The activation of the Arabidopsis P-ATPase 1 by the brassinosteroid receptor BRI1 is independent of threonine 948 phosphorylation

The plasma membrane-spanning receptor brassinosteroid insenstive 1 (BRI1) rapidly induces plant cell wall expansion in response to brassinosteroids such as brassinolide (BL). Wall expansion is accompanied by a rapid hyperpolarization of the plasma membrane, which is recordable by measuring the fluorescence lifetime (FLT) of the green fluorescent protein (GFP) fused to BRI1. For the BL induction of hyperpolarization and wall expansion, the activation of the plasma membrane P-type H⁺-ATPase is necessary. Furthermore, the activation of the P-ATPase requires BRI1 kinase activity and appears to be mediated by a BL-modulated association of BRI1 with the proton pump. Here, we show that BRI1 also associates with a mutant version of the Arabidopsis P-ATPase 1 (AHA1) characterized by an exchange of a well-known regulatory threonine for a non-phosphorylatable residue in the auto-inhibitory C-terminal domain. Even more important, BRI1 is still able to activate this AHA1 mutant in response to BL. This suggests a novel mechanism for the enzymatic activation of the P-ATPase by BRI1 in the plasma membrane. Furthermore, we demonstrate that the FLT of BRI1-GFP can be used as a non-invasive probe to analyze long-distance BL signaling in Arabidopsis seedlings.Key words: BRI1, fluorescence lifetime, membrane potential, P-ATPase, cell wall expansionUsing spectro-microscopic technologies, we recently started the quantitative analysis of the properties and subcellular function of GFP fusion of the plasma membrane-localized brassinosteroid (BR) receptor, BRI1, in living plant cells of Arabidopsis thaliana and tobacco (Nicotiana benthamiana) leaf cells.^1,2 Brassinosteroids, such as brassinolide (BL), are involved in responses to biotic and abiotic stresses and developmental processes, including cell elongation.³ The present model of the BR response pathway includes the binding of BRs to BRI1, resulting in the autophosphorylation of the receptor and the subsequent recruitment of the co-receptor BRI1-associated receptor kinase 1 (BAK1). This association is followed by trans-phosphorylation between BRI1 and BAK1 and results in the activation of downstream BR signaling processes leading to differential gene expression and, finally, to the execution of the specific responses.⁴ However, the molecular events that take place in the plasma membrane immediately after the perception of BL and initiate cell elongation still have to be included in this model.⁵ We recently reported a rapid BRI1-GFP-dependent cell wall expansion in Arabidopsis seedlings, which is attributed to wall loosening and water incorporation into the wall, and precedes cell elongation.^1,2 This expansion response was accompanied by a change in the FLT of BRI1-GFP, which reflects an alteration in the plasma membrane potential (E_m).^2,6 For both the FLT change in BRI1-GFP and the wall expansion, the activity of the plasma membrane P-ATPase is crucial. Notably, H⁺-pump activation was shown to depend on the kinase activity of BRI1.² This suggests a fast BRI1-dependent response pathway in the plasma membrane which links BL perception via P-ATPase activation and E_m hyperpolarization to wall expansion. In this report, we demonstrate that the phosphorylation of a conserved threonine in the auto-inhibitory domain of AHA1 is not required for the enzymatic activation by BRI1 suggesting a novel mechanism by which BRI1 may initiate the activation of the P-ATPase. Furthermore, we show that the FLT of BRI1-GFP is a useful and senstitive probe for the non-invasive analysis of systemic signaling processes in living plants.