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Plant Physiol. (1992) 99, 235-238 0032-0889/92/99/0235/04/$01 .00/0

Received for publication August 13, 1991 Accepted December 11, 1991

Changes in the Ascorbate System during Seed Development of Vicia faba L.' Oreste Arrigoni*, Laura De Gara, Franca Tommasi, and Rosalia Liso Istituto di Botanica, Universita di Bari, Bari, Italy obtain maturing seeds of controlled age, flower at the ninth node of plants were labeled when the first flower of the truss was fully open and the age of the seeds was reckoned from this date. Pods were collected at 2- to 5-d intervals from a very early stage of their development (10 d after anthesis), during the whole growth stage, until the end of seed desiccation. At each stage, pods from the ninth node were picked and two to three seeds from the central portion of each pod were selected to form three batches. All of these batches were then utilized to determine enzyme activities, AA, DHA, and dry weight. In the early stages, until 25 d from anthesis, the analyses were performed on samples composed of about 40 seeds; in the late stages, the analyses were made on samples of 4 to 10 seeds.

ABSTRACT Large changes occur in the ascorbate system during the development of Vicia faba seed and these appear closely related to what are generally considered to be the three stages of embryogenesis. During the first stage, characterized by embryonic cells with high mitotic activity, the ascorbic acid/dehydroascorbic acid ratio is about 7, whereas in the following stage, characterized by rapid cell elongation (stage 2), it is lower than 1. The different ascorbic/dehydroascorbic ratio may be correlated with the level of ascorbate free radical reductase activity, which is high in stage 1 and lower in stage 2. Ascorbate peroxidase activity is high and remains constant throughout stages 1 and 2, but it decreases when the water content of the seed begins to decline (stage 3). In the dry seed, the enzyme disappears together with ascorbic acid. Ascorbate peroxidase activity is observed to be 10 times higher than that of catalase, suggesting that ascorbate peroxidase, rather than catalase, is utilized in scavenging the H202 produced in the cell metabolism. There is no ascorbate oxidase

Enzyme Assays Seeds were homogenized at 4°C in a porcelain mortar with 4 volumes of grinding medium composed of 50 mM TrisHCI, pH 7.2, 0.3 M mannitol, 1 mM EDTA, 0.1% BSA, and 0.05% cysteine. The cytosolic fraction obtained during centrifugation at 45,000g was used to determine enzyme activities. Enzyme assay procedures were performed at 25°C.

in the seed of V. faba. V. faba seeds acquire the capability to synthesize ascorbic acid only after 30 days from anthesis, i.e. shortly before the onset of seed desiccation. This suggests that (a) the young seed is fumished with ascorbic acid by the parent plant throughout the period of intense growth, and (b) it is necessary for the seed to be endowed with the ascorbic acid biosynthetic system before entering the resting state so that the seed can promptiy synthesize the ascorbic acid needed to reestablish metabolic activity when germination starts.

AFR Reductase Activity

This was tested by measuring the oxidation rate of NADH at 340 nm in a reaction mixture (3 mL) composed of 0.2 mm NADH, 1 mm ascorbate, and 0.1 M Tris-HCl, pH 7.2. The reaction was started by adding 0.2 unit ascorbate oxidase to generate saturating concentrations of AFR.

In a previous paper (7), we reported that dry seeds of Vicia faba L. are devoid of AA2 and contain only a small amount of DHA. On the other hand, in the extensive literature on the subject (16), it is reported that, before desiccation, seeds of V. faba contain a large amount of AA. Because these data regard two distinct periods of seed development, it seems possible that changes occur in the AA system when the desiccation period starts and the seed enters the resting state. Thus, the aim of the present paper is to analyze the AA system during the main developmental stages of V. faba seeds.

DHA Reductase

This was tested by following the increase in absorbance at 265 nm due to the (GSH)-dependent production of ascorbate. The assay mixture consisted of 1 mM DHA, 1 mM GSH, and 100 mm potassium phosphate buffer, pH 6.3. Ascorbate Peroxidase This was determined using a reaction mixture composed of 50 tAM ascorbate, 90 jAM hydrogen peroxide, and 50 mM potassium phosphate buffer, pH 6.5. The hydrogen peroxidedependent oxidation of ascorbate was followed by means of the decrease in absorbance at 265 nm.


Plant Material

Plants of Vicia faba L. var Aprilia were grown in experimental fields under insect-proof net in Valenzano Bari. To 'This work was supported by Consiglio Nazionale delle Ricerche. 2Abbreviations: AA, ascorbic acid; DHA, dehydroascorbic acid; AFR, ascorbic free radical; GL, galactono-y-lactone.

Ascorbic Oxidase

This was estimated according to Arrigoni et al. (2). 235

Plant Physiol. Vol. 99, 1992



Catalase This was determined according to Beers and Sizer (3), using 18 mm H202. Proteins were determined with the Lowry method (14), with BSA as a standard protein. AA Biosynthesis This was estimated by incubating seeds in 5 mM GL, the last precursor in the AA biosynthetic pathway (15). After 6 h incubation, the seeds were ground in 5% metaphosphoric acid and the AA formed by GL oxidation was determined. AA and DHA Assays Seeds were ground in 5% metaphosphoric acid at 4°C to prevent the conversion of DHA to 2,3-diketogulonic acid; in this condition, AA is stable for several hours. The homogenate was centrifuged at l0,OOOg and the supernatant was then used. AA was measured at 265 nm by following the decrease in absorbance induced by adding purified ascorbate oxidase according to the method reported in Liso et al. (13). DHA was determined according to the Roe and Oesterling method (18).

Dry Weight Determination The preweighed fresh samples were dried in an oven at 90 ± 1°C until a constant dry weight was obtained. RESULTS

In plants of V. faba var Aprilia grown in a field under insect-proof net, in the climatic conditions of springtime of southern Italy, the seeds develop until maturity in about 80 d from anthesis (19). The results obtained from analyses carried out in developing seeds demonstrate that 40 d after anthesis, when the seed attains maximum length (3 cm) and maximum fresh weight (3.6 g), the total ascorbate content (AA + DHA) is also at maximum level (144,mol/seed); during the following 40 d, simultaneously with the desiccation process (at the end of which the seed weighs 1.81 g), total ascorbate content gradually decreases to a final value of 1.5 umol/seed. AA and DHA contents per seed, as well as the dry weight/ fresh weight ratio, as analyzed during seed development, are reported in Figure 1. Both AA and DHA gradually increase during the first 40 d after anthesis; however, their rates of change are quite different: during the first 18 d after anthesis, the period that corresponds to first stage of embryogenesis, characterized by embryonic cells with high mitotic activity (17), AA content is high with respect to that of DHA (0.15 ,umol AA and 0.02 ,mol DHA/seed at the 10th d; 0.95 ,umol AA and 0.16 ,mol DHA/seed at the 18th d). In the following period, between 20 and 40 d after anthesis, which corresponds to the second stage of embryogenesis characterized by cells undergoing rapid elongation growth (17), DHA strongly rises, and at 43 d attains the maximum level. At the beginning of the desiccation period (stage 3 of embryogenesis), marked by an abrupt increment of the dry weight/fresh weight ratio, both AA and DHA decrease sharply. At the end of desiccation, only a small amount of DHA remains.



seed-length (m m) 16 32





U) U)


2 E



0 :0 I:






10 20 30 40 50 60 70 days from anthesis


Figure 1. AA and DHA contents in developing seed of V. faba. The values are the mean of three experiments ± SD.

It is worth noting that the AA/DHA ratio changes during the growth period of the seed. Throughout the first 18 d after anthesis, the AA/DHA ratio is very high (6-7), whereas immediately after, a rapid decline in the AA/DHA ratio is observed that could reflect the transition from stage 1 to stage 2 of the embryogenesis. During the progression of stage 2, there is a further shift of the AA/DHA ratio toward the oxidative state. Table I shows the data on the activity of the enzymes of the ascorbate system as analyzed during seed development. There is no AA oxidase in the seed, whereas AA peroxidase is present and exhibits very high activity that remains constant throughout the period of seed-size increase (stages 1 and 2), but when water content declines, the activity of the enzyme also decreases and, at the end of desiccation, no AA peroxidase is detectable in the seed. To obtain information about the physiological role of AA peroxidase in removing the hydrogen peroxide, we have analyzed catalase activity present in developing seeds. During stages 1 and 2, the enzyme activity was about 50 nmol H202 min-' mg-' protein; during desiccation, the activity decreased and was 20 nmol H202 min-' mg-' protein in the dry seed. AFR reductase and DHA reductase, enzymes that catalyze the respective reduction of AFR and DHA (compounds that arise from AA oxidation), are both functional in V. faba seed;



however, the activity of AFR reductase is much higher than that of DHA reductase. AFR reductase activity appears high in the first stages of seed development, but it gradually decreases and, at the end of desiccation, it is about one-sixth of the initial activity. DHA reductase remains constant for a long period, but at the end of seed development its activity is very low. AA biosynthesis was tested during seed development by evaluating the capability of the growing seed to oxidize GL into AA. The data in Table II show that until 30 d after anthesis the embryo of V. faba is not capable of synthesizing AA; the seed acquires the capacity of synthesizing AA just 10 d before the onset of desiccation. DISCUSSION AND CONCLUSIONS

The results of this investigation show that large changes occur in the ascorbate system during the development of V. faba seed and these appear to be closely related to the stages of embryogenesis (17). During stage 1, characterized by cell division, the AA/DHA ratio is about 7, whereas in the following stage, characterized by rapid cell elongation growth, the AA/DHA ratio is very low. The sharp decrease in the AA/DHA ratio observed in the transition from stage 1 to stage 2 cannot be ascribed to a higher level of AA utilization during cell elongation growth with respect to cell division period. Both these stages largely utilize ascorbate. We previously reported that the meristematic cells of V. faba utilize about 4 gmol AA h-' g-' fresh weight (13), and that when the AA content of actively proliferating cells is experimentally lowered with lycorine, a specific inhibitor of AA biosynthesis (1, 8, 10), the cell cycle is arrested in the GI phase (13). Cell expansion also requires ascorbate: we recently reported that prolyl hydroxylase, an enzyme strongly involved in the control of expansion growth (12), specifically utilizes ascorbate in vivo for the hydroxylation of the proline present in polypeptide chains (9). However, the amount of AA utilized during cell expansion is always sevento tenfold lower than meristematic cells (1, 10). Another reason for ruling out a major utilization of AA during cell elongation growth compared with that during cell division is the unchanged activity of AA peroxidase in stages 1 and 2 of V. faba development (see Table I). The different AA/DHA ratio can be correlated with the level of AFR reductase activity in the cells. To understand how AFR re-

Table II. AA Biosynthesis in Developing V. faba Seed Incubated in GL The values are the mean of three experiments ± SD. Days fo AA Fresh



10 12 18 20 25 28 30 32 34 40 43



nmol g-' fresh wt h-1

0.012 ± 0.005 0.023 ± 0.010 0.271 ± 0.042 0.410 ± 0.072 0.672 ± 0.060 0.881 ± 0.092 1.075 ± 0.150 1.505 ± 0.225 2.133±0.410

0 0 0 0 0 0

0 81 ± 12 108±25

3.220±0.120 3.634±0.130

210±41 216±39

ductase activity affects the amount of DHA in the cell, it is appropriate to consider the mechanism of DHA formation. It is well known that cell metabolism utilizes AA as an electron donor and that AA oxidation by AA oxidase or peroxidase is a two-step reaction in which each step removes one electron (23). The first AA oxidation product is a semiquinone-like free radical, i.e. AFR. AFR can be reconverted to AA by AFR reductase (2AFR + NAD(P)H 2AA + NAD(P)+) (4) or --

spontaneously undergoes disproportionation (2AFR


AA +

DHA), thus generating DHA (4, 23). It appears clear that when AFR reductase activity is high, a large quantity of AFR is reduced to AA and a small amount of AFR remains available for disproportionation; conversely, when AFR reductase is low, a smaller amount of AFR is reconverted to AA and most of the AFR undergoes disproportionation, generating DHA in large quantities. According to these findings, it is possible to explain why DHA content is low with respect to that of AA in meristematic cells, which are endowed with a high AFR reductase activity. However, DHA content is high in cells undergoing cell elongation growth and that have lower AFR reductase activity (see Table I). Another point that should be mentioned concerns the behavior of AA peroxidase during the developmental stages of the seed. The activity of this enzyme is high (480 nmol

Table I. Activities of Oxido-Reductive Enzymes of the Ascorbate System in the Main Developmental Stages of V. faba Seed The values are the mean of three experiments ± SD. Stages of Days from Peroxidase AA Oxidase AFR Reductaseb DHA Reductasec Anthesis Development units

0 480 ± 12 Cell division 15 0 485 ± 11 Cell elongation 30 0 475 ± 13 Start of desiccation 45 0 0 80 Dryseed b a 1 unit = 1 nmol AA oxidized min- mg-' protein. 1 unit = 1 = min-1 DHA reduced 1 nmol protein. 1 unit mg-' c protein.

69 ± 5 436 ± 9 65 ± 8 298 ± 11 62 ± 6 232 ± 8 5±2 72±4 nmol NADH oxidized min-




H202 reduced min-' mg-' protein) throughout cell division and cell expansion but, when the water content of the seed begins to decline, the activity of AA peroxidase decreases, and at the end of the desiccation period, when the seed enters the resting state, the enzyme disappears together with ascorbate. Because catalase activity is very low during cell division and cell expansion, it is reasonable to conclude that AA peroxidase, rather than catalase, is utilized to efficiently remove the hydrogen peroxide produced by cell metabolism (see also ref. 5). The data of Groden and Beck (1 1), Shigeoka et al. (20), Tommasi et al. (21), and our recent results (showing that decreases in AA peroxidase occurring during the aging of the caryopses of Dasypyrum villosum can be correlated with the onset of a biochemical pathway leading to morphological anomalies of seedlings and to the loss of seed germination capacity [6]), seem to confirm that AA peroxidase is a key defense enzyme against H202 toxicity. The last point emerging from the results of the present study regards the biosynthesis of ascorbate during seed development. Seeds of V. faba are not capable of synthesizing AA until 30 d after anthesis, i.e. during the period of their intense growth. Because the seed contains a large amount of AA during this phase of embryogenesis, characterized by elevated cell division and cell expansion, it is possible to conclude that the AA present in the young seed is furnished entirely by the parent plant. The fact that the seed acquires the capability to synthesize ascorbate a few days before the onset of desiccation, i.e. when cell metabolism begins to decrease, shows a necessity for the seed to be endowed with the ascorbate biosynthetic system before entering the resting state. In this manner, the seed can promptly start the biosynthesis of AA when germination commences. The synthesis of ascorbate can actually be detected within the first hours of seed imbibition (7, 22). The early availability of AA is necessary to meet the large demands for AA by the restored cell metabolism and because AA is, in some way, required to elicit AA peroxidase (22). Although the precise role played by ascorbate in the elicitation of AA peroxidase remains to be determined, it is clear that AA is the specific electron donor for the enzyme. Therefore, if the germinating seed were not able to synthesize ascorbate, the AA peroxidase could not function and, consequently, the hydrogen peroxide produced in the restored oxidative metabolism could not be efficiently removed. LITERATURE CITED 1. Arrigoni 0, Arrigoni-Liso R, Calabrese G (1975) Lycorine as an inhibitor of ascorbic acid biosynthesis. Nature 256: 513-514 2. Arrigoni 0, Dipierro S, Borraccino G (1981) Ascorbate free radical reductase, a key enzyme of the ascorbic acid system. FEBS Lett 125: 242-244 3. Beers R, Sizer IW (1952) A spectrophotometric method for





8. 9.

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measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 195: 133-140 Bielski BHJ, Allen AO, Schwarz HA (1981) Mechanism of disproportionation of ascorbate radicals. J Am Chem Soc 103: 3516-3518 Dalton DA, Hanus FJ, Russel SA, Envans HJ (1987) Purification, properties, and distribution of ascorbate peroxidase in legume root nodules. Plant Physiol 83: 789-794 De Gara L, Paciolla C, Liso R, Stefani A, Arrigoni 0 (1991) Correlation between ascorbate peroxidase activity and some anomalies of seedlings from aged caryopses of Dasypyrum villosum (L.) Borb. J Plant Physiol 137: 697-700 De Gara L, Tommasi F, Liso R, Arrigoni 0 (1987) I1 sistema dell'acido ascorbico in Viciafaba L. Boll Soc Ital Biol Sper 63: 551-558 De Gara L, Tommasi F (1990) Further researches upon the inhibiting action of lycorine on ascorbic acid. Boll Soc Ital Biol Sper 66: 953-960 De Gara L, Tommasi F, Liso R, Arrigoni 0 (1991) Ascorbic acid utilization by prolyl hydroxylase "in vivo." Phytochemistry 30:

1397-1399 10. De Leo P, Dalessandro, De Santis A, Arrigoni 0 (1973) Metabolic responses to lycorine in plants. Plant Cell Physiol 14: 487-496 1 1. Groden D, Beck E (1979) H202 destruction by ascorbate-dependent system from chloroplasts. Biochim Biophys Acta 546: 426-435 12. Lamport DTA (1969) The isolation and partial characterization of hydroxyproline rich glycopeptides obtained by enzymatic degradation of primary cell walls. Biochemistry 8: 1155-1163 13. Liso R, Calabrese G, Bitonti MB, Arrigoni 0 (1984) Relationship between ascorbic acid and cell division. Exp Cell Res 150: 3 14-320 14. Lowry JH, Rosebrough NJ, Farr AL, Randall RJ (195 1) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275 15. Mapson LW, Isherwood FA, Chen YT (1954) Biological synthesis of L-ascorbic acid: the conversion of L-galactono-y-lactone into L-ascorbic acid by plant mitochondria. Biochem J 56: 21-28 16. Mayer AM, Poljakoff-Mayber A (1975) Chemical composition of seeds. In PF Wareing, AY Galston, eds, Ed 2. Pergamon Press Oxford, New York, pp 10-20 17. Muntz K (1981) Seed development. Encycl Plant Physiol 14: 505-558 18. Roe JH, Oesterling MJ (1944) The determination of DHA and AA in plant tissues by 2,4 dinitrophenylydrazine. J Biol Chem 152: 511-521 19. Scarascia GT, De Pace C (1979) Concepts and goals for Vicia faba breeding in Mediterranean environments. Genet Agric 4: 2 17-244 20. Shigeoka S, Nakano Y, Kitaoka S (1980) Purification and some properties of L-ascorbic acid specific peroxidase in Euglena gracilis Z. Arch Biochem Biophys 201: 121-127 21. Tommasi F, De Gara L, Liso R, Arrigoni 0 (1987) Presenza di ascorbico perossidasi nel regno vegetale. Boll Soc Ital Biol Sper 63: 779-785 22. Tommasi F, De Gara L (1990) Correlazione tra presenza di acido ascorbico e comparsa dell'attivita ascorbico perossidasica in embrioni di Avena sativa L. Boll Soc Ital Biol Sper 66: 357-364 23. Yamazaki I, Piette LH (1961) Mechanism of free radical formation and disappearence during the ascorbic acid oxidase and peroxidase reactions. Biochim Biophys Acta 50: 62-69

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