Control of the Formation of Amylases and Proteases in the Cotyledons of Germinating Peas

a-Amylase activity increased in attached cotyledons of germinated Vigna muago seeds until the 5th day after imbibition and decreased thereafter, whereas in detached and incubated cotyledons the activity continuously increased and, at the 6th day, reached the value more than three times that of the maximum activity of attached cotyledons. Zymograms of the activities and Ouchterlony double inmunodiffusion test on the activities of attached and detached cotyledons showed that the increase of activity in detached cotyledons was due to the identical enzyme as in attached tissues. a-Amylase contents, determined by single radial immunodiffusion method, changed in paraflel with enzyme activity in both attached and detached cotyledons, which also suggested the de novo synthesis of a-amylase in V. mugo cotyledons.The rate of incorporation of the label from I3Hjleucine into a-amylase and the ratios of dpm in a-amylase/dpm in trichloroacetic acid-insoluble fraction did not show significant difference between attached and detached cotyledons. The results indicated that in attached cotyledons fluctuation of a-amylase activity was regulated by both synthesis and degradation of the enzyme, whereas in detached cotyledons a-amylase was synthesized and accumulated, because of low degrading activity during incubation.In cereal seeds, such as barley, it is well known that gibberellin (GA3) produced in the embryos moves to the aleurone layers and promotes the de novo synthesis and secretion of a-amylase into endosperm. Recent reports suggested that this enzyme induction depends on increase of translatable mRNA for a-amylase (7,15,16), and that this increase is due to a direct enhancement of transcription of a-amylase gene by GA3 (1).In contrast, the mechanism of a-amylase formation in cotyledons of germinating leguminous seeds is not known. Many studies on effects of axis removal or plant hormones have been done, but there are some discrepancies. For example, Yomo and collaborators (24,26) reported that a-amylase activity increases in the cotyledons during seedling growth and during incubation of axisfree cotyledons of pea and bean. This was inconsistent with the findings of others that this increase is partially inhibited by axis removal (3,11,23 processes of seed formation between cereal and leguminous seeds (2), we studied the mechanism of a-amylase formation in cotyledons of germinating bean seeds.We previously reported that amylolytic activity increases in Vigna mungo cotyledons during germination and that the increase is not delayed by axis removal (13). Histochemical observation revealed that amylolytic and proteolytic activities appeared to increase in tissue areas farthest from vascular bundles during germination (14). We also obtained pure a-amylase preparation from cotyledons of V. mungo seedlings using affinity chromatography (8). In the present study, using antibody toward the aamylase obtained by immunization of a rabbit with purified enzyme, we investigated whether the increase of a-amylase is due to de novo s...

Section: Methodssupporting

confidence: 56%

mentioning

confidence: 99%

In Vivo Synthesis and Turnover of α-Amylase in Attached and Detached Cotyledons of Vigna mungo Seeds

Koshiba

Minamikawa²

1983

“…A number of researchers have used pea seeds for their experiments, but contradictory results have been reported, possibly because the experimental conditions were not standardized. In the case of a-amylase activity, the consequences of excision of cotyledons include inhibition (6,13), promotion (15), and no appreciable effect (1). A reason for these contradictory results is reported.…”

mentioning

confidence: 99%

Auxins Induce α-Amylase Activity in Pea Cotyledons

Hirasawa

1989

The report documents how the development of a-amylase activity in detached cotyledons of Pisum sativum cv Alaska is accelerated 2-to 12-fold during incubation with 1 micromolar to 10 micromolar 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, or with 4-chloroindoleacetic acid, an endogenous auxin from Pisum sativum. It seems probable that auxin from the embryonic axis induces a-amylase in the attached cotyledons during germination.Mobilization of nutrients in barley, rice, and wheat does not occur in endosperms from which the embryo has been removed. A diffusible factor from the embryo promotes aamylase activity in the aleurone layer and has been identified as GA (10,14). It has also been shown that GA is involved in mobilization of nutrients in these cereals. The same simple method of analysis has been applied to other seeds, and in many cases removal of the embryo has been found to prevent or retard mobilization and to affect adversely the development of the monitored enzyme (2). Knowledge about mobilization of stored nutrients in seeds in which the cotyledons are the storage organs is limited. A number of researchers have used pea seeds for their experiments, but contradictory results have been reported, possibly because the experimental conditions were not standardized. In the case of a-amylase activity, the consequences of excision of cotyledons include inhibition (6, 13), promotion (15), and no appreciable effect (1). A reason for these contradictory results is reported. MATERIALS AND METHODS Plant MaterialPea seeds (Pisum sativum cv Alaska) were immersed in a 1% solution of sodium hypochlorite for 2 min and then rinsed with deionized water for 1 min. To maintain aerobic conditions and a steady supply of water to the cotyledons, the seeds were placed on filter paper that was supported by a small Petri dish in a large dish which contained deionized water or one ofthe test solutions. The seeds which were not submerged could take up water or the solution slowly through the filter paper. Incubations were conducted at 25°C under germ-free conditions in the dark. Preparation of EnzymeAfter incubation, each group of eight pairs of cotyledons was homogenized in a Waring Blendor with 5 volumes of 0.2 M Na-acetate buffer (pH 5.4) containing 3 mm CaC12. After the homogenate was passed through four layers ofcheesecloth, the filtrate was centrifuged at 25,000g for 15 min at 4°C and the supernatant was used for assay of a-amylase activity. The assay was carried out by a modification of procedures described by Rinderknecht et al. (11). With this assay, the determination of a-amylase in crude extracts is possible even in the presence of a-amylase which directly interferes with most assay methods (3). The reaction mixture consisted of 2 mL of 2% (w/v) starch azure (Sigma) which was heated slowly to boiling before the assay, 1 mL of 0.2 M Na-acetate (pH 5.4), 0.12 mL of 0.1 M CaC12, and 0.83 mL of deionized water. The reaction was started by the addition of 0.05 mL of supernatant. After incubation...

“…In contrast, in legume seeds, the mechanism for the development of a-amylase is much less clear; hormonal control of a-amylase development such as seen in cereals has not been demonstrated unambiguously. Although much work has been done on the role of the embryonic axis in the development of the enzyme in reserve tissues (cotyledon), the results are inconsistent (6,10,12,19,20).In a previous report (1 1 no dicot amylase cDNA has been available. In the present study we have cloned cDNA for mung bean a-amylase and examined the levels of a-amylase transcript in cotyledons.…”

mentioning

confidence: 91%

Isolation of a cDNA Clone for α-Amylase in Mung Bean Cotyledons

Koizuka

Tanaka²,

Morohashi³

1990

A cDNA was isolated that codes for a-amylase in mung bean (Vigna radiata) cotyledons, and the nucleotide sequence was determined. The deduced amino acid sequence (421 amino acid residues) is about 65% homologous with those of barley a-amylases. By comparing the deduced sequence with the sequence of the purified a-amylase, it was inferred that 23 Nterminal amino acids of a nascent polypeptide represent a signal peptide. Northern blot analysis showed that the levels of a-amylase mRNA are in parallel with the activities of a-amylase synthesis in cotyledons. Under the conditions where the solute leakage from cotyledons is accelerated during imbibition, a rapid increase in the amount of the a-amylase mRNA occurs. We postulate that a factor(s) which regulates in an inhibitory manner the a-amylase expression at the transcriptional level may be present in dry cotyledons and be removed by leakage.The mechanism for the development of a-amylase in reserve tissues of cereal seeds has been studied in detail; GA secreted by the embryo to the scutellum or the aleurone layer induces the synthesis of the enzyme by causing the synthesis of new a-amylase mRNA (13). In contrast, in legume seeds, the mechanism for the development of a-amylase is much less clear; hormonal control of a-amylase development such as seen in cereals has not been demonstrated unambiguously. Although much work has been done on the role of the embryonic axis in the development of the enzyme in reserve tissues (cotyledon), the results are inconsistent (6,10,12,19,20).In a previous report (1 1 no dicot amylase cDNA has been available. In the present study we have cloned cDNA for mung bean a-amylase and examined the levels of a-amylase transcript in cotyledons. MATERIALS AND METHODS Plant MaterialsSeeds of mung bean, Vigna radiata (L.) Wilczek, were treated as previously described (11). Briefly, H2S04-treated seeds were soaked in water for 6 h and husked, and the two cotyledons of each seed were separated, the embryonic axis remaining attached to one of them. The AC2 and the DC were placed on wet filter paper and incubated at 28°C in the dark. In some experiments, the two cotyledons of a seed were separated prior to imbibition, the axis being detached. The adaxial side of such cotyledons is not covered with the seed coat. The cotyledons were soaked in water for 6 h or 15 min. In the former case (6-h imbibition), the seed coats covering the abaxial surface of cotyledons were removed after imbibition and the cotyledons were incubated as above (DDC). In the latter case, the cotyledons were desiccated after 15-min imbibition, resoaked in water for 6 h, husked, and incubated as above (RIC). In another set of experiments, detached cotyledons separated from dry seeds were allowed to imbibe water by placing them abaxial side down on wet filter paper for 6 h, and then they were husked, transferred onto wet filter paper, and incubated as above (FPC)