Kitahata Nobutaka, Nakano Takeshi, Kuchitsu Kazuyuki, Yoshida Shigeo, Asami Tadao
RIKEN, Hirosawa 2-1, Wako, Saitama 351-0198, Japan.
Bioorg Med Chem. 2005 May 16;13(10):3351-8. doi: 10.1016/j.bmc.2005.03.017.
Plant hormone abscisic acid (ABA) plays important roles in dormancy and stress responses, but its binding sites have not yet been fully elucidated. In this report, we suggest the utility of biotin-labeled abscisic acid (bioABA) as a probe to investigate ABA-binding sites on the plasma membrane of barley aleurone protoplasts. BioABA was approximately 100 times less effective than ABA in inhibiting expression of gibberellin-inducible alpha-amylase and in inducing expression of a reporter gene fused to the dehydrin promoter. To ascertain that bioABA could bind to ABA-binding sites on the plasma membrane, we used fluorescence flow cytometry to measure the fluorescence intensity of aleurone protoplasts treated with a combination of bioABA and fluorescence-labeled streptavidin. Addition of bioABA increased the fluorescence of aleurone protoplasts in a concentration-dependent manner, but addition of non-active bioABA derivatives did not. Furthermore, the increase in fluorescence intensity observed upon addition of bioABA was eliminated by co-treatment with excess ABA, but it was not eliminated by co-treatment with other plant hormones. These results suggest that bioABA binds to ABA-binding sites, and that bioABA should be a valuable probe for investigating ABA-binding sites on the plasma membrane.
植物激素脱落酸（ABA）在休眠和应激反应中发挥重要作用，但其结合位点尚未完全阐明。在本报告中，我们提出生物素标记的脱落酸（bioABA）可作为一种探针，用于研究大麦糊粉层原生质体质膜上的ABA结合位点。BioABA在抑制赤霉素诱导的α-淀粉酶表达以及诱导与脱水蛋白启动子融合的报告基因表达方面，其效果约为ABA的100倍。为确定bioABA能够结合质膜上的ABA结合位点，我们使用荧光流式细胞术来测量用bioABA和荧光标记的链霉亲和素处理的糊粉层原生质体的荧光强度。添加bioABA会使糊粉层原生质体的荧光以浓度依赖的方式增加，但添加无活性的bioABA衍生物则不会。此外，添加bioABA后观察到的荧光强度增加可通过与过量ABA共同处理而消除，但不能通过与其他植物激素共同处理而消除。这些结果表明bioABA能结合到ABA结合位点，并且bioABA应是研究质膜上ABA结合位点的一种有价值的探针。
