Biotin-labeled abscisic acid as a probe for investigating abscisic acid binding sites on plasma membranes of barley aleurone protoplasts.
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.
Kitahata
Nobutaka
N
RIKEN, Hirosawa 2-1, Wako, Saitama 351-0198, Japan.
Nakano
Takeshi
T
Kuchitsu
Kazuyuki
K
Yoshida
Shigeo
S
Asami
Tadao
T
D000040:Abscisic Acid / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D005434:Flow Cytometry; D005453:Fluorescence; D001467:Hordeum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007202:Indicators and Reagents / Q000737:chemistry / Q000378:metabolism; D015335:Molecular Probes; D010937:Plant Growth Regulators / Q000378:metabolism / Q000494:pharmacology*; D011523:Protoplasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D012639:Seeds / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism; D000516:alpha-Amylases / Q000378:metabolism
D007202:Indicators and Reagents; D015335:Molecular Probes; D010937:Plant Growth Regulators; D001710:Biotin; D000040:Abscisic Acid; D019809:Streptavidin; D000516:alpha-Amylases