Evaluation of type-B RR dimerization in poplar: A mechanism to preserve signaling specificity?

Plants possess specific signaling pathways, such as the MultiStep Phosphorelay (MSP), which is involved in cytokinin and ethylene sensing, and light, drought or osmotic stress sensing. These MSP comprise histidine-aspartate kinases (HKs) as receptors, histidine phosphotransfer (HPts) proteins acting as phosphorelay proteins, and response regulators (RRs), some of which act as transcription factors (type-B RRs). In previous studies, we identified partners of the poplar osmosensing signaling pathway, composed of two HKs, three main HPts, and six type-B RRs. To date, it is unresolved as to how cytokinin or osmotic stress signal specificity is achieved in the MSP in order to generate specific responses. Here, we present a large-scale interaction study of poplar type-B RR dimerization. Using the two-hybrid assay, we were able to show the homodimerization of type-B RRs, the heterodimerization of duplicated type-B RRs, and surprisingly, a lack of interaction between some type-B RRs belonging to different duplicates. The lack of interaction of the duplicates RR12-14 and RR18-19, which are involved in the osmosensing pathway has been confirmed by BiFC experiments. This study reveals, for the first time, an overview of type-B RR dimerization in poplar and makes way for the hypothesis that signal specificity for cytokinin or osmotic stress could be in part due to the fact that it is impossible for specific type-B RRs to heterodimerize.

Highlighting type A RRs as potential regulators of the dkHK1 multi-step phosphorelay pathway in Populus.

In previous studies, we highlighted a multistep phosphorelay (MSP) system in poplars composed of two hybrid-type Histidine aspartate Kinases, dkHK1a and dkHK1b, which interact with three Histidine Phosphotransfer proteins, dkHPt2, 7, and 9, which in turn interact with six type B Response Regulators. These interactions correspond to the dkHK1a-b/dkHPts/dkRRBs MSP. This MSP is putatively involved in an osmosensing pathway, as dkHK1a-b are orthologous to the Arabidopsis osmosensor AHK1, and able to complement a mutant yeast deleted for its osmosensors. Since type A RRs have been characterized as negative regulators in cytokinin MSP signaling due to their interaction with HPt proteins, we decided in this study to characterize poplar type A RRs and their implication in the MSP. For a global view of this MSP, we isolated 10 poplar type A RR cDNAs, and determined their subcellular localization to check the in silico prediction experimentally. For most of them, the in planta subcellular localization was as predicted, except for three RRAs, for which this experimental approach gave a more precise localization. Interaction studies using yeast two-hybrid and in planta BiFC assays, together with transcript expression analysis in poplar organs led to eight dkRRAs being singled out as partners which could interfere the dkHK1a-b/dkHPts/dkRRBs MSP identified in previous studies. Consequently, the results obtained in this study now provide an exhaustive view of dkHK1a-b partners belonging to a poplar MSP.

Arabidopsis ABA response gene ABI1: features of a calcium-modulated protein phosphatase.

The Arabidopsis ABI1 locus is essential for a wide spectrum of abscisic acid (ABA) responses throughout plant development. Here, ABI1 was shown to regulate stomatal aperture in leaves and mitotic activity in root meristems. The ABI1 gene was cloned and predicted to encode a signaling protein. Although its carboxyl-terminal domain is related to serine-threonine phosphatase 2C, the ABI1 protein has a unique amino-terminal extension containing an EF hand calcium-binding site. These results suggest that the ABI1 protein is a Ca(2+)-modulated phosphatase and functions to integrate ABA and Ca2+ signals with phosphorylation-dependent response pathways.