Putrescine Metabolism in a Fully Habituated Nonorganogenic Sugar Beet Callus and its Relationship with Growth

JOUR.ALOF

]. Plant Physiol. Vol. 154. pp. 503-508 (1999)

Plani PhysjoiOIJ

http:/ /www.urbanfischer.de/journals/jpp

© 1999 URBAN & FISCHER

Putrescine Metabolism in a Fully Habituated Nonorganogenic Sugar Beet Callus and its Relationship with Growth CLAIRE KEvERS, BADIA BISBIS, OmLE FAIVRE-RAMPANT,

and THOMAS

GASPAR

Institute of Botany, B 22, University of Liege, Sart Tilman, B-4000 Liege, Belgium Received May 7, 1998 ·Accepted July 25, 1998

Summary

A fully-habituated and nonorganogenic (HNO) sugar beet callus was previously shown to overproduce polyamines, as compared with a normal (N) auxin- and cytokinin-dependent callus of the same strain. Because relationships were established between polyamine levels and metabolism with different growth and development processes, some key enzymes in the metabolic pathways of polyamines were investigated in the HNO callus, and their involvement in growth appraised. Putrescine was found to be the major free and conjugated polyamine in the HNO callus. It was biosynthesised preferentially via ornithine and ornithine decarboxylase (ODC), which is in agreement with the surplus of synthesised ornithine. Diamine (DAO) and polyamine (PAO)-oxidase activities were also highest in the HNO callus, as compared with the normal, with DAO being the more active. Transglutaminase activities (± Ca) were also higher in HNO than in normal callus. Addition of different polyamines or of inhibitors of their biosynthesis to the culture medium of the HNO callus modified the level of endogenous polyamines and affected callus growth. The results thus pointed out a higher polyamine metabolism, particularly of putrescine, in the actively growing auxin- and cytokinin-independent callus than in the normal one. They also provided evidence for the sensitivity of a habituated tissue type towards this class of growth regulators.

Key words: Beta vulgaris, arginine decarboxylase, callus, diamine oxidase, habituation, ornithine decarboxylase, polyamines, putrescine, sugar beet. Abbreviations: ADC = arginine decarboxylase; CHA = cyclohexylamine; DAO = diamine oxidase; DFMA = a-difluoromethylarginine; DFMO = a-difluoromethylornithine; GABA = y-aminobutyric acid; HNO = habituated nonorganogenic callus; MGBG = methylglyoxal-bis-guanylhydrazone; N = normal callus; ODC =ornithine decarboxylase; PAO =polyamine oxidase; SAM= S-adenosylmethionine; TGase = transglutaminase. Introduction

In plant cultures in vitro, habituation to hormones, that is the acquired independence to exogenous feeding with auxins and cytokinins, has been classed as a neoplasic «disease» in the absence of pathogens (Bednar and Linsmaier-Bednar, 1989; Pengelly, 1989). As in the case of animal tissues under neoplasic progression (Porciani et al., 1993), habituated plant cells accumulate more polyamines than do normal tissues

(Audisio et al., 1976; Le Dily et al., 1993; Hagege et al., 1994; Biondi et al., 1993). In a fully-habituated and nonorganogenic (HNO) sugar beet callus, polyamine accumulation appears as the result of three disturbed metabolic pathways that act cooperatively but favour the glutamate-proline pathway-cycle, which provides the ornithine surplus required for polyamine synthesis (Kevers et al., 1997). Changes in polyamine levels and activities of polyamine biosynthetic enzymes have been implicated in a variety of 0176-1617/99/154/503 $ 12.00/0

504

CLAIRE I
plant growth and developmental processes (reviewed in Evans and Malmberg, 1989; Martin-Tanguy et al., 1996, 1997). Compounds resulting from the catabolism of polyamines, such as y-aminobutyric acid (GABA) and H 2 02> have been more recently proposed as secondary messengers (Mehdy, 1994; Ramputh and Brown, 1996; Hausman et al., 1997). Because the above mentioned fully habituated sugar beet callus is an actively growing tissue (Le Oily et al., 1993), the above data led us to hypothesise a higher polyamine metabolism than in a normal one. This is why we investigated the main aspects of polyamine metabolism in the fully habituated and nonorganogenic (HNO) callus. We also took this opportunity to appraise the role of polyamines in the growth regulation of an auxin- and cytokinin-independent tissue. In plants, two pathways lead to putrescine formation. The first involves ornithine decarboxylase (ODC; EC 4.1.1.17), which catalyses the removal of a carboxyl group from ornithine to putrescine, and the other the decarboxylation of arginine by arginine decarboxylase (ADC, EC 4.1.1.19). The existence of two alternative routes (ADC/ODC) for the synthesis of putrescine is explained by the differential compartmentation of the two enzymes, resulting in the specific regulation of different plant processes (Galston et al., 1997). As in the case of any plant growth regulator, the intracellular free polyamine pool not only depends on synthesis of the polyamines but also on several other processes, including degradation, conjugation and transport (Bagni and Torrigiani, 1992; Martin-Tanguy, 1997). The degradation of polyamine is carried out principally by two enzymes. The first, diamine oxidase (DAO; EC 1.4.3.6), catalyses the oxidation of putrescine. The resulting y-aminobutyraldehyde spontaneously cyclises to form A1-pyrroline and y-aminobutyric acid (GABA) (Flores and Filner, 1985). The second, the polyamine oxidases (PAO; EC 1.5.3.3), oxidise spermidine and spermine. The oxidation of spermine leads to hydrogen peroxide, diaminopropane and y-aminobutyraldehyde, with the resulting aldehyde forming A1-pyrroline and GABA (Flores and Filner, 1985) by cyclisation. Polyamines can form amide conjugates with different molecule types by covalent binding through their primary amine (Tabor and Tabor, 1985). Covalent linkage of polyamines to proteins is catalysed by a class of enzymes known as transglutaminases (TGase; EC 2.3.2.13), which have been localized both intra- and extracellularly (Folk, 1980). The main characteristics of plant transglutaminases were recently reviewed by Serafini-Fracassini et al. (1995). Similar to transglutaminases of animal origin, some plant TGases seem related to structural functions (Tiburcio et al., 1997; Martin-Tanguy, 1997). A possible new function ofTGases in the photosynthetic process was proposed (Del Duca et al., 1994). Futhermore, one possible mechanism through which polyamines might regulate cell division and related growth processes involves their binding to specific regulatory proteins, possibly through TGases (Martin-Tanguy, 1997). In the present paper, the levels of free and conjugated polyamines, the activities of the related biosynthetic (ADC, ODC) and catabolic (DAO, PAO) enzymes, TGase, and the effect of metabolic inhibitors are investigated in the course of proliferation of normal (N) and habituated, nonorganogenic (HNO) sugar beet callus. A higher metabolism of polyami-

nes, particularly of putrescine, was found in the habituated callus, indicating a higher degree of sensitivity of this type of tissue towards the polyamines.

Materials and Methods

Material and culture conditions Experimental conditions for obtaining normal (N) and nonorganogenic (HNO) callus of sugar beet (Beta vulgaris L. ssp. altissima) and for maintaining them as stock cultures under light (16-h photoperiod at 25 ·c, Sylvania Grolux fluorescent lamps providing an irradiation of I7W m- 2) have been reported by De Greef and Jacobs (I979). Callus was usually subcultured every 2 weeks on fresh basal medium supplemented with 0.45 Jlmol!L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.44 Jlmol!L N6-benzyladenine (BAP) (N tissue only). For chemical treatments, samples were transferred on medium containing the compound(s) to be tested. These were filtersterilised and added to the previously autoclaved medium. Callus growth was expressed as an increase in relative fresh mass (FM): (final FM-initial FM) X 100/initial FM. For fresh mass measurements, callus from a Petri dish was rapidly blotted, placed on aluminium foil, and weighed. Initial fresh mass was calculated by the difference between full and empty Petri dishes at the time of subculture. The measurements were repeated at least three times on different series.

Extraction and determination ofpolyamines Samples were harvested at intervals during the I4 days of culture. They were weighed and stored at -40 ·c. Extraction of free and conjugated (soluble and non-soluble in HCl04 4 o/o) polyamines, separation, identification and measurement by direct dansylation and HPLC were done as described by Walter and Geuns (1987).

Enzymatic extract Samples were ground in a chilled mortar at a ratio of 0.5 g fresh weight mL -I of 100 mmol!L Tris-HCl (pH 7.5) buffer containing 5 mmol/L EDTA, 10 mmol!L mercapthoethanol, I mmol!L pyridoxal phosphate, 5 mmol/L dithiothreitol, 0.5 mol!L KCl and IO o/o (w/v) activated charcoal to absorb phenolics. The extract was centrifuged 30 min at 30,000 g.,. The supernatant was saturated at 50 o/o with (NH4hS04 for I h with gentle stirring. The pellet was collected by 20 min centrifugation at 25,000 g., and resuspended in a minimum (1 mL g-I fresh weight) of extraction buffer. This fraction was then dialysed against two liters of IO mmol!L Tris-HCl (pH 8) containing I mmol!L EDTA and IO mmol!L mercaptoethanol for 8 h. All procedures were carried out between 0 and 4 ·c.

ADC and ODC activities The enzyme assays were performed with some modifications according to Martin-Tanguy et a!. (I997). The dialysed extract was used to determine ADC and ODC activities. To assay ODC and ADC, 50 JlL of enzymatic extract were mixed with 12.8 JlL of, respectively, (1-' 4 C) ornithine (7.46 kBq) or (U-' 4C) arginine (7.46 kBq), 45 JlL 100 mmol!L Tris-HCl (pH 7.5), containing 10 mmol!L mercaptoethanol, O.I mmol!L pyridoxal phosphate and 5.5 mmol!L cold ornithine or arginine. Reaction mixtures were incubated for I h at 30 ·c. The reactions were stopped with IO JlL of 5 mol!L acetic acid. For blanks, 10 11L of 5 mol!L acetic acid were added at t = 0. Denatured proteins were removed after 5 min at I5,000 g., centrifugation. Ten JlL aliquots were analysed by thin layer

505

Putrescine Metabolism in Sugar Beet Callus electrophoresis on cellulose plates (MN CCM CEL 300-10). Electrophoresis was performed in acetic acid-pyridine-water (5 : 1 : 94) for 1 h at 250 V. Unlabelled agmatine and putrescine used as standards were also spotted on the plates and developed with ninhydrin (5% [w/v] in methylcellosolve). The cellulose was scraped off and transferred to scintillation vials and the radioactivity determined.

Table 1: Putrescine, spermidine, spermine and total free and conju-

gated (soluble and non-soluble) polyamine levels (nmol/L g- 1 fresh weight) in normal and habituated sugar beet callus after 14 days of culture. Values are the mean ± SE of 3 experiments.

Polyamines

Free

Putrescine

49±5 66± 8 12±3 17± 3 1±1 0

DAO and PAO activities

e

The dialysed extract was used to determine DAO and PAO activities by a radiochemical method that measures the 4c] r.yrroline formation from [1,4) 4C] putrescine (DAO) or from [1,4- 1 C] spermidine (PAO). After 15 min preincubation at room temperature, aliquots (50 JlL) of concentrated supernatant were incubated at 30 "C for 1 h in a final volume of 0.1 mL, and adjusted by adding 100 mmol/L Tris-HCl buffer (pH 8) containing 7.4 KBq of [1,414C] putrescine or [1,4- 14C] spermidine, 5 mmol!L cold putrescine or 2.5 mmol/L cold spermidine, and 30 Jlg catalase. Mter incubation, 0.2 mL 1 mol!L sodium carbonate was added to the mixture and the 4CJ pyrroline was immediately extracted in 4 mL of toluene by vortexing for 20 sec and centrifuging for 5 min. at 2,000 gn. Mter this, 1 mL of the toluene phase was added to scintillation vials containing 4 mL of scintillation liquid (Optiphase, Wallac) for counting.

Habituated callus

Normal callus

Spermidine Spermine

Non-soluble Free

Soluble conjugated conjugated

74±10 22± 4 2± I

98±15 62±9 83±11 Total General total 243±35

e

Non-soluble Soluble conjugated conjugated

149±13 118± 10 101± 8 76± 9 4± I 5± 2

321±27 164±15 8± I

493±43 255±23 198±20 946±86

300

[

250

'o

...J

Transglutaminase-like activity The enzyme assay was performed with some modifications according to Haddox and Russell (1981). The assay mixture (100 JlL) in 100 mmol!L Tris-HCl (pH 8.5) contained 50 f,L of extract, 10mg·mL- 1 ofN, N'-dimethylcasein, 7.4kBq [1,4_1 C] putrescine, 3 mmol!L cold putrescine and 2 mmol!L sodium chloride (TGase Ca-independent) or calcium chloride (TGase Ca-dependent). The final pH optima of the mixtures measured at 30 "C were 8.5. The mixtures were incubated for 1 h at 30 "C. At the end of incubation, 200 JlL of 0.5 mmol/L cold putrescine was added. The reaction was stopped with 4 mL of 20% TCA. The mixture was filtered using a multifilter apparatus (Millipore) and then repeatedly washed with 5% TCA, also containing 0.5 mol!L KCl, H 20, 0.1 mol!L HCl, and 1 % ethanol. The dry filters were put in vials containing 4 mL of scintillation cocktail (Optiphase, Wallac). The controls contained all of the constituents except cellular proteins (which were replaced by buffer).

Protein analyses Soluble protein was determined according to Bradford (1976). Bovine serum albumin was used as standard. All results are the mean of at least 3 separate experiments.

Results

Polyamine levels The free aliphatic amines, putrescine, spermidine and spermine, were determined in the two types of callus after 14 days of culture (Table 1). Their total levels were higher in HNO than in N callus. In all cases, spermine levels were very small and putrescine constituted the major part (58 % and 79 %, respectively) of the free polyamines. Comparatively, the spermidine level was most enhanced in the habituated callus. The content of conjugated polyamine (soluble and non-soluble)

'6

f200 ~

·~

~ 150 :l a. 100

50L___J __ _~_ _ _ _L __ _-L--~----~---L~ 12 14 8 10 4 6 2 0

Days

Fig. 1: Variations in free (e) and conjugated (soluble (•) and nonsoluble (•)) putrescine levels in habituated callus during the culture. Values are the mean ± SE of 3 experiments.

was also higher in HNO than inN callus. In all cases, putrescine constituted the major part of the conjugated polyamines. Contents of free or conjugated polyamines displayed small variations during the 14 days of culture. The most important modifications were observed for putrescine in HNO callus (Fig. 1). The free putrescine level decreased between days 3 and 6 and increased to a maximum at day 10 before it declined. The level of insoluble conjugated putrescine changed in parallel except at the end of the culture, when it continued to increase slowly. No variations were observed in conjugated soluble putrescine.

ADC and ODC activities In the two types of callus, both acnvttles decreased between days 3 and 6. In HNO callus, no significant variations of ODC and ADC activities were detected afterwards. InN callus, the activities increased to a maximum at day 10 and

506

CLAIRE KEVERS, BADIA BISBIS, OorLE FAIVRE-RAMPANT, and THOMAS GASPAR

c:

ADC

~ 1,6 a.

i

'O>

~ 1,2

u

\

\

~

e "' ~

ODC

0,8

"'

(.)

8 0,4

g <(

2

4

6

10

12

Days

14 0

\

\

~ 6

8

10

12

14

Days

Fig.2: ADC and ODC activities in 2 types of callus (e, normal,., habituated) of Beta vulgaris cultured for 14 days. Values are the mean ± SE of 3 experiments.

crease in the free polyamines (± 50%). An increase of the added polyamines (10- 5 mol/L) reduced growth and the free polyamine content. a-Difluoromethylarginine (DFMA) and a-difluoromethylornithine (DFMO), inhibitors of putrescine synthesis, induced a rapid decrease in the polyamine levels (particularly noticeable with DFMO) and increased growth (25 to 50%). The inhibition of spermidine and spermine synthesis by cyclohexylamine (CHA, inhibitor of spermidine synthase) had no effect on growth while methylglyoxal-bis-guanylhydrazone (MGBG), an inhibitor of SAM decarboxylase, reduced it. These two inhibitors reduced the polyamine content.

Discussion

The results showed overaccumulation of polyamines by the habituated callus compared with the normal one, with putrescine as the major free and conjugated compound (Table

" 5

j

TGase (Ca indep.)

TGase (Ca dep.)

~ 4

·~

Days

Days

Fig. 3: DAO and PAO activities in 2 types of callus (e, normal, . , habituated) of Beta vulgaris cultured for 14 days. Values are the mean ± SE of 3 experiments.

declined afterwards (Fig. 2). ODC activity was always higher than ADC activity.

DAO and PAO activities Activities of both DAO and PAO were at a very low level in N callus during the 14-day culture (Fig. 3) and remained so with a small peak at day 10. In HNO callus, the DAO activity changed during the 14 days of culture, and displayed 2 maxima at days 6 and 14. In contrast, PAO activity declined between days 3 and 6 and increased gradually afterwards.

Transglutaminase activities No TGase activities, either Ca-independent or -dependent, were detected in N callus during culture. There were no TGase activities at day 3 in HNO callus but Ca-independent TGase developed rapidly to a maximum at day 10 before declining (Fig. 4), while the Ca-dependent TGase slowly increased between days 10 and 14.

Effect ofpolyamines and inhibitors oftheir biosynthesis on growth and polyamine content ofHNO callus As shown in Table 2, the addition of putrescine or of spermidine (lo- 6 mol/L M) increased growth but induced a de-

~ 3

e "' ~

2

!

1

~

~

~--i

I

I

I

I

I

I

I

I \

\

\

\

\

\

\

•

6

10

-.

•

12

14 0

6

Days

10

12

14

Days

Fig.4: Transglutaminase (Ca-dependent and -independent) activities in 2 types of callus (e, normal, •, habituated) of Beta vulgaris cultured for 14 days. Values are the mean± SE of 3 experiments.

Table 2: Effects of polyamines and polyamine biosynthesis inhib-

itors applied during 14 days of culture on growth index and total free polyamines of habituated callus. Values are the mean ± SE of 3 experiments. Polyamine or inhibitor

Control PUT (10- 6 mol/L) PUT (1 0- 5 mol!L) SPD (1 o- 6 mol!L) SPD (10- 5 mol!L) DFMA (I0- 6 mol!L) DFMO (lo- 6 moi!L) DFMO+ DFMA (10- 6 moi!L) MGBG (lo-s mol!L) CHA (10- 5 moi!L) MGBG+CHA (lo- 5 mol!L)

Growth index (o/oFW)

Polyamine content (nmol g- 1 FW)

7 days

14 days

7 days

41.4±4.1 37.5±6.2 32.8±3.5 47.3±6.8 47.0±2.4 48.6±5.6 33.7±4.0 32.9±4.6 33.3±2.7 43.3±3.9 23.5± 1.9

126.0± 13.1 245±41 152.2±20.2 138± 16 48.5± 5.1 91±12 176.9±26.5 124±31 59.8± 3.0 169±21 162.2± 14.6 168±48 192.3± 16.9 95±14 155.1±10.9 22± 3 53.3± 4.3 106±54 138.0± 15.4 178±45 53.5± 6.6 45± 9

14 days 255±52 136± 9 134±60 130± 17 114±11 153±23 87± 7 32± 5 98±26 142± 12 38±11

Putrescine Metabolism in Sugar Beet Callus 1). ODC activity was also shown to be higher than ADC (Fig. 2). The high ODC activity of the HNO callus could be associated with the provision of greater amounts of ornithine in this tissue (Le Dily et al., 1993). The DAO and PAO oxidase activities were also higher in HNO callus (Fig. 3), with DAO always more active than PAO. Such a relationship berween a higher biosynthesis and higher oxidation of putrescine was also found in other animal (Perin eta!., 1985) and plant (Martin-Tanguy eta!., 1997) tissues. The function of the amine oxidase is possibly as a detoxifYing agent of excessive amounts of putrescine and its conjugates, allowing the recycling of nitrogen and carbon as suggested by Flores and Filner (1985) and Bisbis eta!. (1997). Transglutaminase activities (± Ca) were also higher in HNO than inN callus (Fig. 4) and the content of conjugated polyamines was higher in HNO callus (Table 1). The active polyamine metabolism, especially that of putrescine, was thus higher in HNO callus as evidenced by high anabolism (ODC), catabolism (DAO and PAO), and conjugation (TGases). The addition of polyamines (10- 6 mol!L) to the culture medium increased the growth of the HNO tissue but decreased the polyamine content (Table 2), as was the case when inhibitors of synthesis (DFMO and DFMA) were added. The reduction of active putrescine metabolism by exogenous polyamines and inhibitors of putrescine biosynthesis resulted in an increase of growth in HNO callus. In contrast, the addition of polyamines at mol!L concentration as well as the addition of inhibitors of spermidine and spermine synthesis (MGBG, CHA) had no beneficial effect on growth although the polyamine content was reduced. This question will be further examined in relation to polyamine metabolism. As a conclusion, the actively growing HNO callus was shown to be dependent on its polyamines and especially on its putrescine for its growth through active metabolism (high biosynthesis via ODC, high oxidation via DAO), as was hypothesised. The growth sensitivity of auxin- and cytokininindependent tissues towards polyamines is a new fact. This means and recalls that the term habituation concerns auxins and cytokinins and does not exclude sensitivity to other growth regulators, for instance polyamines. The high turnover of putrescine might be questioned in relation to the high rate of cell division in the habituated tissues. Indeed, considerable evidence now indicates that both polyamine biosynthesis and catabolism are modified in various developmental processes, involving active or reactivation of cell divisions, such as rooting (Hausman et a!., 1997; Martin-Tanguy et a!., 1997) and flowering (Martin-Tanguy eta!., 1996). The question is whether polyamines are involved directly in the control of the cell divisions or through their metabolites (see introduction). In animals, the activity of ODC, which is the only enzyme for putrescine biosynthesis, is strictly regulated during the normal cell cycle: it peaks in G 1 and rises again in G 2/M. Furthermore, expression of this enzyme is transiently increased by growth factors but becomes constitutively activated during cell transformation induced by carcinogens, virus or oncogenes (Auvinen et a!., 1992 and references therein). These authors suggested that the gene encoding ODC is a proto-oncogene involved in regulating cell growth and transformation.

w-s

507

Acknowledgements

The technical help of M. Quoilin and of the Prime and AR 258 personnel (provided to CEDEVIT by the <
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