Polyamine Pattern and Biosynthesis in Zygotic and Somatic Embryo Stages of Vitis vinifera

J.PlantPhysiol. Vol. 138.pp. 545-549(1991}

Polyamine Pattern and Biosynthesis in Zygotic and Somatic Embryo Stages of Vitis vinifera OLIVIER FAURE', MARISA MENGOLI I

2

2

,

ARLETTE NOUGAREDE',

and NELLO BAGNI2

Laboratoire de C ytologie experimentale et morphogenese vegetale, Universite Pierre et Marie Curie, Bk N2, 4,place Jussieu, F-75252 Paris Cedex 05 (France) Universita di Bologna, Dipartimento di Biologia e. s., Via Irnerio 42, 40126 Bologna (ltalia)

Received D ecember 28,1990 . Accepted April 15, 1991

Summary A comparative study of free polyamine levels, arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) activities was carried out in the different stages of somatic embryos of Vitis vinifera cv. Grenache noir, and in zygotic embryos of the same cultivar, before and after germination. Somatic embryos showed a high level of free polyamines and a high putrescine/spermidine ratio. On a per unit basis, an accumulation of putrescine (Put) and spermidine (Spd) occurred in the late torpedo stage, which coincided with the beginning of abnormal growth and disorganized cell proliferation. In globular, heart-shaped and torpedo somatic embryos, ADC activity was higher than ODC activity. Later, in giant somatic embryos, a very high ADC and an even higher ODC activity occurred, except when expressed on a dry weight basis. By contrast, zygotic embryos showed a low level of free polyamines and a Put/Spd ratio approximately equal to one. These data suggest that the abnormal behaviour of grape somatic embryos and their low rate of development into plantlets « 5 %) could be due to the high free polyamine content and/or to an inadequate Put/Spd ratio.

Key words: Vitis vinifera, arginine decarboxylase, ornithine decarboxylase, polyamines, somatic embryo, zygotic embryo. Abbreviations: ADC = arginine decarboxylase; BAP = benzylaminopurine; 2,4-D = 2,4-dichlorophenoxyacetic acid; DTT = dithiothreitol; ODC = ornithine decarboxylase; Put = putrescine; Spd = spermidine; TCA = trichloroacetic acid; TLC = thin layer chromatography. Introduction In grape, somatic embryos can be produced with high frequency (Srinivasan and Mullins, 1980; Bouquet et al., 1982) but difficulties arise when attempting to obtain a high number of plantlets. Somatic embryos of grape are strongly teratologic (Gray and Mortensen, 1987; Faure, 1990) and often have a very low conversion rate into plantlets (Bouquet et al., 1987; Lebrun and Branchard, 1987). As they are unable to achieve the transition from embryogenic development to «germination», a great number of these embryos show an accentuated growth, resulting in the formation of giant organs (hypocotyl, cotyledons) and in the disorganized cell proliferation of their superficial cells (Faure, 1990). © '99' by G ust av F si cher Verlag, Stutt gart

Among the different factors that could be involved in this proliferation and abnormal growth, growth regulators and particularly polyamines (Slocum et aI., 1984; Smith, 1985; Evans and Malmberg, 1989) could playa major role. The content and synthesis of the three main polyamines (putrescine, spermidine and spermine) have been studied during somatic embryogenesis in carrot (Montague et al., 1978; 1979; Fienberg et al., 1984; Mengoli et al., 1989; Robie and Minocha, 1989; Altman et al., 1990); they have also been determined during the development and germination of zygotic embryos in other species (Bagni, 1970; Sen et al., 1981). Up to now, however, only one comparative study (Litz and Schaffer, 1987) has been carried out on zygotic and somatic embryos of the same species, i.e. Mangifera indica L.

546

OLIVIER FAURE, MARISA MENGOLI, ARLETTE NOUGAREDE, and NELLO BAGNI

Arginine (EC 4.1.1.19) and ornithine decarboxylase (EC 4.1.1.17) activity assay The method used for the enzyme assays has been described previously (Bagni and Mengoli, 1985). The extraction medium consisted of 100 mM Tris-HCI (PH 8.3) buffer, containing 50 pM ethylenediaminetetraacetic acid (EDTA), 25 pM pyridoxal phosphate and 2.5 mM dithiothreitol (DTT). ODC and ADC activities were determined by incubating 0.3 mL aliquots of the 26,000 g supernatant or of the resuspended and sonicated pellet for 60 min with 14.8 kBq in 24 pL [1-14C] ornithine (1.998 GBq mmol- 1, Amersham, England) or [1-14C] arginine (1.698 GBq mmol- I , CEA, France), respectively. Cold ornithine or arginine (13.5 mM) were added to the incubation medium.

Protein determination and replication of experiments Fig. 1: Different stages of development in Vitis vinifera cv. Grenache noir somatic embryos (globular, G; heart-shaped, H; torpedo, T; further developmental stages, T + and giant). Scale bar = 500 pm.

Materials and Methods

Proteins of somatic and zygotic embryos (or of the 26,000 g supernatant and pellet separately) were measured using the Bradford method (Bradford, 1976). In fact the DTT present in the extraction buffer did not interfere with this assay, as it did with the Lowry method. All measurements were performed at least in triplicate. Results are given as means with standard errors calculated from three to five replicates.

Plant material Somatic embryos were obtained from callus derived from the connective of cultured anthers of Vitis vinifera cv. Grenache noir cl 6662 GF, according to the method of Rajasekaran and Mullins (1979). One hundred yellow pale translucent anthers (microspore stage) were cultivated for 1 month in 100 mL Erlenmeyer flasks containing 25mL of Nitsch and Nitsch (1969) liquid medium supplemented with 4.5 p.M 2,4-D and 1.1 pM BAP. The cultures were placed at 24 ± 1 °C in the dark and were shaken at 100 rpm. Calli were then transferred to the same basal medium without growth regulators, kept in the light (16 h illumination regime, 3 W m - 2) and subcultured weekly to fresh medium. After 4 - 6 weeks, each culture contained more than one thousand embryos in different developmental stages. The somatic embryos were sampled manually and separated under a dissecting microscope into five characteristic developmental phases (Fig. 1): globular (G), heart-shaped (H), torpedo (T, 1 to 1.5 mm long) and two further stages called torpedo + (T + , 2 to 4 mm long) and giant (5 to 10 mm long). The conversion rate of torpedo and torpedo + embryos into plandets was less than 5 %. Zygotic embryos were extracted from stratified (3 months at 4°C) mature seeds and their germination rate was more than 95 %. For the obtention of germinated zygotic embryos, seeds were placed on moist filter paper in Petri dishes at 24°C in darkness. The embryos were sampled at the time of radicle emergence. All samples were immediately frozen with liquid nitrogen, freeze dried and stored in darkness until needed.

Assay ofpolyamine content The somatic and zygotic embryo stages were homogenized in a mortar with liquid nitrogen in 5 % TCA and the extracts were centrifuged for 15 min at 1,500 g. Free polyamines were dansylated according to Smith and Best (1977) and separated by using TLC precoated plates of silicagel 60 with a concentrating zone (Merck). Cyclohexane: ethylacetate (3: 2, v/v) was used as solvent. Spots, visualized under UV light, were scraped off, eluted in 2 mL acetone and their fluorescence measured using a Jasco FP-550 spectrofluorimeter (excitation 360 nm, emission 506 nm). The results were compared with standards dansylated in the same way.

Results

In all the samples examined, conjugated polyamines were not detectable either in the TCA-soluble or -insoluble fraction. The results presented here are, therefore, referred to as free polyamines. Changes in polyamine content during the development of zygotic and somatic embryos were expressed on a dry weight and protein content basis, and on a per embryo basis (unit-I). In stratified and germinated zygotic embryos, the polyamine content (Fig. 2 a, 2 b, 2 c) was relatively low. In both cases the Put/Spd ratio was approximately equal to one, whereas the content of spermine was about 3 to 4 times lower than that of putrescine or spermidine. Polyamine content expressed on a per unit basis (Fig. 2 a) increased from stratified to germinated zygotic embryos. In germinated zygotic embryos the content of the three polyamines was 6-fold higher than that of stratified zygotic embryos. However, when expressed on a basis of protein content (Fig. 2 b) or dry weight (Fig. 2 c), the polyamine content was not significantly different. In these last two cases it must be emphasized that protein content and dry weight were both 6-fold higher in germinated zygotic embryos than in stratified zygotic embryos. When compared with zygotic embryos, somatic embryos had a very high polyamine (Fig. 2 a, 2 b, 2 c) and, particularly, putrescine content. The Put/Spd ratio was always higher than in zygotic embryos; it progressively decreased from the globular stage (Put/Spd) = 29) to the giant stage (Put/Spd =

6).

When expressed on a per unit basis (Fig. 2 a), polyamine content did not change from globular to heart-shaped stages but it dramatically increased from heart-shaped to torpedo and from torpedo to the later developmental stages.

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In line with the results on polyamine content, ADC and ODC activities expressed on a per unit basis (Fig. 3 a) increased during the progression from globular to torpedo stages, and even more in giant embryos. When expressed on a protein content basis (Fig. 3 b), total ADC and ODC activities decreased from globular to heartshaped stages, but these decreases were due to an increase in protein content. On the other hand, asharp increase in both ADC and ODC activities and protein content was observed in the torpedo stage. This increase in enzymatic activities preceded the increase in polyamine levels found in the T + stage (Fig. 2 b). Finally, a dramatic peak in ADC, and even more in ODC activities, characterized the giant embryos . When expressed on a dry weight basis (Fig. 3 c), ADC and ODC activities decreased during the progression from the globular to the torpedo to the giant stage. The decrease observed in the first three stages was correlated to the decrease in polyamine content (Fig. 2 c) but both were due to a marked increase in dry weight. Moreover, in giant embryos the high polyamine levels, in spite of the dramatic decrease in ODC and ADC activities, could be due to an accumulation phenomenon and (or) a decrease of diamine oxidase activity. Since the percentage of water in the various embryo stages was practically the same (data not shown), a similar trend

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Fig. 2: Free polyamine content (putrescine, _; spermidine, (~); spermine, D) in stratified (St) and germinated (Ger) zygotic embryos of Vitis vinifera cv. Grenache noir and in the different stages of somatic embryos of the same cultivar (globular, G; heartshaped, H; torpedo, T; further stages of development, T+ and Giant). Results expressed as a function of (a) one unit, (b) protein content, (c) dry weight. In b, (0), Ilg of protein per unit; in c, (D), Ilg of dry weight per unit. (SE were omitted when their values were too small for the scales used).

Conversely, when expressed on a protein content basis (Fig. 2 b), polyamine levels decreased from the globular to the heart-shaped stage and remained unaltered from heartshaped to torpedo embryos. However, this apparent decrease in polyamine level between the globular and torpedo stages was only due to a significant increase in protein content, respectively 2- and 4-fold higher in torpedo than in heart-shaped and globular somatic embryos. In the T + embryos, a dramatic increase in both polyamine and protein contents was found; this fact clearly indicated that synthesis and accumulation of polyamines had occurred. In the giant embryos, a decrease in polyamine content, due to a sharp increase in protein content, was observed. Arginine- (ADC) and ornithine decarboxylase (ODC), the two major enzymes involved in putrescine synthesis, displayed higher activities in the 26,000 g supernatant than in the pellet (data not shown). In the first three stages (Fig. 3), ADC activity was always higher than ODC activity and the ADC/ODC ratio progressively increased from the globular (ADC/ODC = 2) to the heart-shaped (ADC/ODC = 3) and finally to the torpedo stage (ADC/ODC = 4). On the contrary, in giant embryos ODC activity was higher than ADC.

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548

OLIVIER FAURE, MARISA MENGOLI, ARLETIE NOUGAREDE, and NELLO BAGNI

was obtained when the results were calculated on a fresh weight basis.

Discussion The present study is the first one dealing with a comparison between polyamine content in zygotic embryos and somatic embryos of grape. It is noteworthy that somatic embryos of Vitis vini/era cv. Grenache noir are characterized by a very high level of polyamines and by a high Put/Spd ratio. Previous studies carried out on the carrot model (Montague et aI., 1978; Feirer et al., 1984; Mengoli et aI., 1989) and in woody species (El Hadrami et aI., 1989 a and b) have also shown that the occurrence of somatic embryos is closely correlated with high levels of polyamines. However, the importance of the Put/Spd ratio in embryogenesis is as yet unknown. In the present study we found a progressive decline in the Put/Spd ratio, from the globular to the giant stage. In line with these results, Feirer et aI. (1984) reported a higher level of putrescine than of spermidine in embryogenic cell cultures of carrot, but the different stages of embryonic development were not studied. In the same type of culture, however, Montague et al. (1978) found much more spermidine than putrescine. Finally, studying the different stages of carrot somatic embryos, Mengoli et aI. (1989) found more putrescine than spermidine in the heart-shaped stage and more spermidine than putrescine in the torpedo stage. In our study, a pronounced increase of putrescine and spermidine levels occurred in the T + stage (Fig. 2 a, b, c). This stage of development coincided with the beginning of abnormal growth and disorganized cell proliferation, which characterizes somatic embryos of grape (Faure, 1990). This abnormal behaviour can be compared with that of plant cell tumors in which polyamine levels, particularly putrescine and spermidine, are unusually high (Audisio et aI., 1976; Bagni and Serafini-Fracassini, 1979). It must also be emphasized that accumulation of putrescine seems to occur in response to various stresses, notably potassium (Klinger et al., 1986) and magnesium deficiencies (Smith, 1984) or ammonium ion excess (Le Redulier and Goas, 1975). Thus, it could be supposed that in an inadequate mineral medium somatic embryos might be stressed and react by accumulating putrescine, leading to excessive cell proliferation. In the first three stages of grape somatic embryo development, our results on ADC and ODC activities were consistent with those of Montague et al. (1979) and Feirer et aI. (1984) in embryogenic cultures of carrot. They differ from those of Mengoli et al. (1989) who reported that in the different stages of carrot somatic embryos, ADC and ODC activities were similar but decreased from globular to torpedo stages. The possibility that a strong arginase activity might have affected the evaluation of ADC activity by conversion of arginine to ornithine was checked. Cold ornithine was added to the enzyme assay at a 10-fold higher concentration than that of the arginine used for the determination of ADC activity. Arginase activity, determined in the supernatant fraction of grape giant embryos, accounted for 38 % of the total CO2 released. Thus the ADC/ODC ratio turns out to

be lower, and this is more in line with the data obtained by Mengoli et aI. (1989), who also checked arginase activity, but only in the pre-embryogenic phase. Unlike somatic embryos, zygotic embryos of grape displayed a low level of polyamines and a Put/Spd ratio approximatively equal to one. However, torpedo somatic embryos and stratified zygotic embryos were morphologically in the same developmental stage. These two types of embryos differed mainly in their germination rate and in their polyamine content; putrescine was 22-fold higher, spermidine 2.5-fold higher and spermine 1.5-fold higher in torpedo somatic embryos than in stratified zygotic embryos. Thus, it could be proposed that the abnormal behaviour of somatic embryos and their low rate of conversion into plantlets were due to an excessively high polyamine content and/or an inadequate Put/Spd ratio. In fact, it has been shown that treatments that decrease polyamine levels, like the addition of ODC inhibitors (Mengoli et aI., 1989) or treatments that modify the Put/Spd ratio, such as the addition of spermidine (Altman et aI., 1990), increased the conversion rate of somatic embryos. Moreover, the addition of abscisic acid, which inhibits ADC activity in Cucumis sp. zygotic embryos (Suresh et aI., 1978), also increases the conversion rate of somatic embryos into plantlets (Ammirato, 1977; Dunstan et aI., 1988; Prado and Berville, 1990; Roberts et aI., 1990). The results presented here open the way to a less empirical study of some treatments that could enhance plantlet formation from somatic embryos of grape. Some encouraging results have already been obtained in our laboratory by using inhibitors of polyamine synthesis.

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