In vitro reconstitution of an abscisic acid signalling 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 development. Several proteins have been reported to function as ABA receptors, and many more are known to be involved in ABA signalling. However, the identities of ABA receptors remain controversial and the mechanism of signalling from perception to downstream gene expression is unclear. 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 ABAresponsive 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. Several ABA receptors have been reported, although many of them remain unconfirmed. Recently, a family of novel START domain proteins, known as PYR/PYLs (also known as RCARs), were identified as ABA receptors. Several of the PYR/PYLs were shown to interact with and inhibit clade-A PP2Cs. The PP2Cs (ABI1, ABI2, HAB1 and PP2CA/AHG3) negatively regulate ABA responses. In contrast, a subfamily of ABA-activated SnRK2s are positive regulators of ABA signalling. Through unknown mechanisms, the inhibition of the negatively acting PP2Cs leads to the successful activation of a subfamily of SnRK2 kinases (SnRK2.2, SnRK2.3 and SnRK2.6 in Arabidopsis), which phosphorylate the basic leucine zipper (bZIP) transcription factors called ABFs/AREBs. The ABFs bind to ABA-responsive promoter elements (ABRE) to induce the expression of ABA-responsive genes. The present study was aimed at defining the core components of the ABA response pathway that are both necessary and sufficient for ABA perception, signalling, and finally ABA-responsive gene expression. It has been suggested that ABA-dependent phosphorylation of ABF2 at amino-acid residues S26, S86, S94 and T135 is important for stress-responsive gene expression in Arabidopsis. We used transient activation analysis with protoplasts from the snrk2.2/2.3/2.6 triple mutant to determine the role of ABF2 phosphorylation and its dependence on SnRK2s for ABA-responsive gene expression. We have shown previously that the snrk2.2/2.3/2.6 triple mutant is deficient in ABA responses. As expected, transfection of snrk2.2/2.3/2.6 protoplasts with ABF2 did not induce RD29B–LUC (luciferase reporter gene driven by the ABA-responsive RD29B promoter) expression even in the presence of ABA, but co-transfection of ABF2 with SnRK2.6 resulted in the induction of RD29B–LUC in an ABA-dependent manner (Fig. 1a). Furthermore, ABF2 with alanine substitutions at all of the four phosphorylation sites was inactive, whereas aspartic acid substitutions at these sites led to a constitutively active ABF2, resulting in the induction of RD29B–LUC expression even without ABA treatment (Fig. 1a). Co-transfection of Alasubstituted ABF2 with SnRK2.6 led to only a very low level of RD29B–LUC induction (Fig. 1a). Replacement of lysine 50, a conserved residue critical for ATP-binding and kinase activity, with asparagine (K50N) inactivates SnRK2.6 in phosphorylation assays in vitro (H.F. and J.-K.Z., unpublished observations). Co-transfection of ABF2 with SnRK2.6 did not induce RD29B–LUC expression (Fig. 1a), demonstrating that the kinase activity is necessary for ABF2 activation. Transfection of ABF2 alone in wild-type protoplasts induced a low level of RD29B–LUC expression under ABA treatment, which is consistent with the presence of a low basal level of endogenous ABA-signalling components in the protoplasts (Supplementary Fig. 1a). These results show that SnRK2.6 mediates ABF2 activation in an ABA-dependent manner, and that ABF2 phosphorylation is sufficient for the induction of RD29B–LUC expression by ABA. We next tested the effect of ABI1 and PYR1 on the induction of RD29B–LUC expression by ABA. Transfection of ABI1 together with ABF2 and SnRK2.6 resulted in inhibition of RD29B–LUC expression (Fig. 1b and Supplementary Fig. 1a). This shows that ABI1 negatively regulates the activation of RD29B–LUC expression that is dependent on SnRK2.6 and ABF2. Addition of PYR1 together with ABI1, SnRK2.6 and ABF2 enabled the ABA-dependent induction of RD29B–LUC expression (Fig. 1b and Supplementary Fig. 1a). However, addition of PYR1, which is defective in interaction with and inhibition of PP2Cs, did not enable the ABA-dependent induction of RD29B–LUC expression. The dominant abi1-1 mutation (G180D) disrupts the interaction between ABI1 and PYR1 (ref. 12). Like the wild-type ABI1, ABI1 also inhibited the effect of SnRK2.6 and ABF2 on RD29B–LUC expression in response to ABA, but this antagonistic effect could not be overcome by expression of PYR1 (Fig. 1b and Supplementary Fig. 1a). This suggests that the ABI1 mutant protein retains the inhibitory activity but can no longer be regulated. Thus reconstitution with PYR1, ABI1, SnRK2.6 and ABF2 is sufficient