Results of the first funding period

Detailed descriptions of the results achieved in the projects are provided in the Progress Reports and in the Preliminary Work parts of the individual reports and proposals of all partners. Therefore, here we only provide an overview of the achievements and describe the emerging models that should be addressed in the requested funding period.

One of the major goals during the last funding period was the advancement and application of Ca2+ imaging techniques based on ratiometric Ca2+ reporter proteins. A better description and understanding of the Ca2+ dynamics in responses to specific cues like ABA or nodulation factors is an essential basis for linking Ca2+ signaling events to Ca2+ decoding steps. This project part has been pursued in close collaboration of the groups of M. Parniske, K. Schumacher and J. Kudla. We have succeeded in identifying Ubi10 promoter driven Cameleon expression cassettes as being most suitable for Ca2+ analysis in Arabidopsis wild type and T-DNA mutant lines, in transient assays in N. benthamiana as well as an stably transformed Lotus japonicus. Moreover, we succeeded in generating Cameleon reporter proteins that are specifically targeted to the nucleus (NLS) or excluded from the nucleus (NES) or alternatively targeted to the plasma membrane or to the chloroplasts. Using these reporter lines we have been able to investigate Ca2+ dynamics at non-preceded sub-cellular and tissue-specific resolution.

Most interesting results from the group of M. Parniske highlight the importance of specific Ca2+-dependent phosphorylation for the regulation of complex biological processes like organogenesis. Rhizobial-induced calcium spiking in root cells is decoded by the Calcium- and Calmodulin-dependent Kinase CCaMK that interacts with and phosphorylates its target protein CYCLOPS. Most excitingly, a CYCLOPS version carrying serine residues 50 and 154 replaced with phosphomimic residues (‘CYCLOPS double D’) not only restored symbiosis in the cyclops mutant background, but spontaneously triggered nodule organogenesis independent of CCaMK and in the absence of rhizobia.

The identification of interactions between Ca2+-regulated kinases and ion channels and the characterization of the functional consequences of these interactions were a major goal on which several groups of the RU collaborated. Here, the groups of R. Hedrich, D. Becker and J. Kudla analyzed the effects of heteromerization of the CBL/CIPK-regulated ion channel AKT1 with its regulatory subunit KC1 and found that assembly of AKT1 and AtKC1 shifted the activation threshold of the root inward rectifier voltage gate negatively by approximately -70 mV and thereby contributes to preventing K+ loss through the uptake channels under physiological conditions (Geiger et al., 2009a). The groups of R. Hedrich and T. Romeis identified the anion channel SLAC1 as target of positive regulation by the SnRK2 kinase OST1 that is counteracted by the function of the PP2C ABI1 (Geiger et al, 2009b). In collaboration with E. Grill both groups extended our understanding of SLAC 1 regulation by the finding that CPK23 further contributes to SLAC 1 activation mediating a distinct Ca2+ sensitivity. Taken together, these findings suggest that the CPK and OST1 branches of ABA signal transduction in guard cells seem to converge on the level of SLAC1 under the control of the ABI1/ABA-receptor complex (Geiger et al., 2010). An additional very interesting level of SLAC1 regulation is suggested by the work of the groups of R. Hedrich and J. Kudla that observed phosphorylation of SLAC1 by CIPK23. This raises the possibility that CPKs and CIPKs exert an antagonistic function on SLAC1 regulation and that the prevalent mode of action may depend on the cellular Ca2+ concentration. Alternatively, only full phosphorylation of SLAC1 by both CPKs and CIPKs may allow for maximal activation of SLAC1. Dual regulation of ion channels by CIPKs and CDPKs appears to become an emerging general scheme that is suggested by the results of our additional studies. The outward rectifying K+ channel GORK appears to be positively regulated by CDPKs and CIPKs and negatively regulated by the PP2C ABI2 but not by ABI1 (collaboration D. Becker, T. Romeis, J. Kudla). Similarly, the tonoplast channels TPK1 (collaboration D. Becker, J. Kudla) and TPC1 (collaboration P. Dietrich, T. Romeis, J. Kudla) appear to be subject to regulation by CDPKs and CIPKs. Taken together, these results already point to a complex functional interplay of CDPKs, CIPKs, SnRK2s and PP2Cs. The identification of the transcription factor HB6, that plays a critical role in mediating ABA-induced transcriptional regulation, as a target of the kinase CIPK15 (and the phosphatase ABI1) extended the role of Ca2+-regulated kinases beyond the regulation of ion fluxes towards the regulation of transcriptional processes (collaboration E. Grill and J. Kudla).

Work by M. Hippler was focused on the establishment and improvement of in vitro and in vivo mass-spectrometric analyses of peptide and protein phosphorylation. In the course of these efforts e.g. novel in vivo phosphorylation sites of CPK23 (GCA2) were identified (collaboration E. Grill and M. Hippler). The studies by E. Grill not only identified the RCARs as true ABA receptors and uncovered the RCAR-PP2C-SnRK2 regulatory module as most important components of early ABA signaling. In recent work he also found that the CDPK GCA2 physically interacts with the ABA receptor components RCAR1 and 3 and is a constituent of the regulatory module controlling ABA responses. Moreover, GCA2 activity is controlled by reversible phosphorylation recruiting OST1 and the PP2C ABI2. OST1 inactivates GCA2 in the presence of Ca2+ by phosphorylation of GCA2 within the autoinhibitory domain while ABI2 dephosphorylates the site. This suggest that the reversible control of GCA2 by OST1 and the protein phosphatase ABI2 depending on Ca2+ levels provides a rheostat function, which integrates Ca2+ signals via GCA2 into early steps of ABA signaling.

Additional work of this RU further increased the complexity of interactions in early ABA and Ca2+ signaling and support the existence of a dynamic supra-molecular protein complex that provides a convergence point of Ca2+ and ABA signaling:

Work by K. Schumacher identified the protein kinase WNK8 as a critical component of ABA responses that interacts with the phosphatase PP2CA, which dephosphorylates the kinase WNK8. In addition, preliminary data suggest an interaction of WNK8 with the kinase CIPK10 (collaboration K. Schumacher and J. Kudla). Moreover, SnRK2s cannot only interact with CDPKs like GCA2 but also with CIPKs, like observed for OST1 and CIPK18 (J. Kudla). Finally, work in the group of J. Kudla has established that PPC2s and CIPKs not only interact with each other, but also that, for example, PP2CA reduces the autophosphorylation of CIPK24 and that, upon inhibition of the phosphatase activity of PP2CA, CIPK24 efficiently phosphorylates PP2CA. These results suggest the existence of a faithfully balanced phosphorylation/dephosphorylation interplay between CIPKs and PP2Cs that brings about the coordinated modulation of ABA signaling by Ca2+-dependent CIPKs right after ABA-signal perception by ABA-bound RCAR/PP2C complexes and at the same time allow for modulation of CIPK-dependent Ca2+ signaling by ABA-regulated PP2Cs.

This complex network of interactions that was identified during the last funding period of this RU should now be investigated by complementary genetic, biochemical, cell biological and electrophysiological approaches in a highly coordinated and collaborative effort of our research consortium

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