Abscisic acid analogs as chemical probes for dissection of abscisic acid responses in Arabidopsis thaliana.

Abscisic acid (ABA) is a phytohormone known to mediate numerous plant developmental processes and responses to environmental stress. In Arabidopsis thaliana, ABA acts, through a genetically redundant family of ABA receptors entitled Regulatory Component of ABA Receptor (RCAR)/Pyrabactin Resistant 1 (PYR1)/Pyrabactin Resistant-Like (PYL) receptors comprised of thirteen homologues acting in concert with a seven-member set of phosphatases. The individual contributions of A. thaliana RCARs and their binding partners with respect to specific physiological functions are as yet poorly understood. Towards developing efficacious plant growth regulators selective for specific ABA functions and tools for elucidating ABA perception, a panel of ABA analogs altered specifically on positions around the ABA ring was assembled. These analogs have been used to probe thirteen RCARs and four type 2C protein phosphatases (PP2Cs) and were also screened against representative physiological assays in the model plant Arabidopsis. The 1'-O methyl ether of (S)-ABA was identified as selective in that, at physiologically relevant levels, it regulates stomatal aperture and improves drought tolerance, but does not inhibit germination or root growth. Analogs with the 7'- and 8'-methyl groups of the ABA ring replaced with bulkier groups generally retained the activity and stereoselectivity of (S)- and (R)-ABA, while alteration of the 9'-methyl group afforded an analog that substituted for ABA in inhibiting germination but neither root growth nor stomatal closure. Further in vitro testing indicated differences in binding of analogs to individual RCARs, as well as differences in the enzyme activity resulting from specific PP2Cs bound to RCAR-analog complexes. Ultimately, these findings highlight the potential of a broader chemical genetics approach for dissection of the complex network mediating ABA-perception, signaling and functionality within a given species and modifications in the future design of ABA agonists.

Action of natural abscisic acid precursors and catabolites on abscisic acid receptor complexes.

The phytohormone abscisic acid (ABA) regulates stress responses and controls numerous aspects of plant growth and development. Biosynthetic precursors and catabolites of ABA have been shown to trigger ABA responses in physiological assays, but it is not clear whether these are intrinsically active or whether they are converted into ABA in planta. In this study, we analyzed the effect of ABA precursors, conjugates, and catabolites on hormone signaling in Arabidopsis (Arabidopsis thaliana). The compounds were also tested in vitro for their ability to regulate the phosphatase moiety of ABA receptor complexes consisting of the protein phosphatase 2C ABI2 and the coreceptors RCAR1/PYL9, RCAR3/PYL8, and RCAR11/PYR1. Using mutants defective in ABA biosynthesis, we show that the physiological activity associated with ABA precursors derives predominantly from their bioconversion to ABA. The ABA glucose ester conjugate, which is the most widespread storage form of ABA, showed weak ABA-like activity in germination assays and in triggering ABA signaling in protoplasts. The ABA conjugate and precursors showed negligible activity as a regulatory ligand of the ABI2/RCAR receptor complexes. The majority of ABA catabolites were inactive in our assays. To analyze the chemically unstable 8'- and 9'-hydroxylated ABA catabolites, we used stable tetralone derivatives of these compounds, which did trigger selective ABA responses. ABA synthetic analogs exhibited differential activity as regulatory ligands of different ABA receptor complexes in vitro. The data show that ABA precursors, catabolites, and conjugates have limited intrinsic bioactivity and that both natural and synthetic ABA-related compounds can be used to probe the structural requirements of ABA ligand-receptor interactions.

Closely related receptor complexes differ in their ABA selectivity and sensitivity.

The recent discovery of a variety of receptors has led to new models for hormone perception in plants. In the case of the hormone abscisic acid (ABA), which regulates plant responses to abiotic stress, perception seems to occur both at the plasma membrane and in the cytosol. The cytosolic receptors for ABA have recently been identified as complexes between protein phosphatases 2C (PP2C) and regulatory components (RCAR/PYR/PYL) that bind ABA. Binding of ABA to the receptor complexes inactivates the PP2Cs, thereby activating the large variety of physiological processes regulated by ABA. The Arabidopsis genome encodes 13 homologues of RCAR1 and approximately 80 PP2Cs, of which six in clade A have been identified as negative regulators of ABA responses. In this study we characterize a novel member of the RCAR family, RCAR3. RCAR3 was identified in a screen for interactors of the PP2Cs ABI1 and ABI2, which are key regulators of ABA responses. RCAR3 was shown to repress ABI1 and ABI2 in vitro, and to stimulate ABA signalling in protoplast cells. RCAR3 conferred greater ABA sensitivity to the PP2C regulation than RCAR1, whereas stereo-selectivity for (S)-ABA was less stringent with RCAR3 as compared with RCAR1. In addition, regulation of the protein phosphatase activity by RCAR1 and RCAR3 was more sensitive to ABA for ABI1 than for ABI2. Based on the differences we have observed in transcriptional regulation and biochemical properties, we propose a model whereby differential expression of the co-receptors and combinatorial assembly of the receptor complexes act in concert to modulate and fine-tune ABA responses.