60 o/o of total PA. The free PA form was most dominant in STSIV (about 60 o/o of total PA), in STSV (about 50 o/o of total PA), and in STSIII (about 40 o/o of total PA), except for spermidine. In STSIV, the high level of PA (52!-lmol/g dry weight including about 43!-lmol/g dry weight of putrescine) is due to all of the forms of putrescine (about 25!-lmol/g dry weight free putrescine, 11!-lmol/g dry weight for the conjugated form and 8!-lmol/g dry weight for the bound form). It has been suggested in the literature (Aribaud et al., 1994; Burtin et al., 1989) that such high levels of free and conjugated PAs (especially putrescine and spermidine) could inhibit cell multiplication and supress differentiation in leaf explants of tobacco and Chrysanthemum morifolium cultured in vitro. In spindle trees, it has been shown that a high intracellular level of putrescine supresses somatic embryogenesis (Bonneau et al., 1995). Moreover, the formation of somatic embryo is associated with a significant decrease in putrescine level. From these results, we decided to evaluate the growth and the embryogenic ability of the studied cell lines.
Growth ofPapaver somniferum calluses In a preliminary work (data not shown), we observed that the growth of the culture during a 21-day period was linear
for all cell lines. Growth weight was estimated by the ratio of the dry weight after 21 days of culture and dry weight of the inoculum, for each cell line (Table2). It is remarkable to note that STSIV, the cell line that accumulated the highest level of PA, presented the lowest growth rate.
Somatic embryogenesis ofPapaver somniferum calluses Some of the cell lines displayed an irregular and spontaneous somatic embryogenesis. In order to know whether this
·.·
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----·---·
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_______
•
Fig. 4: Somatic embryogenesis in Papaver somniferum calluses. Cell line STSV was cultured for 21 days in LSHF medium; (a): Globular embryos (G), Torpedo embryo (T) (x 8), (b): Torpedo stage (x 20), (c): Cotyledonary stage (x20).
Somatic Embryogenesis in Papaver Cells
Table 3: Somatic embryogenesis of Papaver somniferum cell lines:
20~-----------------------.
lliiil Spermine
the embryogenic ability is expressed by the number of embryogemc structures per g of fresh weight. PS: untransformed calluses; STSI, STSII, STSIII, STSIV, STSV: transgenic cell lines. Cell lines
Embryo somatic number/g fresh weight
PS STSI STSII STSIII STSIV STSV
64± 2 90±14 169±42 16± 6 0 300±67
capacity was linked to the transgene expression and/or to the intracellular PA levels, the cell lines were transferred to a hormone-free medium. This condition is known to facilitate somatic embryogenesis (Ribnicky eta!., 1996). Within a cell line, the morphology of the embryo-like structures varied greatly; globular, torpedo and heart cotyledonary stage embryos (Fig. 4). In all cases, except for the STSIV, embryo-like structures appeared about 10 days after subculture. The number of embryo-like structures regularly increased during 3 weeks of culture, but with large variations in the values depending on the cell line. After 3 weeks (Table 3), 3 cell lines (STSI, STSII, STSV) were more embryogenic than the control (PS) and the other two (STSIII, STSIV) were less embryogenic. STSV appeared to be the most embryogenic. Surprisingly, STSIV, which exhibited the highest level of SAM-S activity and the highest putrescine level, was the least embryogenic cell line. This result is in agreement with those obtained by Martin-Tanguy (1997) relating an inhibitory effect of free and conjugated putrescine on embryogenic capacity.
Polyamine content in calluses cultured in LSHF medium In order to correlate PA and somatic embryogenesis, PA content was measured after 21 days of culture in a hormonefree medium (Fig. 5). Similarly to cells cultivated in LS medium, we noted a cell line-dependent variability of the PA content. Some of the cell lines (STSIII, STSIV) contained more PA than the control, while the other ones, such as STSI, STSII, STSV, contained less than the control. However, in this condition, only the control accumulated putrescine, which was the major compound detected in all cell lines cultured in LS. After transfer of the cells to LSHF medium, spermidine was the major compound; spermine could also be detected. Only traces of DAP could be detected in all cell lines. A decrease in the total polyamine level (about 4-fold) could be noted, independently of the cell line (transformed or not). Among polyamines, putrescine was the most affected. These results are similar to the results reported by Tiburcio et a!. (1985), Santanen and Simola (1992), and Bonneau et a!. (1995). Indeed, they observed a significant decrease in putrescine levels associated with the formation of somatic embryos in tobacco, Picea abies and Euonymus europeaus L., respectively. This decrease could reflect a conversion of putrescine to spermidine (Men-
733
•
Spermidine
ILl Putrescine
PS
STSI
STSII STSIII STSIV STSV Cell lines
Fig. 5: Polyamine content (~mol/g dry weight) of Papaver somniferum calluses cultured in a hormone free-medium (LSHF); PS: untransformed calluses; STSI, STSII, STSIII, STSIV, STSV: transgenic cell lines.
goli et al., 1989). Spermidine and spermine did not decrease so dramatically as putrescine. The spermidine content decreased slightly in PS, STSI and STSIII, which were less embryogenic and decreased 2-fold in STSII and STSV, which were the most embryogenic cell lines. It can even be noted that the spermidine content of STSIV was higher than that of STSIV cultured in an LS medium (about lliJ.mol/g dry weight versus 61J.mol/g dry weight). Our results, except for STSIV, confirm those of Amarasinghe eta!. (1996) who observed a decrease in spermidine level in interior spruce calluses cultured on a medium for differentiation (without hormones). The free, conjugated and bound forms of spermidine, the most dominant PAin all of the cell lines cultured in LSHF medium, are given in Table 4. In all of the transformed cell lines, the main part of spermidine was accumulated in bound form (Table 4). This form represents about 60% (STSIII) to 93% (STSIV, STSV) of the total spermidine. It is worth noting that spermidine was accumulated mainly in free and conjugated forms when the cells were cultured in LS medium. Table 4: Free, conjugated and bound spermidine content (~mollg dry weight) of Papaver somniferum calluses cultured in a LSHF medium; PS: untransformed calluses; STSI, STSII, STSIII, STSHT, STSV: transgenic cell lines (mean values of2 replicates). PS
STSI
STSII
STSIII
STSIV
STSV
0.33 2.88 2.30 5.51
0.11 0.86 3.94 4.91
0.28 1.24 2.29 3.81
0.55 2.98 5.10 8.63
0 0.75 10.18 10.93
0 0.16 2.28 2.44
Spermidine Free Conjugated Bound Total
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S. NABHA, F. lAMBLIN, F. GILLET, D. LAuRAIN, M. FuNIAUX, A. DAVID, and A. ]ACQUIN
Concerning the embryogenic ability of the cell lines, a negative correlation was observed with the polyamine content. The most embryogenic cell line (STSV) had the lowest PA level and the non-embryogenic (STSIV) had the highest PA level. These results differ from those reported by numerous authors (Montague et al., 1978; Robie and Minocha, 1989; El Hadrami et al., 1989).
Conclusion
We noted an increase in the transgene expression (enzyme assays and Northern blots), as the cell lines were ageing. Morevee, the polyamine level and particularly that of putrescine was very heterogenous from one cell line to another one. This PA level, which decreased when calluses were transferred to a differentiation medium, was negatively correlated to embryogenesis in the various cell lines. Acknowledgements
We are grateful to Pro£ J. Martin-Tanguy for useful suggestions and critical reading of the manuscript. We thank «Le Biopole Vegetal>> Amiens-France for its financial support.
References
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