Biochem. Physiol. Pflanzen 173, 160-166 (1978)
Metabolism of Ageing Seed: Glutamic Acid Decarboxylase and Succinic Semialdehyde Dehydrogenase Activity of Aged Wheat Embryos L. GALLES CHI and C. FLORIS Institute of Botany, University of Pisa, Pisa, Italy Key Term Index: ageing seeds, ripening conditions, glutamic acid decarboxylase, succinic semialdehyde dehydrogenase; Triticum durum.
Summary The GAD1) and SSA-DH activities of embryos from progressive aged lots of wheat seeds were compared and correlated to germination capacity of seeds. GAD activity was also studied on old seeds having different germination capacity and provenience (dry and wet ripening conditions). The behaviour of the enzymes during seed ageing is quite different: the level of GAD activity is high up to the 10th year of age while the SSA-DH activity, high in the 1 and 2 years old embryos, declines after 3 years of age. The GAD appears to be negatively influenced by wet ripening conditions of seeds. The importance of these enzymes in the glutamate metabolism and their possible influence during the life-span of seed in relation to energy-dependent processes are discussed.
Considerable attention has been given to the changes that occur when seeds pass from the viable to the non viable state. Storage of seed and its conditions are the cause of different changes in ageing seeds which have been determined by physiological (D'AMATO and HOFFMANN-OSTENHOF 1956; ROBERTS 1961; HARRISON 1966; FLORIS 1970; AsPINALL and PALEG 1971) citological (D'AMATO 1964; ORLOVA and NIKITINA 1968; KAUL 1969; INNOCENTI and AVANZI 1971; FLORIS and ANGUILLESI 1974) genetical (GISQUET et al. 1951; SAX 1962; FLORIS and MELETTI 1972) and biochemical parameters (LINKO and SOGN 1960; CHING and SCHOOLCRAFT 1968; KOOSTRA and HARRINGTON 1969; GRZESIUK and KULKA 1971; CHING 1972; HALLAM et al. 1972; ANGUILLESI et al. 1974). The study of enzymatic activities of natural aged seeds is of primary interest for the possible correlations between enzymatic changes and the reduction or loss of germination capacity. Some enzymes have been considered, amylase (FRENCH 1959; ASPINALL and PALEG 1971), catalase (CROCHER et al. 1918), dehydrogenase (ASPINALL and PALEG 1971), glutamic acid decarboxylase (LINKO and SOGN 1960; GRABE 1964), but no many studies treat the enzymatic activity of progressively ageing seeds, up to the loss of germination capacity, or the alternative metabolic pathways which, during the ageing process, could be utilized. With this in mind, our problema area is to study the 1) Following abbreviations are used in the text: GAD = glutamic acid decarboxylase; GAB A = y-aminobutyric acid; GABA-T = y-aminobutyric acid transaminase; SSA = succinic semialdehyde; SSA-DH = succinic semialdehyde dehydrogenase.
GAD and SSA-DH Activity of ageing wheat seeds
glutamic acid metabolism during the seed life-span in relation to the particular glutamate pathway known as GABA-shunt. This pathway utilizes glutamic acid decarboxylation, catalyzed by GAD (EO 126.96.36.199), and giving GABA which, in turn, is transaminated with IX-ketoglutaric acid. This reaction is catalyzed by GABA-T (EO 188.8.131.52) to give glutamic acid and SSA and is followed by the oxidation of SSA, catalyzed by SSA-DR (EO 184.108.40.206). The present research has the object to investigate the GAD and SSA-DR activities of ageing wheat seeds with the aim to correlate these activities with the progressive ageing and to analyze the consequence of different ripening condition of seeds in this regard. Material and Methods Triticum durum c.v. Cappelli stock seeds from different crops (harvested in the year 1976, 1975, 1974,1973, 1972, 1971, 1970, 1969, 1968, 1967, 1966, 1965, 1964, 1963, 1961) were utilized. Storage of all seeds was carried out into sealed glass containers at 10°C in the dark and isolated embryos, excised from dry seeds sterilized in 1 % NaClO, were utilized as enzymatic source. Germination capacity was evaluated on samples of 50 seeds (3 replicates) allowed to imbibe on water moistened filter paper at 23°C in the dark. Dry seeds used for germination assay were before sterilized by 1 % NaClO for 10 min and repeatedly washed in deionized water. GAD was extracted and evaluated as previously described (GALLESCHI et al. 1975, 1976). The SSA-DH was determined from homogenates of ground wheat embryos suspended in 0.1 M potassium phosphate buffer (pH 7.35) containing 0.014 M mercaptoethanol. The homogenates were centrifuged (3,500 . g for 10 min) at 4°C and supernatant was precipitated with 40 % liaturation with (NH4)2S04 (10,000 . g for 10 min). This precipitate was discarded and the following precipitate at 60 % of saturation was dissolved in 0.1 M tris-HCl buffer (pH 9.0) and used for enzymatic assay. The SSA-DH was determined spectrophotometrically at 340 nm; the reaction mixture (in a total volume of 1 ml) contained NAD (l,umole), 2-mercaptoethanol (3,umoles), SSA (0.5 ,umoles), tris-HCl buffer pH 9.0 (80,umoles) and an appropriate amount of enzyme extract. Proteins were evaluated by biuret method. GAD activity was also evaluated during the early germination of same age old seeds which differ for provenience and germination capacity at 3 days [Pisa (PI) seeds = 26 %; Cagliari (CA) seeds = 96 %]. The seeds were allowed to imbibe, up to 30 h, on distilled water moistened paper, in the dark, and samples were colle.cted at different times to have isolated embryos as enzymatic source.
Germination assay, carried out to evaluate germination capacity of seeds to be utilized for extraction procedure, shows (Fig. 1) that seed viability, when seeds are stored at 10 °0, remains high up to the 10th year of age after which it declines strongly. Germination capacity of 12 years old seeds and the following ones is about 0 % in our storage conditions. GAD activity evaluated on dry seeds (Fig. 2) is constant during the 10 years of cold storage and its values are high; in the more old seeds, between the 10th and the 11th year of age, this enzymatic activity declines of about 5 fold and reachs very low values in the oldest seeds. Oomparative studies of GAD activity of embryos from the same age seeds but differing for provenience and germination capacity, when evaluated during the 30 h 11 Bioohem. Physio!. Pflanzen, Bd. 173
of imbibition, show that the seeds with high germination capacity (CA seeds) exhibit a high GAD activity during the initial 24 h, which decreases at 30 h; on the contrary, the seeds with low germination capacity (PI seeds) exhibit a very low GAD activity which remains constant during all the 30 h considered (Fig. 3).
III .f seed (JUII)
Fig. 1. Germination capacity (%), at 3 days, of Triticum durum seeds.
lie of seed(yulI)
Fig. 2. GAD specific activity of embryos from aged seeds of Triticum durum.
GAD and SSA-DH Activity of ageing wheat seeds
The SSA-DH activity of embryos from seeds of increasing age is high in the 1 year old seeds, decreases in the second year of ageing and reduces strongly in the 4 years old seeds. Seeds from 5 to 16 years of age exhibit a constant, very low, SSA-DH activity (Fig. 4). 12 11
""" - - 1 - T ------I-------.t\ ~----
\ \\ \ ''y
time ,I illbibition (hull)
Fig. 3. GAD specific activity of wheat embryos during 30 h of imbibition of Triticum durum 1966 seeds from Gagliari (- - -) and Pisa (--).
Fig. 4. SSA-DH specific activity of embryos from seeds of Triticum durum. 11*
Germination behaviour may be considered an expression of a lot of structural and functional changes which occur in the seed during its life-span. Storage conditions, in particular adverse storage of seed at high temperature and moisture content as well as many factors of external environment during ripening of seeds, can modify the course of these changes and piay a significant role in affecting the viability of seeds. Since these factors influence the intermediary metabolism, many studies on enzymatic activity during the seed ageing were carried out. ASPINALL et al. (1972) showed a decline of IX-amylase activity in endosperms of aged wheat seeds which, reduced to low levels in 7 years old seeds, may be restored to young seed level by adding gibberellic acid .. Biochemical lesions in the protein-synthesising system in rye (ROBERTS and OSBORNE 1973), pea (BRAY' and C;HOW 1976), the loss of albumins of high molecular weight in oat (GRzESIUK and KULKA 1971) and the reduction of polyamines in wheat embryos (ANGUILLESI and FLORIS 1975) were demonstrated to be associated with a decline or loss of seed viability. In our material the GAD activity, which is constant during the initial 10 years, reduces significantly and remains at very low values in the following years. The SSADH activity seems to be more sensitive to the progress of ageing: it reduces in the 2nd year of storage reaching very low values at the 5th year of seed life. By this, the GAD activity appears to be positively correlated to germination in terms of loss of germination capacity while no strict correlation appears to exist as to SSA-DH activity, which, clearly, is rapidly reduced or lost as a consequence of the changes occurring in the early period of storage. LINKO et al. (1960) reported differences in the GAD activity of seeds from the same age but of different variety and provenience. In durum wheat, GAD activity was assayed during the early phases of germination of old seeds which differ for provenience but have the same age. The seeds were choosen so as to examine materials in a critical period of ageing, CA seeds still having a high germination percentage and PI seeds showing the negative incidence of .age on germination (about 30 % at 3 days) in the same storage conditions. Data exhibited confirm the influence, during seed ripening, of environmental conditions which are dry at Cagliari and wet at Pisa. These conditions, which determine a longer life-span of CA seeds than the PI seeds (FLORIS et al. 1972), can modify the GAD activity, very high in the former in confront to the latter seeds. Preliminary expe:r:iments to evaluate the nature of these differences were carried out on partially purified preparations (GALLEscHI et al.1976) from both seeds and the obtained data showed that no differences exist as to the ATP regulation and pyridoxal-5'-phosphate reactivation. Differences of GAD activity, as determined on young wheat seeds, do not seem to attribute to changes of enzyme regulation (G ALLESCHI et al. 1976) and might be consequences of different mechanisms of synthesis and breakdown of apoenzyme which, in mammals, appears to be influenced by group-specific proteases (KATANUMA et al. 1972). Similar mechanisms could occur also in CA and PI seeds.
GAD and SSA-DH Activity of ageing wheat seeds
The experimental data reported in the present paper evidenced, at different times of seed life-span, the loss of two enzymatic activities connected to a particular pathway of glutamate metabolism, the G;\BA-shunt (DIXON and FOWDEN 1961). This fact could be a proof of the loss of this pathway in the old unviable seeds. Since the end product of GABA-shunt in higher plants is connected with Krebs cycle (DIXON and FOWDEN 1961) and, in Triticum, the GAD is regulated by ATP (GALLESC:S:I et al. 1976), it is possible to hypothesize the energetic role of the enzymatic pathway. In the old seeds, the failure of this energy-generating system could be of foundamental importance in energy-dependent processes and an additional factor limiting or precluding protein synthesis (BRAY and C:s:ow 1976) and repair mechanisms (BERJAK and VILLIERS 1972) during the early period of germination. Acknowledgements The authors acknowledge Mr. G. MARRAZZINI and F. SAVIOZZI for their expert technical assistance.
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L. GALLESCHI and C. FLORIS, GAD and SSA-DH Activity of ageing wheat seeds
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Authors' address: LUCIANO GALLESCHI and CARLO FLORIS, Institute of Botany, University of Pisa, Pisa, Italy.