ABA Receptor Rumbled?: The plant hormone abscisic acid (ABA) is critical for normal development and for mediating plant responses to stressful environmental conditions. Now, two papers present analyses of candidate ABA receptors (see the news story by Pennisi ). Ma et al. (p. 1064; published online 30 April) and Park et al. (p. 1068, published online 30 April) used independent strategies to search for proteins that physically interact with ABI family phosphatase components of the ABA response signaling pathway. Both groups identified different members of the same family of proteins, which appear to interact with ABI proteins to form a heterocomplex that can act as the ABA receptor. The variety of both families suggests that the ABA receptor may not be one entity, but rather a class of closely related complexes, which may explain previous difficulties in establishing its identity.

The phytohormone abscisic acid (ABA) plays an important role in several physiological responses such as stomatal conductance and seed dormancy and also in protecting plants against stress conditions such as drought and cold. Recently a family of proteins known as PYR/PYL/RCAR were identified as ABA receptors, but details of the signalling pathway downstream of ABA binding remained unclear. In this paper, Zhu and colleagues reconstitute the ABA-mediated signalling in vitro and test key observations in vivo. This is the first reported reconstitution of a plant hormone signalling pathway. The plant hormone abscisic acid (ABA) is a regulator of plant growth, development and responses to environmental stresses. Although several proteins have been reported to function as ABA receptors and many more are known to be involved in ABA signalling, the identities of ABA receptors remain controversial and the mechanism of signalling unclear. ABA-mediated signalling is now reconstituted in vitro, defining a minimal set of core components of the pathway. The phytohormone abscisic acid (ABA) regulates the expression of many genes in plants; it has critical functions in stress resistance and in growth and development1,2,3,4,5,6,7. Several proteins have been reported to function as ABA receptors8,9,10,11,12,13, and many more are known to be involved in ABA signalling3,4,14. However, the identities of ABA receptors remain controversial and the mechanism of signalling from perception to downstream gene expression is unclear15,16. Here we show that by combining the recently identified ABA receptor PYR1 with the type 2C protein phosphatase (PP2C) ABI1, the serine/threonine protein kinase SnRK2.6/OST1 and the transcription factor ABF2/AREB1, we can reconstitute ABA-triggered phosphorylation of the transcription factor in vitro. Introduction of these four components into plant protoplasts results in ABA-responsive gene expression. Protoplast and test-tube reconstitution assays were used to test the function of various members of the receptor, protein phosphatase and kinase families. Our results suggest that the default state of the SnRK2 kinases is an autophosphorylated, active state and that the SnRK2 kinases are kept inactive by the PP2Cs through physical interaction and dephosphorylation. We found that in the presence of ABA, the PYR/PYL (pyrabactin resistance 1/PYR1-like) receptor proteins can disrupt the interaction between the SnRK2s and PP2Cs, thus preventing the PP2C-mediated dephosphorylation of the SnRK2s and resulting in the activation of the SnRK2 kinases. Our results reveal new insights into ABA signalling mechanisms and define a minimal set of core components of a complete major ABA signalling pathway.

Abscisic acid (ABA) is an important phytohormone regulating seed dormancy, germination, seedling growth, and plant transpiration. We report here an Arabidopsis triple mutant that is disrupted in 3 SNF1-related protein kinase subfamily 2 (SnRK2s) and nearly completely insensitive to ABA. These SnRK2s, SnRK2.2, SnRK2.3, and SnRK2.6 (also known as OST1), are activated by ABA and can phosphorylate the ABA-responsive element binding factor family of b-ZIP transcription factors, which are important for the activation of ABA-responsive genes. Although stomatal regulation of snrk2.6 and seed germination and seedling growth of the snrk2.2 / 2.3 double mutant are insensitive to ABA, ABA responses are still present in these mutants, and the growth and reproduction of these mutants are not very different from those of the WT. In contrast, the snrk2.2 / 2.3 / 2.6 triple mutant grows poorly and produces few seeds. The triple mutant plants lose water extremely fast when ambient humidity is not high. Even on 50 μM ABA, the triple mutant can germinate and grow, whereas the most insensitive known mutants cannot develop on 10 μM ABA. In-gel kinase assays showed that all ABA-activated protein kinase activities are eliminated in the triple mutant. Also, the expression of ABA-induced genes examined is completely blocked in the triple mutant. These results demonstrate that the protein kinases SnRK2.2, SnRK2.3, and SnRK2.6 have redundant functions, and suggest that ABA signaling is critical for plant growth and reproduction.

Cold temperatures trigger the expression of the CBF family of transcription factors, which in turn activate many downstream genes that confer freezing tolerance to plants. It has been shown previously that the cold regulation of CBF3 involves an upstream bHLH-type transcription factor, ICE1. ICE1 binds to the Myc recognition sequences in the CBF3 promoter. Apart from Myc recognition sequences, CBF promoters also have Myb recognition sequences. We report here that the Arabidopsis MYB15 is involved in cold-regulation of CBF genes and in the development of freezing tolerance. The MYB15 gene transcript is up-regulated by cold stress. The MYB15 protein interacts with ICE1 and binds to Myb recognition sequences in the promoters of CBF genes. Overexpression of MYB15 results in reduced expression of CBF genes whereas its loss-of-function leads to increased expression of CBF genes in the cold. The myb15 mutant plants show increased tolerance to freezing stress whereas its overexpression reduces freezing tolerance. Our results suggest that MYB15 is part of a complex network of transcription factors controlling the expression of CBFs and other genes in response to cold stress.

Abscisic acid (ABA) is an important phytohormone regulating various plant processes, including seed germination. Although phosphorylation has been suggested to be important, the protein kinases required for ABA signaling during seed germination and seedling growth remain elusive. Here, we show that two protein kinases, SNF1-RELATED PROTEIN KINASE2.2 (SnRK2.2) and SnRK2.3, control responses to ABA in seed germination, dormancy, and seedling growth in Arabidopsis thaliana. A snrk2.2 snrk2.3 double mutant, but not snrk2.2 or snrk2.3 single mutants, showed strong ABA-insensitive phenotypes in seed germination and root growth inhibition. Changes in seed dormancy and ABA-induced Pro accumulation consistent with ABA insensitivity were also observed. The snrk2.2 snrk2.3 double mutant had a greatly reduced level of a 42-kD kinase activity capable of phosphorylating peptides from ABF (for ABA Response Element Binding Factor) transcription factors. ABA-induced expression of several genes whose promoters contain an ABA response element (ABRE) was reduced in snrk2.2 snrk2.3, suggesting that the mechanism of SnRK2.2 and SnRK2.3 action in ABA signaling involves the activation of ABRE-driven gene expression through the phosphorylation of ABFs. Together, these results demonstrate that SnRK2.2 and SnRK2.3 are redundant but key protein kinases that mediate a major part of ABA signaling in Arabidopsis.