Effect of Exogenous Arginine, Ornithine, Methionine and γ-Amino Butyric Acid on Maize (Zea Mays L.) Embryogenesis, and Polyamine Content

J Plant Physiol.

Vol. 142. pp. 74-80 (1993)

Effect of Exogenous Arginine, Ornithine, Methionine and -y-Amino Butyric Acid on Maize (Zea Mays L.) Embryogenesis, and Polyamine Content M.

SANTOS*,

I.

CLAPAROLS,

and]. M.

TORNE

Laboratory of Plant Tissue Culture, Department of Agrobiology, Centre d'Investigacio i Desenvolupament de Barcelona (e.S.I.e.), Jordi Girona 18-26, 08034 Barcelona, Spain Received November 25,1992 . Accepted March 5, 1993

•

Summary

The influence of four exogenous amino acids related to polyamine metabolism (-y-aminobutyric acid, arginine, methionine and ornithine) on maize (Zea mays L.) somatic embryogenesis was investigated. The endogenous polyamine contents of the treated calli were analyzed, and arginine and ornithine decarboxylase activities were determined. An established embryogenic callus (Type 1) of the inbred W64Ao2 was used. The endogenous polyamine content of calli was increased by addition of all four amino acids tested and the levels of spermidine plus spermine were higher than those of putrescine in all cases. Upon the addition of 2 mM arginine to the culture medium a 25 % increase in embryogenesis was observed. Moreover, arginine decarboxylase (ADC) activity was significantly improved and ornithine decarboxylase (ODC) activity was also raised. The addition of 1.5 mM ornithine also increased embryogenic callus production, and ODC and ADC activities. Nevertheless, this increase was not as marked as in the case of arginine. This study indicates that the addition of amino acids, which are precursors of polyamine synthesis (especially Arg but also Orn), may be used to improve the rate of embryogenic callus production in an auxin-established maize culture system. A possible explanation of this effect is also dicussed.

Key words: Amino acids; maize embryogenesis; polyamines; Zea mays L. Abbreviations: EC ~ embryogenic callus; NEC = non-embryogenic callus; Arg = arginine; Orn = ornithine; Met = methionine; Gaba = -y-amino butyric acid; PA = polyamines; Put = putrescine; Spd = spermidine; Spm = spermine; DAP = diaminopropane.

Introduction

Somatic embryogenesis in maize can be induced from immature embryos of various genotypes (Hodges et aI., 1986). Given the importance of this technique for molecular studies and genetic manipulation (Gordon-Kamm, 1990), sev" Author to whom correspondence should be addressed. © 1993 by Gustav Fischer Verlag, Stuttgart

eral experiments were carried out in order to maximize this capacity. With regard to organic nitrogen supply, several reports demonstrated stimulation of somatic embryogenesis from maize immature embryos by i-proline (Armstrong and Green, 1985). Trigiano and Conger (1987) reported that a combination of proline and serine stimulated somatic embryogenesis in suspension cultures of Dactylis glomerata. The

Amino acids on maize embryogenesis and polyamines

positive effect of other exogenous amino acids (Asn, Gly and Ser) on the improvement of somatic embryogenesis in a established maize embryogenic system have also been observed (Claparols et al., 1992, in press). The polyamines Put, Spd and Spm were considered as plant growth regulators due to their effects on cell growth, cell division and differentiation at low concentrations (Galston and Kaur-Sawhney, 1987). The biosynthesis of common polyamines can be divided into two main steps: synthesis of Put and formation of Spd and Spm. In higher plants two alternative pathways for Put biosynthesis coexist: 1) direct via ornithine decarboxylase and 2) indirect through a series of intermediates, following arginine decarboxylation. The influence of polyamines and their biosynthetic enzymes (especially arginine and ornithine decarboxylases) on growth rate and morphogenesis in many plants has already been shown (Bagni et al., 1983; Altman et al., 1982; Robie and Minocha (1989). In these plant systems Spd and Spm are generally synthesized from Put and Met. The source of the propylamino group (S-adenosylmethionine) for the biosynthesis of the polyamines Spd and Spm is also the precursor of aminocyclopropane carboxylic acid (ACq, an important source of ethylene in higher plants (Smith, 1990). Ethylene is known as a senescence hormone whereas polyamines promote growth and retard senescence. For this reason, the effect of these two classes of compounds has been thought to be antagonistic (Altman, 1989). Finally, the oxidative degradation of polyamines by amine oxidases proceduces diaminopropane (DAP) and /,-aminobutyric acid (GABA), (Tiburcio et al., 1990). The present paper describes the influence of four exogeneous amino acids related to Put synthesis (Arg and Orn), polyamine degradation (Gaba) or S-adenosylmethionine synthesis (Met) on the maintenance of maize somatic embryogenesis. An established Type 1 embryogenic callus of the W64Ao2) inbred line was used. Furthermore, the endogenous polyamine content (Put, Spd and Spm) and DAP in the treated callus were investigated. Arginine and ornithine decarboxylase activities were also determined.

Material and Methods In vitro culture

For these experiments, an embryogenic cell line of Type 1 maize callus of the inbred W64Ao2 was used (Fig. 1). This compact embryogenic callus (EC) was isolated from certain sectors of an organogenic callus cultured under the conditions previously described (Torne et al., 1984). Embryogenic callus grows together with a friable and non-embryogenic (NEC) tissue that is more or less abundant depending on the medium utilized (see Fig. 1). The EC was cultured routinely on a basal N6 medium (Chu et al., 1975) supplemented by the organic components of the same medium plus 9 11M 2,4-D. The organic nitrogen was supplied with 0.03 mM asparagine, 0.1 mM glycine and 6 mM L-proline. The medium was solidified with 2 gil Gelrite (R) and the pH was adjusted to 5.8 before autoclaving. This medium was considered to be the control medium (C medium, Table 1) for all the experiments. The regeneration capacity of the calli was tested by periodically subculturing a representa-

75

Fig. 1: Embryogenic (EC) and non-embryogenic (NEC) W64Ao2 callus cultured on control medium. Bar = 2 mm.

tive. sample in a MS modified medium (Torne et al., 1984) without aUXIn.

Six replicates each containing 500 to 600 mg of 3 year-old callus were used for each treatment. Calli were cultured in control medium with the addition of the respective amino acid concentrations (see Table 1) and subcultured every 30 days for three consecutive months. At the end of this period, fresh weight of the embryogenic and non-embryogenic fractions of the calli, cultured in the different media, was measured separately in sterile Petri dishes containing disks of filter paper in order ot absorb the gelified medium. The results are presented as percentage of embryogenesis and as total fresh weight of EC and NEC fractions per culture.

Polyamine analysis

Samples were homogeneized in chilled mortars at a ratio of 300 mg fresh weight/mL of 5 % (v/v) cold perchloric acid (PCA). The homogenates were centrifuged at 27,000 g for 20 min, after which aliquots from the supernatant and the pellet fractions (the latter resuspended in IN NaOH) were hydrolyzed with 12N HCl as described by Tiburcio et al. (1985). The unhydrolyzed PCA supernatant containing the free PAs (S fraction), as well as the hydrolyzed PCA supernatant (SH fraction) and the hydrolyzed pellet containing PA liberated from conjugates (PH fraction), were dansylated, solvent-purified, separated by thin layer chromatography (TLC) and quantified using a Perkin-Elmer spectrophotofluorimeter. Three replicates were done for each analysis. Determination ofADC and ODC activities

Samples were extracted as detailed in an earlier report (Tiburcio et al., 1985). For determination of ADC and ODC activities, the reaction mixture consisted of 160 ilL of extract, 20 ilL of dialysis buffer (PH 8.0) and 20 ilL of a solution containing 74 x 104Bq/ mL L-(UYC) arginine or ornithine (11.28 x 109 Bq/mmol and 20.94 x 108 Bq/mmol, respectively) diluted with unlabeled arginine or ornithine to yield a final concentration of 10 mM. The reaction mixture was incubated for 45 min with gentle shaking at 37 oC, after the reaction was stopped by adding 0.2 mL 10 % perchloric acid. Radioactivity was measured by means of 14C02 given off in a scin-

76

M. SANTOS, I. CUPAROLS, and J. M. TORNE

Table 1: Final FW of EC and NEC and percentage of EC production (%E) obtained after 3 months of culture in the different media. C = control medium; G = Gaba media; A = Arg media; M = Met media; 0 = Om media. 1 = 0.25 mM; 2 = 0.5 mM; 3 = 1 mM; 4 = 2 mM. V = vitrified callus. Significance levels (with respect to C medium): *p < 0.05, **p < 0.01, ***p < 0.001. Medium

EC (g)

NEC (g)

Total FW

%E

C

3.89

3.35

7.24

53.72

G1 G2 G3 G4

5.88 3.24 1.71 0.32

4.03 4.14 7.06 2.21

9.91 7.38 8.77 2.53

58.99 43.90* 19.52*** 12.52***

A1 A2 A3 A4

2.81 4.24 5.97 10.70

7.48 5.86 4.55 2.60

10.29 10.10 10.52 13.30

27.31*** 41.96** 56.72 80.44***

M1

0.15 0.42 0.94

2.85 3.01 4.73 3.81

3.00 3.43 5.67 3.81

6.10*** 12.23*** 16.52***

1.59 (V) 1.75 (V) 7.53 5.50 (V)

2.76 5.47 3.76 5.60

4.35 7.22 11.29 11.10

36.58*** 24.24*** 66.69** 49.58

M2

M3 M4 01 02 03

04

A rginine media: The highest percentage of EC production in these media was the one obtained with A4 medium (2mM Arg), which was significantly higher than that of the C medium. The lowest concentration assayed (0.25 mM) produced a significant reduction in EC production with respect to C medium. The increase in EC production was proportionate to the rise in Arg concentration in the medium. In this media, total callus growth was always higher than in C medium and non-embryogenic calli were not abundant. Methionine media: This amino acid had a very negative effect on EC production. The four tested concentrations reduced embryogenesis significantly and produced callus necrosis, which was practically total at 2 mM concentration (M 4 medium). The analyses of EC cultured in Met media are not presented for this reason. Ornithine media: This amino acid produced an increase in total callus growth in 03 and 04 media but only calli obtained in 03 medium (1 mM) were really embryogenic. The other calli were vitrified and non-embryogenic as verified subsequently (data not presented). Polyamine content Total PA contents (S+SH+PH fractions) of EC and NEC cultured in the different media are presented in Figs. 2 and 3. Table 2 shows Put/Spd+Spm ratios.

tillation counter Beckman LS 6000 Sc. Three replicates were down for each analysis. Protein content

The total protein content of calli was determined according to Bradford (1976) using bovine gamma-globulin (Sigma) as a standard.

Embryogenic calli It may be observed that EC growing in C medium (see Figs. 2 and 3) was characterized by similar DAP, Put and Spd contents. The Put/Spd+Spm ratio (Table 2) was 0.505, indicating that the content of Spd + Spm was approximately double that of the Put content.

Statistical analyses

Statistical assays were performed by the Student's t-test, with six calli for each treatment. Values lower than 0.05 % were considered to be significant. Results

Embryogenesis and callus growth Production of EC and significance levels of the different media with respect to C medium are given in Table. 1. The highest percentage of embryogenic callus production between the 17 assayed media was obtained in A4 medium (80.4%), followed by 03 (66.6%) and G1 (58.9%). Control medium presented 53.7 % of the EC production. In general, the increases in total callus growth (g FW) were directly related to embryogenesis, the most embryogenic media having higher total callus growth than C medium. Gaba media: Between the four Gaba concentrations assayed, 0.25 mM (G 1 medium) was the only concentration with respect to EC production that showed a slight increase with respect to C medium. Higher Gaba concentrations produced a significant reduction in EC production. Callus growth was similar or slightly higher than in C medium. In general, friable and non-embryogenic calli were abundant.

Gaba media The levels of Spd and Spm with respect to G 1 and G 2 media were always significantly higher than in C medium (see Table 2: Put/Spd+Spm ratios of the embryogenic and nonembryogenic calli cultured on the different media. Put/Spd + Spm

Media C

G1 G2 G3 G4 A1 A2 A3 A4

M1

Embryogenic

Nonembryogenic

0.505 0.348 0.454 0.458

0.489 0.363 0.511 0.340 0.615 0.508 0.557 0.377 0.363 0.401 0.741 1.260 1.160 0.567 0.454 0.507 0.346

0.506 0.696 0.591 0.531

M2

M3 M4 01 02 03

04

0.662 0.703 0.470 0.499

Amino acids on maize embryogenesis and polyamines 1400

nEC

nmol. PAa/glw

1400

12 00

12 00

1000

1000

EC

nmols PAs / gl"

77

CJ

DIp

~ Put _ Spd _Spm

"Total. 800

800

600

60 0

400

400

200

200

C

1600

QABA 1

nmol. PAs/gfw

GABA 2

GAltA 3

GABA ..

Media

nEC

C

16 0 0

1400

14 00

1200

12 00

1000

100 0

800

800

600

6 00

400

400

200

2 00

c

Arg 1

"ra

2

Mg3

Afa -4

Media

D r-ol.

GABA 3

QABA 2

GAB" 1

Media

EC

nmol. PAs / gfw

c=J DIP ~ Put

Il!ffi

Sp d

Il!ffi

Spm

_Tot.,. c=J r.oIP

c

Arg 1

Ara 2

Arg 3

Arg"

Media

Fig. 2: Total PA content (S+SH+PH fractions) of EC and NEC cultured on Gaba and Arg media. Bars represent SE of 9 replicates.

Fig. 2). PA variations were less marked in G3 medium but also significantly higher than in C medium. The level of Put in Gl medium (the most embryogenic one) decreased slowly with respect to C medium (non-significant). The Put level was increased in G2 and G3 media but it was only significant in G2 medium. DAP content in Gl and G2 media was significantly lower than that of C medium. The Put/Spd + Spm ratio was slightly lower than that of C medium in all the G media (see Table2).

Arg media All of the Arg doses utilized increased slightly the Put, Spd and Spm contents in the EC, although the significance level varied (see Fig. 2). The level of DAP was always significantly lower than that of C medium, except for A4 medium. The Put/Spd+Spm ratio in A4 medium (the most embryogenic one) was similar to C medium (see Table2).

Met media This amino acid produced alterations in all of the doses utilized and the production of EC was minimal, as explained

above. The PA analysis of this type of callus was not possible for this reason.

Om media All of the Om concentrations tested produced a significant increase in total PAs with respect to C medium (see Fig. 3), although this increase was less marked in 04 medium. DAP content in Om media was irregular, being in 03 and 04 more similar to that of C medium. The Put/Spd + Spm ratio in the best Om medium (03) was slightly different from that of C medium (see Table 2).

Non embryogenic calli The NEC cultured in C medium had 50 % of the total PA content with respect to that of EC cultured in the same medium (see Figs. 2 and 3). However, the Put/Spd+Spm ratio of NEC was similar to that of the EC, both cultured in C medium, indicating that the three polyamines were reduced proportionally with respect to that of EC. In most cases, this ratio was lower in the treated-NEC than in the treated-EC (see Table 2). Moreover, it may be observed (Figs. 2 and 3) that NEC-PA content in all of the tested Gaba, Arg and Om media was higher than that of C medium. These increases

78 2000

' ',r'" -",.

M. SANTOS, I. CLAPAROlS, and]. M. TORNE nmol. PAs/glw

nEG

1800

o

1800

Spd

1400

D

Spm

1200

_Tot.11

1600

1400 1200 1000

1000

"'J

.., j

800 600 400

400

200

DIp

~ Put

_

1600

200 i

O

T.Cap

,I

~J

a ] -<~

Orn 1

C

HOO

EG

8

PA./glw

Orn 2

Orn 3

Orn"

Media

Me' 3

Me' 4

Media

c

Orn 1

Orn 2

Orn 3

Orn 4

Media

nEG

2200 2000 1800 1600 1400 1200 1000 800 600 400 200

c

Met 1

Met 2

Table 3: Enzymatic activities (pmolsC02 h- 1 mg- I prot) and protein content (mgprot g-I fw) of calluses cultured in C, A4 and 03 media. Medium

Control (EC)

Control (NEC)

A4

ADC activity ODC activity

64.9± 9.9 1355.6±39.7 0.4± 0.03

29.5± 3.6* 438.8±S3.S** O.S± 0.01

162.9±11.0** 95.9± 8.1 1730.1 ±61.4* 1526.8 ± 40.5* O.S± 0.03 0.4± 0.02

Protein content

03

Fig.3: Total PA content (S+SH+PH fractions) of EC and NEC cultured on Om and Met media. Bars represent SE of 9 replicates. crease over the control and ODC activity was also significantly raised. When the medium was supplemented with 1.5 mM of Orn (03 medium) ODC and ADC activities increased. However, in this case, only ODC activity was significantly higher than that of the control. Total protein content remained stable in all cases.

• p
Discussion

were not very significant in the M 1 and M2 but highly significant in M3 and M4 (up to 6-fold that of C medium), Put content being the most noteworthy. This pattern was reproduced in the other media, although the increases were less marked than those of certain Met media. Enzymatic activities

Table 3 expresses the in vivo ADC and ODC activities and the protein content of the calli cultured in the most embryogenic media (A4 and 03). Enzymatic activities of EC and NEC cultured in C medium are also included. As may be seen, ODC activity was markedly higher than ADC in all cases. Both activities were significantly higher in EC than in NEC when both were cultured in C medium. Moreover, when the medium was supplemented with 2 mM of Arg (A 4 medium), ADC activity of the EC showed a significant in-

With respect to the organic nitrogen supply, the positive effect of 2 mM Arg (A4) on EC production (25 % embryogenesis over C medium) should be pointed out. The fresh weight of EC was also increased when compared with that of NEC, when the doses of Arg were enhanced. Moreover, the Nred/NOr ratio in A4 medium was higher (0.7) than that of all other media (from 0.49 to 0.56), including control medium (0.47) (data not presented), and this ratio may be important for enhancing nitrogen assimilation by the calli cultured in this medium. It is well known that Arg is a beneficial nitrogen source for protein biosynthesis. It has been reported that Arg accelerated division of giant ChIorella cells in heterotrophic cultures (Thinh and Griffiths, 1974). Moreover, Arg stimulated spikelet development in cultured maize tassels (Pareddy and Greyson, 1989). In addition to providing a high level of reduced nitrogen, Arg might playa role in plant development by conversion through Put to PAs,

Amino acids on maize embryogenesis and polyamines

which have been implicated in regulation of cell growth and development (Slocum et aI., 1984). The in vivo ADC activities detected in the calli cultured in 2 mM Arg-medium indicate that this enzyme is activated by exogenous Arg with a simultaneous increase in embryogenesis. Moreover, ADC activity of EC is higher than that of NEC, both being cultured in C medium. These effects corroborate the fact that ADC could be implicated in maize embryogenesis, as has been demonstrated in other plant species (Feirer et aI., 1984; Mengoli et aI., 1989). The positive effect of l.SmM Orn (03) on EC production should be noted. Furthermore, ODC activity in this medium increases with respect to that of C medium. However, this increase is not as marked as in the case of Arg. Although increasing doses of Arg enhanced embryogenesis proportionally, this phenomenon does not occur in the case of Orn (see Table 1). As regards ODC, it should be noted that this enzyme is especially active in our embryogenic calli, its activity being always higher than that of ADC. Results similar to those of the present study were obtained in maize organogenic calli cultured in a medium with 2,4-D (Torne et aI., in preparation). Hiatt (1989), investigating the initiation of PA synthesis with a maize cell line and using a hormone-medium, also observed that ODC activities were higher than ADC activities. He attributed this phenomenon to a different subcellular compartmentation of ODC and ADC. The latest evidence (Galston and Sawhney, 1990) suggests that ODC is localized in DNA-containing organelles whereas ADC is cytosolie. Despite the few references involving ODC and embryogenesis, EI-Hadrami et al. (1992), using Hevea brasilensis, obtained a significant decrease in embryogenesis after inhibition with DFMO (suicide inhibitor of ODC activity). The observed increase in ODC activity associated with the addition of 2 mM Arg might be attributed to a reduction in the activity of the metabolic citrulline pathway, which converts Orn to Arg. In higher plants, it is well known that the conversion of Orn to Arg is necessary for Arg formation (Thompson, 1980). In our case, the exogenous addition of Arg could reduce this conversion and accumulate Orn, resulting in the activation of the ODC pathway. All of these data could indicate that ADC and ODC pathways are differently involved in the embryogenic processes. With respect to the influence of Gaba in embryogenesis, a decrease in EC production was observed. This decrease is related to the increase in the concentration of this amino acid in the medium. This phenomenon could be associated with the fact that Gaba is a polyamine degradation product whose accumulation in the medium may interfere with the adequate functioning of the PA synthesis pathways. In the case of Met, the marked decrease in EC production at all the doses tested may be attributed to a senescence phenomenon induced by this amino acid, which is also associated with ethylene synthesis. The negative influence of ethylene on maize embryogenesis has already been shown (Vain et al., 1989). The influence of callus age on embryogenesis must also be considered. The maize EC used in the present work were 3 years old at the time of the experiment and their embryogenic potential was lower than that of younger maize EC

79

proceeding from the same cell-line utilized before (Claparols et aI., 1992, in press). The rejuvenation effect of DFMA (suicide inhibitor of ADC activity) pretreatments on maize organogenic calli has been previously demonstrated (Tiburcio et aI., 1991). This effect consisted in increasing the regeneration capacity of the inhibitor-pretreated-calli with respect to that of the non-treated calli and it was accompanied by an increase in PA synthesis. The raised embryogenic callus production and PA synthesis induced by exogenous Arg in this experiment could be related to a similar rejuvenation phenomenon. It has been suggested that PAs may act as antisenescent compounds due to their rapid binding to the membranes (Altman et al., 1977). The effect of PAs interfering with ethylene biosynthesis at the membrane level has also been shown (Altman et aI., 1982; Slocum et aI., 1984). Previous studies carried out on carrot (Feirer et aI., 1984; Mengoli et aI., 1989), woody species (El Hadrami et aI., 1989) and on Vitis (Faure et aI., 1991) have shown that somatic embryogenesis is closely related to high levels of PAs. These results agree with the higher PA content observed in the control-EC (approximately the double) with respect to that of the control-NEC in our experiment. As may be expected, the addition of a PA precursor increases the endogenous PA content of the calli in all cases, but EC production is only incremented in A4 and 03 medium, where the Put/Spd+Spm ratio is similar to that of C medium. These observations suggest that not only an increase in total PA synthesis but also a high level of Spd + Spm could encourage maize embryogenesis. Nevertheless, the importance of Put/Spd + Spm ratio in embryogenesis has not yet been elucidated. In our maize EC cultured in the control medium, this ratio is less than one. This value is also obtained in the non-embryogenic part of the callus, although in this case total PAs are lower than in EC. In conclusion, the results presented here indicate that the addition of amino acids, which are precurcors of PA synthesis (especially Arg but also Orn), at suitable concentrations may be used to increase the rate of embryogenic callus production in an auxin-established maize culture system. The involvement of the enzymes ADC and ODC in this phenomenon should also be noted. Acknowledgements

This study was financed by a grant from CICYT: BIO 88-215.

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M. SANTOS, I. CLAPAROLS, and]. M. Tow

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