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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 H(2)O(2) produced in the cell metabolism. There is no ascorbate oxidase 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 furnished 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 promptly synthesize the ascorbic acid needed to reestablish metabolic activity when germination starts.

By using lycorine, a specific inhibitor of ascorbate biosynthesis, it was possible to demonstrate that plant cells consume a high quantity of ascorbate (AA). The in vivo metabolic reactions utilizing ascorbate are the elimination of H2O2 by ascorbate peroxidase and the hydroxylation of proline residues present in the polypeptide chains by means of peptidyl-proline hydroxylase. Ascorbate acts in the cell metabolism as an electron donor, and consequently ascorbate free radical (AFR) is continuously produced. AFR can be reconverted to AA by means of AFR reductase or can undergo spontaneous disproportion, thus generating dehydroascorbic acid (DHA). During cell division and cell expansion ascorbate consumption is more or less the same; however, the AA/DHA ratio is 6-10 during cell division and 1-3 during cell expansion. This ratio depends essentially on the different AFR reductase activity in these cells. In meristematic cells AFR reductase is very high, and consequently a large amount of AFR is reduced to AA and a small amount of AFR undergoes disproportionation; in expanding cells the AFR reductase activity is lower, and therefore AFR is massively disproportionated, thus generating a large quantity of DHA. Since the transition from cell division to cell expansion is marked by a large drop of AFR reductase activity in the ER, it is suggested here that AFR formed in this compartment may be involved in the enlargement of the ER membranes and provacuole acidification. DHA is a toxic compound for the cell metabolism and as such the cell has various strategies to counteract its effects: (i) meristematic cells, having an elevated AFR reductase, prevent large DHA production, limiting the quantity of AFR undergoing disproportionation (ii) Expanding cells, which contain a lower AFR reductase, are, however, provided with a developed vacuolar system and segregate the toxic DHA in the vacuole. (iii) Chloroplast strategy against DHA toxicity is efficient DHA reduction to AA using GSH as electron donor. This strategy is usually poorly utilized by the surrounding cytoplasm. DHA reduction does play an important role at one point in the life of the plant, that is, during the early stage of seed germination. The dry seed does not store ascorbate, but contains DHA, and several DHA-reducing proteins are detectable. In this condition, DHA reduction is necessary to form a limited AA pool in the seed for the metabolic requirements of the beginning of germination. After 30-40 h ascorbate ex novo synthesis starts, DHA reduction declines until a single isoform remains, as is typical in the roots, stem, and leaves of seedlings.(ABSTRACT TRUNCATED AT 400 WORDS)

O. 1997, Ascorbate synthesis and ascorbate peroxidase activity during the early stage of wheat germination. -Physiol. Plant. 100: 894-900.Embryos from dry caryopses of wheat {Triticum durum L. cv. Norba) are completely devoid of ascorbate (ASC) but contain a low amount of dehydroascorbate (DHA). The de novo biosynthesis of ASC starts in the wheat embryos after 8-10 h of germination but before the ASC biosynthetic pathway is completely restored the embryos can provide themselves with ASC by the reduction of the stored DHA. Three different proteins having DHA-reducing capability are present in the embryos during the early stages of germination. However, when the de novo ASC biosynthesis from sugar is completely restored, the DHA reduction capability largely drops and only one DHA-reducing protein remains active. The presence of three proteins having DHA-reducing capability and their behaviour during germination is discussed. Dry embryos are also devoid of ASC peroxidase (EC 1.11.1.11); this hydrogen peroxide scavenger enzyme appears after the same lag as ASC and increases during germination in parallel with the rise in ASC. When ASC biosynthesis is experimentally induced, the ASC peroxidase also appears earlier; moreover the affinities for ASC of the three ASC peroxidase isoenzymes that progressively appear during germination depend on the ASC available in the embryos: highest in the first isoenzyme, that appears when the ASC content is very low, lowest in the isoenzyme that is expressed last, when the ASC content is 10-11 times higher.

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