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The recalcitrance to rooting of the micropropagated shoots of the rac tobacco mutant: Implications of polyamines and of the polyamine metabolism
Plant Physiol. Biochem., 2000, 38 (6), 441−448 / © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0981942800007683/FLA
The recalcitrance to rooting of the micropropagated shoots of the rac tobacco mutant: Implications of polyamines and of the polyamine metabolism Odile Faivre-Rampant*, Claire Kevers, Jacques Dommes, Thomas Gaspar Hormonologie Fondamentale et Appliquée, Institute of Botany B 22, University of Liège - Sart Tilman, B-4000 Liège, Belgium * Author to whom correspondence should be addressed (fax +32 4 366 38 59; e-mail [email protected])
(Received 28 January 2000; accepted 28 February 2000) Abstract – Rooting of wild-type tobacco (Nicotiana tabacum cv. Xanthi) shoots raised in vitro was promoted by polyamines in the absence of any other growth regulator and was inhibited by two inhibitors of polyamine metabolism. The auxin insensitive and recalcitrant to rooting rac mutant shoots did not respond to the same treatments. The activities of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), diamine oxidase (DAO), polyamine oxidase (PAO) and transglutaminases (TGases), and the titres of free and conjugated polyamines were estimated in the whole shoots and the basal parts of the stems of both tobaccos in the course of multiplication in vitro. The rac shoots grew at a lower rate. The wild-type rooted from the 7th day without special treatment. During the second week of culture, the shoots of both tobaccos were actively growing and showed an increase in ADC, ODC, DAO, PAO and TGase activities. Afterwards all these activities declined. These changes were concomitant with an increase in the polyamine contents (free and conjugated). Biosynthesis and oxidation of polyamines apparently occurred simultaneously and seemed directly correlated. In the basal part of the mutant stems however, the accumulation of free and conjugated putrescine as well as the transient increase in biosynthetic enzyme activities were delayed compared to the wild-type. These results are discussed in relation to growth behaviour and to root formation. © 2000 Éditions scientifiques et médicales Elsevier SAS Polyamines / rooting / tobacco mutant ADC, arginine decarboxylase / AG, aminoguanidine / BAP, benzylaminopurine / CHA, cyclohexylamine / DAO, diamine oxidase / GABA, γ-aminobutyric acid / IAA, indole-3-acetic acid / IBA, indole-3-butyric acid / NAA, naphthaleneacetic acid / ODC, ornithine decarboxylase / PAO, polyamine oxidase / Put, putrescine / Spd, spermidine / Spm, spermine / TGases, transglutaminases
1. INTRODUCTION Tobacco mesophyll protoplasts resistant to the toxic effect of NAA have been selected by Muller et al. [24] among UV-mutagenized populations. The mutant protoplasts had the ability to divide in media containing NAA concentrations which were toxic for the wild-type protoplasts. Auxin-resistant mutant shoots were derived from the protoplasts and called rac mutants due to their inability to root, even in response to an auxin treatment. The auxin resistance of the mutant cells and tissues was not correlated with an increased rate of conjugation or breakdown of auxins or with a perturbation of auxin transport [9]. Using the auxin-induced hyperpolarization response, the mutant protoplasts were shown to be ten times less sensitive to auxin than the
wild-type protoplasts [10]. Histological analyses indicated that 3–4 d after seed germination, the root meristem degenerated and was transformed into a callus [26]. In stem cuttings treated with IBA, perivascular cells divided, but never formed adventitious root meristems [21]. According to these authors, the rac mutation did not disrupt the auxin concentrations, but implied changes in the receptor affinity for auxin and/or in the efficiency of the transduction pathway. However, a reappraisal of the auxin concentrations did not lead to the same conclusions: the tobacco rac mutant showed hyperauxiny as compared to the wildtype (preliminary results in a preceding paper by Faivre-Rampant et al. [12]). This would result from an over-accumulation of phenolic compounds inhibiting the auxin catabolism, in the rac mutant.
Plant Physiol. Biochem., 0981-9428/00/6/© 2000 E´ditions scientifiques et médicales Elsevier SAS. All rights reserved
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In the present paper, we investigated the auxin recalcitrance of the rac mutant at the level of polyamine metabolism. Indeed polyamines have been shown to be key factors, indissociable from auxins, in the process of adventitious rooting [14, 19]. Many studies have shown the beneficial effects of applying various polyamines on the rooting process [1, 4]. When poplar shoots were induced to root, an early increase in putrescine (Put) level occurred, concomitant with a transient increase in IAA. The timing of these events corresponded to the termination of the inductive phase [14]. Spermidine (Spd) and spermine (Spm) did not change significantly. Other arguments causally implicated Put in the rooting inductive phase of poplar: (a) the transient increase did not occur in the non-rooting cuttings [16]; (b) it was observed only in the basal rooting zone [16]; (c) inhibitors of the Put biosynthesis, such as DFMO and DFMA (α-difluoromethylornithine and α-difluoromethylarginine, respectively) applied prior to or at the beginning of the inductive phase inhibited rooting [16]; (d) an inhibitor of the Put conversion into Spd and Spm, CHA (cyclohexylamine, an inhibitor of the Spd synthase), which promoted the accumulation of endogenous Put, favoured rooting in the absence of exogenously supplied auxin [17]; (e) exogenously applied Put prior to or at the beginning of the inductive phase stimulated rooting [17]. Additional results point to a major role played by the Put catabolism through its ∆1-pyrroline-GABA (γ-aminobutyric acid) pathway [16, 17]. Indeed the treatment of poplar cuttings with AG (aminoguanidine, an inhibitor of the diamine oxidase, DAO, which converts Put to GABA) inhibited their rooting [17]. Similar results [19] were obtained with walnut shoot cuttings in rooting inductive phase where endogenous IAA and peroxidase had been also implicated [18, 28]. Furthermore, a low activity of the amine-oxidases and an accumulation of the conjugated polyamines negatively affected organogenic programmes, including rooting, in Chrysanthemum morifolium leaf disc explants [3]. In the in vitro shoot-forming cultures of the wildtype and rac tobaccos [11], the shoots of the wild tobacco rooted spontaneously at the end of the growth cycle; the rac shoots did not even in response to an auxin treatment. The present work was undertaken to assess the role of polyamines and of their catabolism in rooting induction in tobacco. We evaluated the levels of free and conjugated polyamines and the activities of the related biosynthetic (ADC, ODC) and catabolic (DAO, PAO) enzymes in shoots of rac and wild-type tobacco. Plant Physiol. Biochem.
TGase activities (catalysing covalent linkages of polyamines to numerous substances including proteins, see review by Serafini-Fracassini et al. [29]) were also considered. In addition, we devised culture conditions in which wild-type shoots did not spontaneously root. In these conditions, the effect on rooting of exogenous polyamines and inhibitors of their metabolism (AG and CHA) could be further evaluated.
2. RESULTS 2.1. Growth pattern and rooting behaviour On the commonly used micropropagation medium (MS, 3 % sucrose, 0.8 % Roland agar, 0.13 µM BAP), wild-type shoots started to grow 3 ± 1 d after subculture (figure 1). New leaves were observed after 5 ± 1 d, and root formation after 7 ± 1 d with a number of roots per shoot of 6 ± 2. At day 10 ± 1, an average of 78 ± 3 % of wild-type shoots showed adventitious roots. The growth of rac shoots started 5 ± 1 d after subculture with the neoformation of two leaves after 7 ± 1 d. The mutant did not root at the end of the multiplication cycle, nor after any rooting treatment. Both types of shoots continued to grow throughout the culture cycle, but the rac mutant grew more slowly (figure 1). To study the effects of exogenous polyamines on rooting induction, it was necessary to devise culture conditions in which wild-type shoots do not spontaneously root. Such conditions were obtained by modifying the multiplication medium. In comparison with the commonly used micropropagation medium, it did not contain any NH4+, nor any growth regulator, but ten times more KNO3 and 1 % Kobe agar instead of 0.8 % Roland agar. On this medium, neither rac nor wildtype shoots rooted spontaneously (table I).
2.2. PA titres during the multiplication cycle Figure 2 shows the changes of free and conjugated polyamines in the shoots of the wild-type and the rac mutant during the multiplication cycle. Put was by far the major polyamine contained in the shoots of the two types of tobacco with higher levels in rac shoots. A transient accumulation of free and conjugated Put was observed with the maximum value on day 14. Spd and Spm also showed a transient increase on day 14, but to a lesser extent than Put, with levels not significantly different between the wild-type and the mutant. In the basal parts (where roots form, if they do) of the wild-type and mutant tobacco stems, Put was the
Polyamine metabolism of the rac mutant
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Figure 2. Changes in free and conjugated polyamine levels during a multiplication cycle (21 d) in the whole shoots of wild-type (D8) and rac mutant tobaccos. (●), Put; (■), Spd; (▲), Spm.
Figure 1. Shoot growth in vitro during a multiplication cycle (21 d) of the wild-type D8 (●) and mutant rac (■) tobaccos. Top, Changes in shoot height; bottom, changes in node number per shoot.
major polyamine as in whole shoots (figure 3). Level of free Put in the basal parts of the rac stems and in the rac whole shoots showed a similar time course. In the basal parts of the wild-type stems, Put showed an earlier transient accumulation with higher level observed between the 7th and the 14th day. The levels and variations of free and conjugated Spd and Spm were low compared to Put.
peak at day 14 as for polyamine level, followed by a marked decrease (figure 4 A). ADC activity was lower than ODC in the two tobaccos throughout the culture period and only showed very small changes. ADC activity was always higher in rac shoots than in wild-type shoots.
2.3. ADC and ODC activities ODC activity increased in the whole shoots of the two tobaccos during the second week of culture with a Table I. Effects of polyamines on the rooting of tobacco wild-type shoots grown in a non-rooting medium (MS without NH4+, ten times more KNO3, 3 % glucose, 1 % Kobe agar, no growth regulator). The percentage of rooting shoots was determined after two subcultures (mean of five independent assays ± SD). The rac shoots did not root in any of these conditions. Polyamines added Put Spd Spm
Concentrations (M) 0 0 0 0
10
–6
10 ± 5 0 0
10–5
10–4
10–3
28 ± 7 0 0
52 ± 7 0 0
68 ± 6 33 ± 6 0
Figure 3. Changes in free and conjugated polyamine levels during a multiplication cycle (21 d) in the basal part of the stems of wild-type (D8) and rac mutant tobaccos. (●), Put; (■), Spd; (▲), Spm.
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Figure 4. ADC and ODC activities during a multiplication cycle (21 d) in whole shoots (A) and the basal parts of stems (B) of wild-type (D8) (●) and rac (■) mutant tobaccos.
In the basal parts of the wild-type stems, ADC and ODC activities showed a transient increase at day 7 (figure 4 B). In the rac mutant, these activities were relatively stable, and always higher than in wild-type shoots.
2.4. DAO and PAO activities In the whole shoots of the two tobaccos, DAO and PAO activities showed about the same changes as ADC and ODC activities with a maximum at day 14 but to a lesser extent for the wild-type (figure 5 A). DAO activity was lower than PAO activity in the wild-type and mutant tobaccos. In the basal parts of the stems, PAO activity did not change significantly during the culture. Its activity was somewhat higher in the mutant. DAO activity reached a maximum at day 14 in both wild-type and rac tobaccos (figure 5 B), like in the whole shoots. 2.5. TGase activities In the wild-type and mutant tobaccos, TGase activity showed parallel changes and about the same level, with a maximum at day 14 (figure 6) for the whole shoots. There were no significant differences in the basal parts of the stems between the two types of tobacco. 2.6. Effects of exogenous PAs and inhibitors on rooting None of the three polyamines tested, at concentrations varying from 10–6 to 10–3 M, was able to induce Plant Physiol. Biochem.
Figure 5. DAO and PAO activities during a multiplication cycle (21 d) in whole shoots (A) and the basal parts of stems (B) of wild-type (D8) (●) and rac (■) mutant tobaccos.
rooting of the rac mutant whatever the medium used. On the medium designed to avoid spontaneous rooting, exogenously applied Put stimulated rooting of wild-type shoots to 68 % in the 1–1 000 µM range in the absence of any growth regulator (table I). In the presence of 1 000 µM Spd, 33 % of the shoots rooted. Inhibition of the conversion of Put into ∆1-pyrrolineGABA by AG and to a lesser extent, inhibition of the conversion of Put into Spd by CHA led to a reduction in rooting (table II).
3. DISCUSSION In both wild-type and rac shoots, a transient increase in the level of polyamines, mainly Put, was observed 14 d after subculture. Thus differentiation of the shoots appears to be accompanied by higher levels of free and conjugated Put throughout the multiplication cycle. It was reported that in tobacco, high intracellular levels of conjugated Put inhibited cell proliferation and suppressed bud formation [7]. Thus, higher polyamine levels in the rac mutant might be responsible for its lower growth rate. In the wild-type tobacco cuttings, rooting was stimulated by application of exogenous polyamines. Treatments with inhibitors of polyamine metabolism, on the contrary, had a negative effect on rooting. These
Polyamine metabolism of the rac mutant
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transient increase in Put occurred during the first week of culture; in the rac mutant, this increase was delayed to the second week. Hausman et al. [16] have shown that Put was a good marker for adventitious rooting. This delay in the Put transient accumulation in the basal parts of the rac stems might be correlated with the absence of root formation.
Figure 6. TGase activities during a multiplication cycle (21 d) in the whole shoots and the basal part of the stems of wild-type (●) and rac mutant (■) tobaccos.
results provide additional support for the involvement of polyamines in the rooting process. The auxin insensitive mutant rac shoots did not root even in response to polyamine application. This observation supports a previous claim that auxins and polyamines are indissociable factors in the rooting process. Indeed, in poplar cuttings [16, 17], rooting depended on a concomitant variation in the levels of both regulators. A time course study of polyamine levels in the basal parts of the stems showed important differences between wild-type and rac tobacco shoots. In wild-type, a
Table II. Effects of inhibitors of polyamine metabolism on the rooting percentage of wild-type after three subcultures on growth medium (MS supplemented with 0.13 µM BAP). Results are means of five independent assays ± SD. The rac shoots did not root in any of these conditions. AG, Aminoguanidine; CHA, cyclohexylamine. Inhibitors added AG CHA
Concentrations (M) 0 100 100
10
–6
89 ± 8 100
10–5
10–4
10–3
83 ± 5 100
82 ± 6 100
80 ± 5 80 ± 8
In whole shoots, ODC activity and polyamine levels showed parallel changes. ADC activity was always lower than ODC activity and remained relatively constant throughout the growth period. Considerable evidence now indicates that both ADC and ODC relative contributions to polyamine biosynthesis are dependent upon the type of tissue and the developmental process [13]. It seems likely that ADC and ODC have different roles in cell proliferation, expansion growth and differentiation. In several plants, ADC seems to be involved in vegetative development, whereas ODC may be required for floral induction, sexual differentiation, root formation and callus formation [22]. More recently, an oat ADC gene under the control of an inducible promoter, the tetracyclinerepressor system, has been expressed in transgenic tobacco plants [6, 23]. Transgenic plants treated with tetracycline during the vegetative stage displayed increased ADC activity and increased Put levels, corresponding to different degrees of altered phenotype, characterized by shorter internodes, thinner stems and leaves, leaf chlorosis and necrosis and reduced root growth. In the basal parts of the wild-type stems, the higher ODC and ADC activities corresponded to a maximum in Put level. In the rac mutant, these activities hardly changed but were always higher than in wild-type stems. This might also be related to the absence of root formation for the rac mutant. In cultures of wild-type and rac whole shoots, DAO and PAO activities increased during the second week. Thereafter the levels declined. The maximum values coincided with those of ADC and ODC activities. These results suggest a direct correlation between the biosynthesis and oxidation of Put in physiological phases such as shoot growth (leaf neoformation, stem elongation and root formation). Similarly, in Helianthus tuberosus tuber, the increase in DAO activity paralleled the accumulation of Put [30]. But in the basal parts of the stems, whatever the tobacco genotype, a maximum in DAO activity was observed on the 14th day, whereas PAO activity hardly changed during the multiplication cycle. Our data are also in agreement with those for animal systems in which DAO vol. 38 (6) 2000
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activity increases concomitantly with or immediately after an increase in ODC activity and Put content [27, 28]. In mammals, it has been established that posttranslational covalent linkage of polyamines to proteins is catalysed by a class of enzymes known as TGases. In our case, time course of TGase activities in whole shoots showed the same pattern than the one of ODC/ADC activities and Put level. However, there is no significant differences in the basal parts of the stems between the wild-type and the mutant. Information on TGase activities in plants is relatively incomplete. Recently, ε-(γ-glutamyl)-lysine, the product of TGase’s protein cross-linking activity, was detected in root and shoot tissue of pea, broad bean, wheat and barley by cation-exchange chromatography [20]. Cloning of genes coding for TGases in plants will in the future allow the study of the mechanisms controlling polyamine conjugation to proteins as well as the molecular mechanism by which these bound polyamines play a role in physiological processes. As a conclusion, the present work provided essential new information for the recalcitrance to rooting of the rac mutant tobacco: it is insensitive to exogenous application of polyamines. This might be a further confirmation of the indissociability of auxins and polyamines in the rooting developmental process. Finally, we showed that the transient increases in both the Put level and in polyamine metabolic enzyme activities occurred in the basal parts of the rac stems, but with a delay in comparison to wild-type stems. These latter observations suggest that rooting of the rac mutant might be induced but arrested at the expression phase.
In such conditions, the wild-type shoots rooted spontaneously at the end of each multiplication cycle, but the rac ones did not. The effect of two inhibitors of polyamine metabolism, AG and CHA, was tested by addition to the same growth medium. For some experiments, the micropropagation medium was modified by eliminating the NH4+ ions, using ten times more KNO3, 3 % glucose, 1 % Kobe agar (Marseille), and no growth regulator. In this medium, the shoots were subcultured every 10 d. The three polyamines to be tested, i.e. Put, Spd and Spm, were filter-sterilised and added to the previously autoclaved medium.
4.2. Determination of free and conjugated PAs Free polyamine extraction, separation, identification and measurement by direct dansylation and HPLC were done as described by Walter and Geuns [31]. Free polyamines were extracted in 5 % (v/v) cold HClO4 at a ratio of 100 mg fresh tissues·mL–1. The techniques which were used to extract, separate and measure the levels of polyamine conjugates have been described elsewhere [8]. Water-soluble conjugates from the aqueous extracts were eluted from an Amberlite (Serva CG 50, H+ form) column with 3 M acetic acid. Non-watersoluble conjugates were extracted from the pellet in 100 % MeOH and purified in ethyl acetate. For the quantification of polyamine conjugates, extracts were boiled in 6 M HCl, followed by measurement of free amines. The compounds were chromatographed on a Bondpack C18 reverse phase column (particle size 5 µM, 4 × 125 mm, Waters Assoc.) with an acetonitrile/ water (72/28 v/v) solvent gradient at a flow rate of 1 mL·min–1.
4.3. Determination of ADC and ODC activities 4. METHODS 4.1. Plant material The wild-type, D8, and homozygous rac mutant seeds of Nicotiana tabacum cv. Xanthi were surface sterilized for 3 min in a 10 % (v/v) commercial bleach solution, placed on MS medium [25], pH 5.7, containing 0.8 % (w/v) Roland agar (Brussels), 3 % (w/v) sucrose and 0.13 µM BAP. Shoot apices or stem nodes were explanted for continued multiplication through 3-week cycles on the same medium. Cultures, in 600-mL cylindrical glass jars containing 100 mL medium covered by a glass lid maintained with a sheet of transparent plastic film, were placed under a 16-h photoperiod (Sylvania Grolux fluorescent tubes providing 15 µE·m–2·s–1), at 28 °C (day) and 25 °C (night). Plant Physiol. Biochem.
Arginine decarboxylase (ADC, EC 4.1.1.17) and ornithine decarboxylase (ODC, 4.1.1.19) activities were determined according to the methods of Aribaud et al. [2] after several modifications. Samples were ground at a ratio of 0.5 g fresh weight·mL–1 0.1 M Tris-HCl (pH 7.5) containing 5 mM EDTA, 10 mM mercaptoethanol, 1 mM pyridoxal phosphate, 5 mM dithiothreitol (DTT) and 0.5 M KCl. The extract was centrifuged for 30 min at 20 000 × g. The supernatant was saturated to 50 % with (NH4)2SO4 for 1 h with gentle stirring. The precipitate was collected by centrifugation at 20 000 × g for 20 min. The pellet was resuspended in a minimum volume of extraction buffer. This fraction was then dialysed against two changes of 10 mM Tris-HCl containing 1 mM EDTA, 10 mM mercaptoethanol for 8 h. All the steps were
Polyamine metabolism of the rac mutant
carried out at 4 °C. The dialysed extract was used to determine the ADC and ODC activities. To assay ODC, 100 µL extract were mixed with 13 µL [1-14C]ornithine (2 GBq·mmol–1, ICN), 45 µL 0.1 M TrisHCl (pH 7.5) containing 10 mM mercaptoethanol, 0.1 mM pyridoxal phosphate and 1 mM cold ornithine. To assay ADC, 100 µL extract were mixed with 13 µL [U-14C]-arginine (11 GBq·mmol–1, ICN), 45 µL 0.1 M Tris-HCl (pH 7.5) containing 10 mM mercaptoethanol, 0.1 mM pyridoxal phosphate and 1 mM cold arginine. Reaction mixtures were incubated for 1 h at 30 °C. The reactions were stopped with 10 µL 17 M acetic acid. Ten-µL aliquots were analysed by thin layer electrophoresis on cellulose plates (MachereyNagel). Electrophoresis was performed in acetic acid/ pyridine/water (5/1/94, v/v/v) for 1 h at 250 V. Unlabelled arginine, ornithine, agmatine and putrescine as standards were also spotted on the plate and developed with ninhydrin (5 % w/v in ethanol). The cellulose (ornithine spots for ODC activity and arginine spots for ADC) was scraped off and transferred to scintillation vials and the radioactivity determined in a LS 5000 scintillation counter (Beckman). We measured the activities by making a difference between the remaining substrate amount in the spots and the loaded amount of substrate.
4.4. Determination of DAO and PAO activities Diamine oxidase (DAO, EC 1.4.3.6) and polyamine oxidase (PAO, EC 1.4.3.4) activities were determined using a modification of the procedure described previously [2]. Extracts were prepared as for the determination of ADC and ODC activities. DAO activity was assayed by measuring the [14C]-∆1-pyrroline formation from [14C]-Put. Aliquots (50 µL) of extract were incubated for 1 h at 30 °C in a final volume of 100 µL containing 7.4 kBq [1,4-14C]-Put (4.11 GBq·mmol–1, Amersham), in 0.1 M Tris-HCl (pH 8), 5 mM Put and 0.3 mg·mL–1 catalase. PAO activity was assayed by measuring the [14C]-∆1-pyrroline formation from [14C]Spd. Aliquots (50 µL) of extract were incubated for 1 h at 30 °C in a final volume of 100 µL containing 7.4 kBq [1,4-14C]-Spd (4.37 GBq·mmol–1, Amersham), in 0.1 M Tris-HCl (pH 8.5), 2.5 mM Spd and 0.3 mg·mL–1 catalase. After incubation, 200 µL 1 M sodium carbonate was added to the mixture and the [14C]-∆1-pyrroline immediately extracted in 4 mL toluene by vortexing for 20 s and centrifuging for 5 min at 2 000 × g. Then, 1 mL of the toluene phase was added to scintillation vials containing 4 mL scintillation liquid and counted in a LS 5000 scintillation counter (Beckman).
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4.5. TGase-like activity Transglutaminase-like activity was determined using the method described by Haddox and Russell [15]. Extracts were prepared as for the determination of ODC and ADC activities. Aliquots (50 µL) of extract were incubated for 1 h at 30 °C in a final volume of 100 µL containing 7.4 kBq [1,4-14C]-Put –1 (4.11 GBq·mmol , Amersham) in 0.1 M Tris-HCl (pH 8.5), 3 mM cold Put and 10 mg·mL–1 dimethylcaseine. After incubation, 200 µL 0.5 M cold Put were added and the reaction was stopped with 4 mL of 20 % (w/v) thrichloroacetic acid (TCA). The mixture was then filtered and the filter was washed with 5 % TCA containing 0.5 M KCl, purified water and finally 0.1 M HCl. The filter was then dried and placed in scintillation vials containing 4 mL scintillation liquid and counted in a LS 5000 scintillation counter (Beckman).
4.6. Protein analysis Soluble proteins were determined by the method of Bradford [5]. Bovine serum albumine was used as standard. All results are means of at least three separate experiments (± SD).
Acknowledgments Odile Faivre-Rampant is gratefully indebted to the European Community for a FAIR research training grant. Provision of the tobacco seeds by Dr Caboche (INRA, Versailles) is also gratefully acknowledged.
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[5] 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 (1976) 248–254. [6] Burtin D., Michael A.J., Overexpression of arginine decarboxylase in transgenic plants, Biochem. J. 325 (1997) 331–337. [7] Burtin D., Martin-Tanguy J., Paynot M., Rossin N., Effects of the suicide inhibitors of arginine and ornithine decarboxylase activities on organogenesis, growth, free polyamine and hydroxycinnamoylputrescine levels in leaf explants of Nicotiana tabacum Xanthi n.c. cultivated in vitro in a medium producing callus formation, Plant Physiol. 89 (1989) 104–110. [8] Cabanne F., Dalebroux M.A., Martin-Tanguy J., Martin C., Hydroxycinnamic acid amides and ripening to flower of Nicotiana tabacum var Xanthi n.c., Physiol. Plant. 53 (1981) 399–401. [9] Caboche M., Muller J.F., Chanut F., Aranda G., Cirakoglu S., Comparison of the growth promoting activities and toxicities of various auxin analogs on cells derived from wild-type and a nonrooting mutant of tobacco, Plant Physiol. 83 (1987) 795–800. [10] Ephritikhine G., Barbier-Brygoo H., Muller J.F., Guern J., Auxin effect on the transmembrane potential difference of wild-type and mutant tobacco protoplasts exhibiting a differential sensitivity to auxin, Plant Physiol. 8 (1987) 801–804. [11] Faivre-Rampant O., Kevers C., Bellini C., Gaspar Th., Peroxidase activity, ethylene production, lignification and growth limitation in shoots of a nonrooting mutant of tobacco, Plant Physiol. Biochem. 36 (1998) 873–877. [12] Faivre-Rampant O., Kevers C., Gaspar Th., Endogenous auxin and cytokinin levels in shoots of a nonrooting tobacco mutant, Biol. Plant. 42 (Suppl.) (1999) S47. [13] Galston A.W., Flores H.E., Polyamines and plant morphogenesis, in: Slocum R.A., Flores H.E. (Eds.), Biochemistry and Physiology of Polyamines in Plants, CRC Press, Florida, 1991, pp. 175–186. [14] Gaspar Th., Kevers C., Hausman J.F., Indissociable chief factors in the inductive phase of adventitious rooting, in: Altman A., Waisel Y. (Eds.), Biology of Root Formation and Development, Plenum Press, New York, 1997, pp. 55–63. [15] Haddox M.K., Russell H.D., Increased nuclear conjugated polyamines and transglutaminase during liver regeneration, Proc. Natl. Acad. Sci. USA 78 (1981) 1712–1716. [16] Hausman J.F., Kevers C., Gaspar Th., Involvement of putrescine in the inductive rooting phase of poplar shoots raised in vitro, Physiol. Plant. 92 (1994) 201–206. [17] Hausman J.F., Kevers C., Gaspar Th., Auxin-polyamine interaction in the control of the rooting inductive phase of poplar shoots in vitro, Plant Sci. 110 (1995) 63–71.
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[18] Heloir M.C., Kevers C., Hausman J.F., Gaspar Th., Changes in the concentrations of auxins and polyamines during rooting of in vitro-propagated walnut shoots, Tree Physiol. 16 (1996) 515–519. [19] Kevers C., Bringaud C., Hausman J.F., Gaspar Th., Putrescine involvement in the inductive phase of walnut shoots rooting in vitro, Saussurea 28 (1997) 50–57. [20] Lilley G.R., Skill J., Griffin M., Bonner L.R., Detection of Ca2+-dependent transglutaminase activity in root and leaf tissue of monocotyledonous and dicotyledonous plants, Plant Physiol. 117 (1998) 1115–1123. [21] Lund S., Smith A., Hackett W., Cuttings of a tobacco mutant, rac, undergo cell divisions but do not initiate adventitious roots in response to exogenous auxin, Physiol. Plant. 97 (1996) 372–380. [22] Martin-Tanguy J., Conjugated polyamines and reproductive development: Biochemical, molecular and physiological approaches, Physiol. Plant. 100 (1997) 675–688. [23] Masgrau C., Altabella T., Farras R., Flores D., Thompson A.J., Besford R., Tiburcio A.F., Inducible overexpression of oat arginine decarboxylase in transgenic tobacco plants, Plant J. 11 (1997) 465–473. [24] Muller J.F., Goujaud J., Caboche M., Isolation in vitro of naphthaleneacetic acid-tolerant mutants of Nicotiana tabacum, which are impaired in root morphogenesis, Mol. Gen. Genet. 199 (1985) 194–200. [25] Murashige T., Skoog F., A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant. 15 (1962) 473–497. [26] Pelèse F., Megnegneau B., Sotta B., Sossountzov L., Caboche M., Miginiac E., Hormonal characterization of a nonrooting naphthaleneacetic acid tolerant tobacco mutant by an immunoenzymic method, Plant Physiol. 89 (1989) 86–92. [27] Perin A., Sessa A., Desiderio M.A., Diamine oxidase in regenerating and hypertophic tissues, in: Mondovi B. (Ed.), Stucture and Function of Amine Oxidase, CRC Press, Florida, 1985, pp. 179–186. [28] Ripetti V., Kevers C., Gaspar Th., Two successive media for the rooting of walnut shoots in vitro. Changes in peroxidase activity and in ethylene production, Adv. Hort. Sci. 8 (1994) 29–32. [29] Serafini-Fracassini D., Del Duca S., Beninati S., Plant transglutaminases, Phytochemistry 40 (1995) 355–365. [30] Torrigiani P., Serafini-Fracassini D., Fara A., Diamine oxidase activity in different physiological stages of Helianthus tuberosus tuber, Plant Physiol. 89 (1988) 69–73. [31] Walter H., Geuns J., High speed HPLC analysis of polyamines in plant tissues, Plant Physiol. 83 (1987) 232–234.
The recalcitrance to rooting of the micropropagated shoots of the rac tobacco mutant: Implications of polyamines and of the polyamine metabolism Odile Faivre-Rampant*, Claire Kevers, Jacques Dommes, Thomas Gaspar Hormonologie Fondamentale et Appliquée, Institute of Botany B 22, University of Liège - Sart Tilman, B-4000 Liège, Belgium * Author to whom correspondence should be addressed (fax +32 4 366 38 59; e-mail [email protected])
(Received 28 January 2000; accepted 28 February 2000) Abstract – Rooting of wild-type tobacco (Nicotiana tabacum cv. Xanthi) shoots raised in vitro was promoted by polyamines in the absence of any other growth regulator and was inhibited by two inhibitors of polyamine metabolism. The auxin insensitive and recalcitrant to rooting rac mutant shoots did not respond to the same treatments. The activities of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), diamine oxidase (DAO), polyamine oxidase (PAO) and transglutaminases (TGases), and the titres of free and conjugated polyamines were estimated in the whole shoots and the basal parts of the stems of both tobaccos in the course of multiplication in vitro. The rac shoots grew at a lower rate. The wild-type rooted from the 7th day without special treatment. During the second week of culture, the shoots of both tobaccos were actively growing and showed an increase in ADC, ODC, DAO, PAO and TGase activities. Afterwards all these activities declined. These changes were concomitant with an increase in the polyamine contents (free and conjugated). Biosynthesis and oxidation of polyamines apparently occurred simultaneously and seemed directly correlated. In the basal part of the mutant stems however, the accumulation of free and conjugated putrescine as well as the transient increase in biosynthetic enzyme activities were delayed compared to the wild-type. These results are discussed in relation to growth behaviour and to root formation. © 2000 Éditions scientifiques et médicales Elsevier SAS Polyamines / rooting / tobacco mutant ADC, arginine decarboxylase / AG, aminoguanidine / BAP, benzylaminopurine / CHA, cyclohexylamine / DAO, diamine oxidase / GABA, γ-aminobutyric acid / IAA, indole-3-acetic acid / IBA, indole-3-butyric acid / NAA, naphthaleneacetic acid / ODC, ornithine decarboxylase / PAO, polyamine oxidase / Put, putrescine / Spd, spermidine / Spm, spermine / TGases, transglutaminases
1. INTRODUCTION Tobacco mesophyll protoplasts resistant to the toxic effect of NAA have been selected by Muller et al. [24] among UV-mutagenized populations. The mutant protoplasts had the ability to divide in media containing NAA concentrations which were toxic for the wild-type protoplasts. Auxin-resistant mutant shoots were derived from the protoplasts and called rac mutants due to their inability to root, even in response to an auxin treatment. The auxin resistance of the mutant cells and tissues was not correlated with an increased rate of conjugation or breakdown of auxins or with a perturbation of auxin transport [9]. Using the auxin-induced hyperpolarization response, the mutant protoplasts were shown to be ten times less sensitive to auxin than the
wild-type protoplasts [10]. Histological analyses indicated that 3–4 d after seed germination, the root meristem degenerated and was transformed into a callus [26]. In stem cuttings treated with IBA, perivascular cells divided, but never formed adventitious root meristems [21]. According to these authors, the rac mutation did not disrupt the auxin concentrations, but implied changes in the receptor affinity for auxin and/or in the efficiency of the transduction pathway. However, a reappraisal of the auxin concentrations did not lead to the same conclusions: the tobacco rac mutant showed hyperauxiny as compared to the wildtype (preliminary results in a preceding paper by Faivre-Rampant et al. [12]). This would result from an over-accumulation of phenolic compounds inhibiting the auxin catabolism, in the rac mutant.
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In the present paper, we investigated the auxin recalcitrance of the rac mutant at the level of polyamine metabolism. Indeed polyamines have been shown to be key factors, indissociable from auxins, in the process of adventitious rooting [14, 19]. Many studies have shown the beneficial effects of applying various polyamines on the rooting process [1, 4]. When poplar shoots were induced to root, an early increase in putrescine (Put) level occurred, concomitant with a transient increase in IAA. The timing of these events corresponded to the termination of the inductive phase [14]. Spermidine (Spd) and spermine (Spm) did not change significantly. Other arguments causally implicated Put in the rooting inductive phase of poplar: (a) the transient increase did not occur in the non-rooting cuttings [16]; (b) it was observed only in the basal rooting zone [16]; (c) inhibitors of the Put biosynthesis, such as DFMO and DFMA (α-difluoromethylornithine and α-difluoromethylarginine, respectively) applied prior to or at the beginning of the inductive phase inhibited rooting [16]; (d) an inhibitor of the Put conversion into Spd and Spm, CHA (cyclohexylamine, an inhibitor of the Spd synthase), which promoted the accumulation of endogenous Put, favoured rooting in the absence of exogenously supplied auxin [17]; (e) exogenously applied Put prior to or at the beginning of the inductive phase stimulated rooting [17]. Additional results point to a major role played by the Put catabolism through its ∆1-pyrroline-GABA (γ-aminobutyric acid) pathway [16, 17]. Indeed the treatment of poplar cuttings with AG (aminoguanidine, an inhibitor of the diamine oxidase, DAO, which converts Put to GABA) inhibited their rooting [17]. Similar results [19] were obtained with walnut shoot cuttings in rooting inductive phase where endogenous IAA and peroxidase had been also implicated [18, 28]. Furthermore, a low activity of the amine-oxidases and an accumulation of the conjugated polyamines negatively affected organogenic programmes, including rooting, in Chrysanthemum morifolium leaf disc explants [3]. In the in vitro shoot-forming cultures of the wildtype and rac tobaccos [11], the shoots of the wild tobacco rooted spontaneously at the end of the growth cycle; the rac shoots did not even in response to an auxin treatment. The present work was undertaken to assess the role of polyamines and of their catabolism in rooting induction in tobacco. We evaluated the levels of free and conjugated polyamines and the activities of the related biosynthetic (ADC, ODC) and catabolic (DAO, PAO) enzymes in shoots of rac and wild-type tobacco. Plant Physiol. Biochem.
TGase activities (catalysing covalent linkages of polyamines to numerous substances including proteins, see review by Serafini-Fracassini et al. [29]) were also considered. In addition, we devised culture conditions in which wild-type shoots did not spontaneously root. In these conditions, the effect on rooting of exogenous polyamines and inhibitors of their metabolism (AG and CHA) could be further evaluated.
2. RESULTS 2.1. Growth pattern and rooting behaviour On the commonly used micropropagation medium (MS, 3 % sucrose, 0.8 % Roland agar, 0.13 µM BAP), wild-type shoots started to grow 3 ± 1 d after subculture (figure 1). New leaves were observed after 5 ± 1 d, and root formation after 7 ± 1 d with a number of roots per shoot of 6 ± 2. At day 10 ± 1, an average of 78 ± 3 % of wild-type shoots showed adventitious roots. The growth of rac shoots started 5 ± 1 d after subculture with the neoformation of two leaves after 7 ± 1 d. The mutant did not root at the end of the multiplication cycle, nor after any rooting treatment. Both types of shoots continued to grow throughout the culture cycle, but the rac mutant grew more slowly (figure 1). To study the effects of exogenous polyamines on rooting induction, it was necessary to devise culture conditions in which wild-type shoots do not spontaneously root. Such conditions were obtained by modifying the multiplication medium. In comparison with the commonly used micropropagation medium, it did not contain any NH4+, nor any growth regulator, but ten times more KNO3 and 1 % Kobe agar instead of 0.8 % Roland agar. On this medium, neither rac nor wildtype shoots rooted spontaneously (table I).
2.2. PA titres during the multiplication cycle Figure 2 shows the changes of free and conjugated polyamines in the shoots of the wild-type and the rac mutant during the multiplication cycle. Put was by far the major polyamine contained in the shoots of the two types of tobacco with higher levels in rac shoots. A transient accumulation of free and conjugated Put was observed with the maximum value on day 14. Spd and Spm also showed a transient increase on day 14, but to a lesser extent than Put, with levels not significantly different between the wild-type and the mutant. In the basal parts (where roots form, if they do) of the wild-type and mutant tobacco stems, Put was the
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Figure 2. Changes in free and conjugated polyamine levels during a multiplication cycle (21 d) in the whole shoots of wild-type (D8) and rac mutant tobaccos. (●), Put; (■), Spd; (▲), Spm.
Figure 1. Shoot growth in vitro during a multiplication cycle (21 d) of the wild-type D8 (●) and mutant rac (■) tobaccos. Top, Changes in shoot height; bottom, changes in node number per shoot.
major polyamine as in whole shoots (figure 3). Level of free Put in the basal parts of the rac stems and in the rac whole shoots showed a similar time course. In the basal parts of the wild-type stems, Put showed an earlier transient accumulation with higher level observed between the 7th and the 14th day. The levels and variations of free and conjugated Spd and Spm were low compared to Put.
peak at day 14 as for polyamine level, followed by a marked decrease (figure 4 A). ADC activity was lower than ODC in the two tobaccos throughout the culture period and only showed very small changes. ADC activity was always higher in rac shoots than in wild-type shoots.
2.3. ADC and ODC activities ODC activity increased in the whole shoots of the two tobaccos during the second week of culture with a Table I. Effects of polyamines on the rooting of tobacco wild-type shoots grown in a non-rooting medium (MS without NH4+, ten times more KNO3, 3 % glucose, 1 % Kobe agar, no growth regulator). The percentage of rooting shoots was determined after two subcultures (mean of five independent assays ± SD). The rac shoots did not root in any of these conditions. Polyamines added Put Spd Spm
Concentrations (M) 0 0 0 0
10
–6
10 ± 5 0 0
10–5
10–4
10–3
28 ± 7 0 0
52 ± 7 0 0
68 ± 6 33 ± 6 0
Figure 3. Changes in free and conjugated polyamine levels during a multiplication cycle (21 d) in the basal part of the stems of wild-type (D8) and rac mutant tobaccos. (●), Put; (■), Spd; (▲), Spm.
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Figure 4. ADC and ODC activities during a multiplication cycle (21 d) in whole shoots (A) and the basal parts of stems (B) of wild-type (D8) (●) and rac (■) mutant tobaccos.
In the basal parts of the wild-type stems, ADC and ODC activities showed a transient increase at day 7 (figure 4 B). In the rac mutant, these activities were relatively stable, and always higher than in wild-type shoots.
2.4. DAO and PAO activities In the whole shoots of the two tobaccos, DAO and PAO activities showed about the same changes as ADC and ODC activities with a maximum at day 14 but to a lesser extent for the wild-type (figure 5 A). DAO activity was lower than PAO activity in the wild-type and mutant tobaccos. In the basal parts of the stems, PAO activity did not change significantly during the culture. Its activity was somewhat higher in the mutant. DAO activity reached a maximum at day 14 in both wild-type and rac tobaccos (figure 5 B), like in the whole shoots. 2.5. TGase activities In the wild-type and mutant tobaccos, TGase activity showed parallel changes and about the same level, with a maximum at day 14 (figure 6) for the whole shoots. There were no significant differences in the basal parts of the stems between the two types of tobacco. 2.6. Effects of exogenous PAs and inhibitors on rooting None of the three polyamines tested, at concentrations varying from 10–6 to 10–3 M, was able to induce Plant Physiol. Biochem.
Figure 5. DAO and PAO activities during a multiplication cycle (21 d) in whole shoots (A) and the basal parts of stems (B) of wild-type (D8) (●) and rac (■) mutant tobaccos.
rooting of the rac mutant whatever the medium used. On the medium designed to avoid spontaneous rooting, exogenously applied Put stimulated rooting of wild-type shoots to 68 % in the 1–1 000 µM range in the absence of any growth regulator (table I). In the presence of 1 000 µM Spd, 33 % of the shoots rooted. Inhibition of the conversion of Put into ∆1-pyrrolineGABA by AG and to a lesser extent, inhibition of the conversion of Put into Spd by CHA led to a reduction in rooting (table II).
3. DISCUSSION In both wild-type and rac shoots, a transient increase in the level of polyamines, mainly Put, was observed 14 d after subculture. Thus differentiation of the shoots appears to be accompanied by higher levels of free and conjugated Put throughout the multiplication cycle. It was reported that in tobacco, high intracellular levels of conjugated Put inhibited cell proliferation and suppressed bud formation [7]. Thus, higher polyamine levels in the rac mutant might be responsible for its lower growth rate. In the wild-type tobacco cuttings, rooting was stimulated by application of exogenous polyamines. Treatments with inhibitors of polyamine metabolism, on the contrary, had a negative effect on rooting. These
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transient increase in Put occurred during the first week of culture; in the rac mutant, this increase was delayed to the second week. Hausman et al. [16] have shown that Put was a good marker for adventitious rooting. This delay in the Put transient accumulation in the basal parts of the rac stems might be correlated with the absence of root formation.
Figure 6. TGase activities during a multiplication cycle (21 d) in the whole shoots and the basal part of the stems of wild-type (●) and rac mutant (■) tobaccos.
results provide additional support for the involvement of polyamines in the rooting process. The auxin insensitive mutant rac shoots did not root even in response to polyamine application. This observation supports a previous claim that auxins and polyamines are indissociable factors in the rooting process. Indeed, in poplar cuttings [16, 17], rooting depended on a concomitant variation in the levels of both regulators. A time course study of polyamine levels in the basal parts of the stems showed important differences between wild-type and rac tobacco shoots. In wild-type, a
Table II. Effects of inhibitors of polyamine metabolism on the rooting percentage of wild-type after three subcultures on growth medium (MS supplemented with 0.13 µM BAP). Results are means of five independent assays ± SD. The rac shoots did not root in any of these conditions. AG, Aminoguanidine; CHA, cyclohexylamine. Inhibitors added AG CHA
Concentrations (M) 0 100 100
10
–6
89 ± 8 100
10–5
10–4
10–3
83 ± 5 100
82 ± 6 100
80 ± 5 80 ± 8
In whole shoots, ODC activity and polyamine levels showed parallel changes. ADC activity was always lower than ODC activity and remained relatively constant throughout the growth period. Considerable evidence now indicates that both ADC and ODC relative contributions to polyamine biosynthesis are dependent upon the type of tissue and the developmental process [13]. It seems likely that ADC and ODC have different roles in cell proliferation, expansion growth and differentiation. In several plants, ADC seems to be involved in vegetative development, whereas ODC may be required for floral induction, sexual differentiation, root formation and callus formation [22]. More recently, an oat ADC gene under the control of an inducible promoter, the tetracyclinerepressor system, has been expressed in transgenic tobacco plants [6, 23]. Transgenic plants treated with tetracycline during the vegetative stage displayed increased ADC activity and increased Put levels, corresponding to different degrees of altered phenotype, characterized by shorter internodes, thinner stems and leaves, leaf chlorosis and necrosis and reduced root growth. In the basal parts of the wild-type stems, the higher ODC and ADC activities corresponded to a maximum in Put level. In the rac mutant, these activities hardly changed but were always higher than in wild-type stems. This might also be related to the absence of root formation for the rac mutant. In cultures of wild-type and rac whole shoots, DAO and PAO activities increased during the second week. Thereafter the levels declined. The maximum values coincided with those of ADC and ODC activities. These results suggest a direct correlation between the biosynthesis and oxidation of Put in physiological phases such as shoot growth (leaf neoformation, stem elongation and root formation). Similarly, in Helianthus tuberosus tuber, the increase in DAO activity paralleled the accumulation of Put [30]. But in the basal parts of the stems, whatever the tobacco genotype, a maximum in DAO activity was observed on the 14th day, whereas PAO activity hardly changed during the multiplication cycle. Our data are also in agreement with those for animal systems in which DAO vol. 38 (6) 2000
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activity increases concomitantly with or immediately after an increase in ODC activity and Put content [27, 28]. In mammals, it has been established that posttranslational covalent linkage of polyamines to proteins is catalysed by a class of enzymes known as TGases. In our case, time course of TGase activities in whole shoots showed the same pattern than the one of ODC/ADC activities and Put level. However, there is no significant differences in the basal parts of the stems between the wild-type and the mutant. Information on TGase activities in plants is relatively incomplete. Recently, ε-(γ-glutamyl)-lysine, the product of TGase’s protein cross-linking activity, was detected in root and shoot tissue of pea, broad bean, wheat and barley by cation-exchange chromatography [20]. Cloning of genes coding for TGases in plants will in the future allow the study of the mechanisms controlling polyamine conjugation to proteins as well as the molecular mechanism by which these bound polyamines play a role in physiological processes. As a conclusion, the present work provided essential new information for the recalcitrance to rooting of the rac mutant tobacco: it is insensitive to exogenous application of polyamines. This might be a further confirmation of the indissociability of auxins and polyamines in the rooting developmental process. Finally, we showed that the transient increases in both the Put level and in polyamine metabolic enzyme activities occurred in the basal parts of the rac stems, but with a delay in comparison to wild-type stems. These latter observations suggest that rooting of the rac mutant might be induced but arrested at the expression phase.
In such conditions, the wild-type shoots rooted spontaneously at the end of each multiplication cycle, but the rac ones did not. The effect of two inhibitors of polyamine metabolism, AG and CHA, was tested by addition to the same growth medium. For some experiments, the micropropagation medium was modified by eliminating the NH4+ ions, using ten times more KNO3, 3 % glucose, 1 % Kobe agar (Marseille), and no growth regulator. In this medium, the shoots were subcultured every 10 d. The three polyamines to be tested, i.e. Put, Spd and Spm, were filter-sterilised and added to the previously autoclaved medium.
4.2. Determination of free and conjugated PAs Free polyamine extraction, separation, identification and measurement by direct dansylation and HPLC were done as described by Walter and Geuns [31]. Free polyamines were extracted in 5 % (v/v) cold HClO4 at a ratio of 100 mg fresh tissues·mL–1. The techniques which were used to extract, separate and measure the levels of polyamine conjugates have been described elsewhere [8]. Water-soluble conjugates from the aqueous extracts were eluted from an Amberlite (Serva CG 50, H+ form) column with 3 M acetic acid. Non-watersoluble conjugates were extracted from the pellet in 100 % MeOH and purified in ethyl acetate. For the quantification of polyamine conjugates, extracts were boiled in 6 M HCl, followed by measurement of free amines. The compounds were chromatographed on a Bondpack C18 reverse phase column (particle size 5 µM, 4 × 125 mm, Waters Assoc.) with an acetonitrile/ water (72/28 v/v) solvent gradient at a flow rate of 1 mL·min–1.
4.3. Determination of ADC and ODC activities 4. METHODS 4.1. Plant material The wild-type, D8, and homozygous rac mutant seeds of Nicotiana tabacum cv. Xanthi were surface sterilized for 3 min in a 10 % (v/v) commercial bleach solution, placed on MS medium [25], pH 5.7, containing 0.8 % (w/v) Roland agar (Brussels), 3 % (w/v) sucrose and 0.13 µM BAP. Shoot apices or stem nodes were explanted for continued multiplication through 3-week cycles on the same medium. Cultures, in 600-mL cylindrical glass jars containing 100 mL medium covered by a glass lid maintained with a sheet of transparent plastic film, were placed under a 16-h photoperiod (Sylvania Grolux fluorescent tubes providing 15 µE·m–2·s–1), at 28 °C (day) and 25 °C (night). Plant Physiol. Biochem.
Arginine decarboxylase (ADC, EC 4.1.1.17) and ornithine decarboxylase (ODC, 4.1.1.19) activities were determined according to the methods of Aribaud et al. [2] after several modifications. Samples were ground at a ratio of 0.5 g fresh weight·mL–1 0.1 M Tris-HCl (pH 7.5) containing 5 mM EDTA, 10 mM mercaptoethanol, 1 mM pyridoxal phosphate, 5 mM dithiothreitol (DTT) and 0.5 M KCl. The extract was centrifuged for 30 min at 20 000 × g. The supernatant was saturated to 50 % with (NH4)2SO4 for 1 h with gentle stirring. The precipitate was collected by centrifugation at 20 000 × g for 20 min. The pellet was resuspended in a minimum volume of extraction buffer. This fraction was then dialysed against two changes of 10 mM Tris-HCl containing 1 mM EDTA, 10 mM mercaptoethanol for 8 h. All the steps were
Polyamine metabolism of the rac mutant
carried out at 4 °C. The dialysed extract was used to determine the ADC and ODC activities. To assay ODC, 100 µL extract were mixed with 13 µL [1-14C]ornithine (2 GBq·mmol–1, ICN), 45 µL 0.1 M TrisHCl (pH 7.5) containing 10 mM mercaptoethanol, 0.1 mM pyridoxal phosphate and 1 mM cold ornithine. To assay ADC, 100 µL extract were mixed with 13 µL [U-14C]-arginine (11 GBq·mmol–1, ICN), 45 µL 0.1 M Tris-HCl (pH 7.5) containing 10 mM mercaptoethanol, 0.1 mM pyridoxal phosphate and 1 mM cold arginine. Reaction mixtures were incubated for 1 h at 30 °C. The reactions were stopped with 10 µL 17 M acetic acid. Ten-µL aliquots were analysed by thin layer electrophoresis on cellulose plates (MachereyNagel). Electrophoresis was performed in acetic acid/ pyridine/water (5/1/94, v/v/v) for 1 h at 250 V. Unlabelled arginine, ornithine, agmatine and putrescine as standards were also spotted on the plate and developed with ninhydrin (5 % w/v in ethanol). The cellulose (ornithine spots for ODC activity and arginine spots for ADC) was scraped off and transferred to scintillation vials and the radioactivity determined in a LS 5000 scintillation counter (Beckman). We measured the activities by making a difference between the remaining substrate amount in the spots and the loaded amount of substrate.
4.4. Determination of DAO and PAO activities Diamine oxidase (DAO, EC 1.4.3.6) and polyamine oxidase (PAO, EC 1.4.3.4) activities were determined using a modification of the procedure described previously [2]. Extracts were prepared as for the determination of ADC and ODC activities. DAO activity was assayed by measuring the [14C]-∆1-pyrroline formation from [14C]-Put. Aliquots (50 µL) of extract were incubated for 1 h at 30 °C in a final volume of 100 µL containing 7.4 kBq [1,4-14C]-Put (4.11 GBq·mmol–1, Amersham), in 0.1 M Tris-HCl (pH 8), 5 mM Put and 0.3 mg·mL–1 catalase. PAO activity was assayed by measuring the [14C]-∆1-pyrroline formation from [14C]Spd. Aliquots (50 µL) of extract were incubated for 1 h at 30 °C in a final volume of 100 µL containing 7.4 kBq [1,4-14C]-Spd (4.37 GBq·mmol–1, Amersham), in 0.1 M Tris-HCl (pH 8.5), 2.5 mM Spd and 0.3 mg·mL–1 catalase. After incubation, 200 µL 1 M sodium carbonate was added to the mixture and the [14C]-∆1-pyrroline immediately extracted in 4 mL toluene by vortexing for 20 s and centrifuging for 5 min at 2 000 × g. Then, 1 mL of the toluene phase was added to scintillation vials containing 4 mL scintillation liquid and counted in a LS 5000 scintillation counter (Beckman).
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4.5. TGase-like activity Transglutaminase-like activity was determined using the method described by Haddox and Russell [15]. Extracts were prepared as for the determination of ODC and ADC activities. Aliquots (50 µL) of extract were incubated for 1 h at 30 °C in a final volume of 100 µL containing 7.4 kBq [1,4-14C]-Put –1 (4.11 GBq·mmol , Amersham) in 0.1 M Tris-HCl (pH 8.5), 3 mM cold Put and 10 mg·mL–1 dimethylcaseine. After incubation, 200 µL 0.5 M cold Put were added and the reaction was stopped with 4 mL of 20 % (w/v) thrichloroacetic acid (TCA). The mixture was then filtered and the filter was washed with 5 % TCA containing 0.5 M KCl, purified water and finally 0.1 M HCl. The filter was then dried and placed in scintillation vials containing 4 mL scintillation liquid and counted in a LS 5000 scintillation counter (Beckman).
4.6. Protein analysis Soluble proteins were determined by the method of Bradford [5]. Bovine serum albumine was used as standard. All results are means of at least three separate experiments (± SD).
Acknowledgments Odile Faivre-Rampant is gratefully indebted to the European Community for a FAIR research training grant. Provision of the tobacco seeds by Dr Caboche (INRA, Versailles) is also gratefully acknowledged.
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