ABSTRACT
Abscisic acid (ABA) is a carotenoid-derived plant hormone known to regulate critical functions in growth, development and responses to environmental stress. The key enzyme which carries out the first committed step in ABA biosynthesis is the carotenoid cleavage 9-cis-epoxycarotenoid dioxygenase (NCED). We have developed a series of sulfur and nitrogen-containing compounds as potential ABA biosynthesis inhibitors of the NCED, based on modification of the sesquiterpenoid segment of the 9-cis-xanthophyll substrates and product. In in vitro assays, three sesquiterpene-like carotenoid cleavage dioxygenase (SLCCD) inhibitor compounds 13, 17 and 18 were found to act as inhibitors of Arabidopsis thaliana NCED 3 (AtNCED3) with K(i)'s of 93, 57 and 87 microM, respectively. Computational docking to a model of AtNCED3 supports a mechanism of inhibition through coordination of the heteroatom with the non-heme iron in the enzyme active site. In pilot studies, pretreatment of osmotically stressed Arabidopsis plants with compound 13 resulted lower levels of ABA and catabolite accumulation compared to levels in mannitol-stressed plant controls. This same inhibitor moderated known ABA-induced gene regulation effects and was only weakly active in inhibition of seed germination. Interestingly, all three inhibitors led to moderation of the stress-induced transcription of AtNCED3 itself, which could further contribute to lowering ABA biosynthesis in planta. Overall, these sesquiterpenoid-like inhibitors present new tools for controlling and investigating ABA biosynthesis and regulation.
Subject(s)
Abscisic Acid/biosynthesis , Arabidopsis/enzymology , Bridged Bicyclo Compounds/chemistry , Bridged Bicyclo Compounds/chemical synthesis , Cyclohexanones/chemistry , Heptanes/chemistry , Heptanes/chemical synthesis , Oxygenases/antagonists & inhibitors , Plant Growth Regulators/chemical synthesis , Sesquiterpenes/chemical synthesis , Bridged Bicyclo Compounds/pharmacology , Computer Simulation , Cyclohexanones/chemical synthesis , Cyclohexanones/pharmacology , Dioxygenases/antagonists & inhibitors , Dioxygenases/metabolism , Down-Regulation , Drug Design , Gene Expression Regulation, Plant , Germination/drug effects , Heptanes/pharmacology , Kinetics , Oxygenases/metabolism , Plant Growth Regulators/chemistry , Plant Growth Regulators/pharmacology , Plant Proteins , Sesquiterpenes/chemistry , Sesquiterpenes/pharmacologyABSTRACT
Bicyclic analogues of the plant hormone abscisic acid (ABA) were designed to incorporate the structural elements and functional groups of the parent molecule that are required for biological activity. The resulting tetralone analogues were predicted to have enhanced biological activity in plants, in part because oxidized products would not cyclize to forms corresponding to the inactive catabolite phaseic acid. The tetralone analogues were synthesized in seven steps from 1-tetralone and a range of analogues were accessible through a second route starting with 2-methyl-1-naphthol. Tetralone ABA 8 was found to have greater activity than ABA in two bioassays. The absolute configuration of (+)-8 was established by X-ray crystallography of a RAMP hydrazone derivative. The hydroxymethyl compounds 10 and 11, analogues for studying the roles of 8- and 9-hydroxy ABA 3 and 6, were also synthesized and found to be active.
Subject(s)
Abscisic Acid/analogs & derivatives , Abscisic Acid/chemistry , Tetralones/chemistry , Tetralones/chemical synthesis , Abscisic Acid/pharmacology , Arabidopsis/drug effects , Arabidopsis/physiology , Germination/drug effects , Models, Molecular , Molecular Conformation , Plant Growth Regulators/chemistry , Seeds/drug effects , Seeds/physiology , Spectroscopy, Fourier Transform Infrared , Tetralones/pharmacologyABSTRACT
An affinity probe has been developed for isolation of receptor proteins that bind the plant hormone abscisic acid (ABA). The structural features required for biological activity have been preserved, and the probe has been demonstrated to bind to known ABA-binding proteins.
Subject(s)
Abscisic Acid/metabolism , Plant Proteins/metabolism , Receptors, Cell Surface/metabolism , Abscisic Acid/analogs & derivatives , Amino Acid Sequence , Brassica/enzymology , Brassica/genetics , Brassica/metabolism , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Molecular Sequence Data , Plant Proteins/analysis , Plant Proteins/genetics , Protein Binding , Receptors, Cell Surface/analysis , Structure-Activity RelationshipABSTRACT
In a biotransformation study to prepare deuterium labelled phaseic acid (PA) from deuterated abscisic acid (ABA), the product contained fewer deuterium atoms than expected. Thus, spectroscopic data of isolated deuterated PA prepared from biotransformation of (+)-5,8',8',8'-d4-ABA in maize (Zea mays L. cv. Black Mexican Sweet) cell suspension cultures showed 83% deuterium incorporation at the 8'-exo position. Also, metabolism studies of (+)-4,5-d2-ABA in maize resulted in the isolation of deuterium labelled ABA derivatives, namely PA, dihydrophaseic acid (DPA), 4'-O-beta-D-glucopyranosylDPA, 8'-hydroxyPA, 8'-hydroxyDPA and 8'-oxoDPA, as deduced from spectroscopic methods. These combined results suggested the presence of an aldehyde intermediate which is either: (a) reduced to 8'-hydroxyABA and cyclized to PA, or (b) is hydrated and cyclized to 8'-hydroxyPA or (c) is further oxidized to the acid and cyclized to 8'-oxoPA. The chemical synthesis of this intermediate, as well as its biotransformation in maize cell cultures is presented.
