Featured Article - November 2009
Short description: After years of controversy, two groups have independently identified the same receptor family for the plant stress hormone abscisic acid.
Abscisic acid (ABA) is a small-molecule hormone involved in plants' responses to stress. Despite reports over the past few years, its receptor has not been identified beyond all doubt, as a trail of retracted papers attests. Now two papers published in Science independently identify members of the same family of proteins as ABA receptors 1, 2 .
Park et al. 1 , in a bid to understand ABA's actions, used a synthetic selective ABA agonist and germination inhibitor, pyrabactin, to overcome the problem of genetic redundancy in members of the ABA pathway that has thwarted researchers so far. Using this inhibitor, they identified 12 PYRABACTIN RESISTANCE 1 (Pyr1) mutant alleles.
Further analysis showed that Pyr1 and Pyl (PYR-like) are necessary for many of the plant's responses to ABA in vivo. Pyr1 encodes a member of the START domain superfamily, and because of this Park et al. hypothesize that pyrabactin stimulates protein–protein interactions between PYR1 and a downstream effector.
Yeast two-hybrid assays using about 2 million cDNA clones indicated that PYR1 interacts with the type 2C protein phosphatase (PP2C) HAB1 in response to pyrabactin. The significance of this interaction was confirmed by control two-hybrid assays that included the use of two mutants PYRS152L and PYR1P88S, which reducesensitivity to pyrabactin sensitivity in plants. Further examination of the PYR and PYL family interactions suggested that at least six members of the family are involved in ABA responses.
Park et al. then used NMR spectroscopy in collaboration with PSI CESG to detect whether ABA binds to PYR1. As the START protein family contains a conserved ligand-binding cavity, Park et al.'s experiments focused on this probable ABA-binding site. Chemical-shift analysis suggested that ABA binds PYR1 and probably induces a conformational change.
The authors also used NMR spectroscopy to look at PYR1's interaction with HAB1 and compare it with the binding of the mutant PYR1P88S. Using this technique, they find that the mutant PYR1P88S still binds ABA, but is unable to bind to HAB1 when ABA is present, explaining its defective phenotype in the plant.
As PP2Cs are negative regulators of ABA signaling, Park et al. investigated whether PYR/PYL interactions with HAB1 inhibit phosphatase activity, and the experiments confirmed their supposition: PYR/PYLs regulate PP2Cs in response to ABA. From the demonstrated specificity of pyrabactin they concluded that PYR/PYLs are ABA receptors that control ABA signaling by inhibiting the PP2Cs that normally inhibit signaling. PP2Cs are thought to inhibit ABA signaling by dephosphorylating and inhibiting the SnRK2 kinases, which are known positively acting factors regulating the pathway.
Ma et al. 2 independently identified the same family of receptors for ABA using a different approach. By examining known negative regulators of ABA signaling — the PP2C proteins ABI1 and AB2 — they discovered a protein they named regulatory component of ABA receptor 1 (RCAR1).
They showed that in the presence of RCAR1, ABA instantaneously blocks the phosphatase activity of ABI2. Further experiments indicated that a single molecule of ABA binds a single RCAR1 molecule, and suggested that RCAR1 has all the characteristics of a receptor. This RCAR1 molecule belongs to the same 14-member PYR/PYL family identified by Park et al.
Two approaches have arrived at the same receptor for ABA, which shows the power of complementary techniques and specific chemical probes in combating the problem of functional redundancy.
S. Y. Park, P. Fung, N. Nishimura, D. R. Jensen, H. Fujii et al. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins.
Science 324, 1068-1071 (2009). doi:10.1126/science.1173041
Y. Ma, I. Szostkiewicz, A. Korte, D. Moes, Y. Yang et al. Regulators of PP2C phosphatase activity function as abscisic acid sensors.
Science 324, 1064-1068 (2009). doi:10.1126/science.1172408