The B-type response regulators are the final output elements of the His-to-Asp phosphorelay system. In spite of a systematic identification and characterization of the different partners belonging to these systems in Arabidopsis, Zea mays, Glycine max, and Oryza sativa, only few members of these systems from herbaceous plants have been identified as drought-related actors. In Arabidopsis, it has been demonstrated that the hybrid Histidine-aspartate Kinase, AHK1, is a positive regulator of osmotic stress signaling and that AHK1 gene expression is regulated by salt stress and dehydration [13, 14]. Moreover, among three authentic AHPs (AHP1, 2 and 3), only AHP2 was shown to be an interacting partner of this osmosensor . However, the B-type RR interacting partners of AHP2 [16, 17, 28] clearly play a pivotal role in the response to cytokinin [29–32] or to ethylene . Until now, very few information is available concerning an interaction network between osmotic stress signaling partners belonging to the multistep phosphorelay system.
The aim of the present study was to determine, in Populus, the potential third partners in osmotic stress signaling by bringing out interactions between B-type RRs and three HPts main interacting partners of the putative osmosensor HK1. Based on a homology approach, we isolated eight cDNAs, from the poplar Dorskamp genotype, encoding RRs sharing the common features of B-type RR family members. A phylogenetic analysis of poplar B-type RRs and several RRs from other model plant species revealed that in most cases, B-type RR family members can be classified independently of species but present pairings within species. In Arabidopsis, based on sequence comparisons and expression patterns, the B-type RRs have been divided into three sub-groups: BI, the largest sub-group that contains ARR involved in cytokinin responses [25, 29], BII and BIII represented respectively by the pairs ARR13/ARR21 and ARR19/ARR20 [27, 33, 45]. Our rooted tree produced the same global scheme: the B-type RRs of the sub-group BI of Arabidopsis clustered together notably with a high number of B-type RRs from the other species and more particularly with isolated poplar B-type RRs.
We did not succeed in isolating RR17 or 20 which belong to the sub-group II, or the RR18 of sub-group I. Nevertheless, as shown in P. trichocarpa genotype, RR17 and RR20 are not expressed in roots , in agreement with our difficulties in isolating them. On the other hand, in the Dorskamp genotype, we isolated RR14 but not RR18, which were undetected and detected respectively in P. trichocarpa genotype. These differential expressions of B-type RRs observed in P. trichocarpa and P. deltoides (Bartr.) Marsh x P. nigra L. Dorskamp genotypes could be explained by their genetic variability.
To identify the partners potentially involved in the osmosensing pathway, we performed a global interaction analysis in yeast between HPts (HPt2, 7, and 9), the main partners of HK1, and the eight isolated B-type RRs. Both qualitative approaches used allowed us to obtain an overview of B-type RR partners of these HPts. Except for RR19, strong interactions between all B-type RRs and the three HPts were detected with different interaction profiles. Based on different activation levels of the second reporter gene (β-galactosidase), three RR groups emerged for HPt2 and HPt9, while the RRs are divided into two groups for HPt7.
To substantiate the partnership observed by Y2H analysis, some HPt/RR interactions were also analyzed in planta using BiFC assays. Among interactions observed in yeast, two HPts and two B-type RRs were chosen: i) HPt2 and HPt9, that present the strongest interaction with HK1 and seem to be key actors of the osmosensing pathway on the basis of their expression profiles in Populus, ii) RR13 and RR16, that display strong or weak β-galactosidase activity with the three HPts, respectively. These BiFC assays demonstrated that HPt2 and HPt9 interact with RR13 or RR16 in the C. roseus cells. In Arabidopsis, an interaction study concerning Cytokinin Response Factors (CRFs) and other members of cytokinin signaling pathway showed that BiFC technique led to a more complete detection of interactions than with the Y2H method . Based on this study and due to the lack of HPt2, 7, 9/RR19 interactions observed in Y2H system, we decided to complete the interaction study by testing the ability of RR19 to interact in planta with HPts. As for the CRFs study, the BiFC analysis showed that RR19 displayed interactions with HPt2, 7 and 9. Cutcliff et al. , speculated that the lack of some interaction in Y2H system can be explained by the absence of plant specific post-translational modifications on protein or plant-specific protein facilitating the interactions of this protein. The whole of HPt/RR interactions in planta occurs in the nucleus. To validate this study, we confirmed that HPt2 and 9 have a nuclear and cytosolic localization while the RR13 and 16 are localized in the nucleus. Although the localization of the second and third partners has already been described in Arabidopsis[22–27], and that the nuclear localization of HPt/B-type RR interactions is expected, the localization of these interactions was until now only putative. Our study shows clearly this localization for the first time in plant cell.
To reinforce the hypothesis of interactions between the second and third partners in planta, an analysis of the concomitant presence of the transcripts of both partners (HPts and B-type RRs) in the same plant organs was performed. A previous analysis of expression of HK1 and HPt genes in four different organs of poplar cuttings under control and osmotic stress conditions revealed that only HK1, HPt2, 7 and 9 are co-expressed in all organs and conditions tested . In the present study, we also analyzed the B-type RR genes expression in the same poplar cuttings organs and conditions, which led us to show that only RR12, 13, 14, 16 and 19 are expressed in the same organs and conditions than HK1 and the three HPts. By contrast to HPts, none B-type RR showed an organo-specific expression. However, the absence of RR15, 21 and 22 transcripts in organs and conditions tested, even though they are able to interact physically with HPts in yeast, allows us to conclude that these B-type RRs are of lesser importance in the osmosensing signaling pathway in these organs.
The five poplar B-type RRs, RR12, 13, 14, 16 and 19, interacting with HPt2, 7 and 9, the predominant partners of HK1, and concomitantly expressed in the main organs of poplar, could be probably involved in a poplar osmosensing pathway. Nevertheless, some Arabidopsis B-type RRs homologous to these five poplar RRs are transcription factors of cytokinin or ethylene signaling pathways, such as ARR2 involved in leaf senescence , ethylene response  and resistance against pathogens . Consequently, these transcription factors would behave as a set of protagonists linking several signaling pathways to coordinate different developmental processes and stress responses . This hypothesis is consistent with the known cross-talk between cytokinin, ABA and abiotic stresses [13, 49, 50].