Characterization of an abscisic acid carrier in suspension-cultured barley cells

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Abstract
The uptake of [3H]-abscisic acid in barley (Hordeum vulgare L. cv. Heartland) cell cultures was found to be mediated through both non-saturable and saturable components. The kinetic parameters of the saturable component, determined at pH 4.5 and 21 °C, showed a Km for natural or (+ )-ABA of 1.3±0.7μM and an apparent Vmex of 7.0 ± 2.8 nmol g−1 cells h−1. The carrier showed a strong preference for the natural enantiomer of ABA as compared to the unnatural one. Other substances tested, e.g. amino acids, organic acids, and other growth regulators, did not appear to interfere with the carrier-mediated uptake of ABA. At low external concentrations of ABA (below 2.0 μM), the saturable component was greater than the diffusion component. Similarly, between pH 4.0 and 6.0, the saturable uptake was responsible for more than 50% of the total uptake. The carrier may be important in vivo for mediating uptake when endogenous levels of ABA are low (c. 1 μM). The carrier specificity was evident in inhibition experiments done with ABA analogues. Our data showed that the carrier could accommodate small modifications in the ABA structure. Four analogues were able to compete efficiently with ( + )-ABA for the binding site of the carrier. Three of these competitors were of the (+)-series. Only one ( −)-analogue, (−)-ABA, was able to inhibit markedly the saturable uptake of ( + )-ABA. The induction of the ABA-respons-ive gene WCS120 (Houde et al., 1992) presented stricter requirements for the ABA molecule than the carrier, although with a similar preference for the ( + )-analogues. Besides ( + )-ABA itself, only two of the analogues tested, both ( + )-series, were able to induce the WCS120 gene after a 24 h incubation period. The absence of correlation between the activity of the analogues as ABA inhibitors in the carrier system, and their capacity to induce the WCS120 gene tend to suggest that the carrier is not directly involved in the signal transduction pathway leading to the induction of this specific gene.

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