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Da-Peng Zhang, Zi-Lian Zhang, Jia Chen, and Jiang Lu

The abscisic acid (ABA) has a key role in the regulation of grapevine fruit ripening, but the cellular and molecular biological mechanism of the hormone action in the fruit ripening remains unknown. By means of differential centrifugation, microsomes were prepared from Kyoho grapevine (Vitis vinifera L. × V. Labrusca L.) berries, and using the microsomes, we have obtained evidence for the occurrence of specific ABA-binding sites on the membranes with the microvolume radio-ligand binding assay. The binding sites had saturability, high affinity, and low capacity. The results of trypsin and dithiothreitol treatments to the microsomes suggested that ABA binding sites had the properties of proteins that might have disulfide group located at or near the binding site. The binding maximum amount of ABA in the microsomes was at pH 6.0 and the activity of ABA binding proteins was higher at 25 than at 0°C (temperature). The amount of ABA bound reached 54% of the ABA binding maximum (Bmax) for 10 minutes of incubation and Bmax reached for 30 minutes. The dissociation constant (Ka) and Bmax of ABA binding proteins in the microsomes were 17.5 nmol/L and 98.4 fmol/mg protein, respectively.

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Da-Peng Zhang, Zi-Lian Zhang, Jia Chen, and Jiang Lu

By using the micro-volume radio-ligand binding essay, the changes in the kinetic characteristics of the abscisic acid (ABA)-binding protein(s) of the Kyhoh grapevine (Vitis vinifera × V. labrusca) fruit during the different stages of fruit development have been studied. The changes in the berry volume growth, concentration of sugar, organic acids, and ABA in fruit mesocarp have been surveyed, especially for studies of ABA-binding protein. The dissociation constant (Kd) and ABA binding maximum (Bmax) were determined by the Scatchard plots for ABA binding in microsomes of the fruit. They are Kd = 17.5, 50.0, 6.3, 13.3 nmol·L–1; Bmax = 98.6, 523.0, 41.6, 85.4 μmol·mg–1 protein, respectively, for the fruit developmental phase I, II, veraison, and phase III. The Scatchard plots showed a rectilinear function for all of the developmental phases including veraison, which suggests the sole ABA-binding site of high affinity for ABA in the fruit microsomes, but this site could either be only one kind of the same protein or consist of more kinds of different proteins for different developmental stages. The binding affinity of ABA-binding protein(s) for ABA was shown to be higher at veraison time than during other developmental phases; this binding affinity increased nearly by 10 times from phase II to veraison, while the concentration (Bmax) of the ABA-binding protein(s) decreased to the minimum at veraison. The very low concentration of ABA at veraison may be able to trigger the onset of fruit ripening due to the increase of the binding affinity of ABA-binding protein(s) for ABA at this time. The possible functions of the ABA-binding protein(s) for fruit development during the different developmental stages were discussed, and it is suggested that the protein(s) detected could be the putative ABA receptor(s) or transporter(s) for the action of this plant hormone in grapevine.