Cold temperatures trigger the expression of the CBF family of transcription factors, which in turn activate many downstream genes that confer chilling and freezing tolerance to plants. We report here the identification ofICE1(inducer ofCBFexpression1), an upstream transcription factor that regulates the transcription ofCBFgenes in the cold. AnArabidopsis ice1mutant was isolated in a screen for mutations that impair cold-induced transcription of aCBF3promoter-luciferase reporter gene. Theice1mutation blocks the expression ofCBF3and decreases the expression of many genes downstream ofCBFs, which leads to a significant reduction in plant chilling and freezing tolerance.ICE1encodes a MYC-like bHLH transcriptional activator. ICE1 binds specifically to the MYC recognition sequences in theCBF3promoter.ICE1is expressed constitutively, and its overexpression in wild-type plants enhances the expression of theCBFregulon in the cold and improves freezing tolerance of the transgenic plants.

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.

The perception of abiotic stresses and signal transduction to switch on adaptive responses are critical steps in determining the survival and reproduction of plants exposed to adverse environments. Plants have stress‐specific adaptive responses as well as responses which protect the plants from more than one environmental stress. There are multiple stress perception and signalling pathways, some of which are specific, but others may cross‐talk at various steps. Recently, progress has been made in identifying components of signalling pathways involved in salt, drought and cold stresses. Genetic analysis has defined the Salt‐Overly‐Sensitive (SOS) pathway, in which a salt stress‐induced calcium signal is probably sensed by the calcium‐binding protein SOS3 which then activates the protein kinase SOS2. The SOS3–SOS2 kinase complex regulates the expression and activity of ion transporters such as SOS1 to re‐establish cellular ionic homeostasis under salinity. The ICE1 (Inducer of CBF Expression 1)–CBF (C‐Repeat Binding Protein) pathway is critical for the regulation of the cold‐responsive transcriptome and acquired freezing tolerance, although at present the signalling events that activate the ICE1 transcription factor during cold stress are not known. Both ABA‐dependent and ‐independent signalling pathways appear to be involved in osmotic stress tolerance. Components of mitogen‐activated protein kinase (MAPK) cascades may act as converging points of multiple abiotic as well as biotic stress signalling pathways. Forward and reverse genetic analysis in combination with expression profiling will continue to uncover many signalling components, and biochemical characterization of the signalling complexes will be required to determine specificity and cross‐talk in abiotic stress signalling pathways.