Scientific Goals

Scientific objectives in this funding period

The initiative for the implementation of this RU was founded on the common interest of individual groups in the understanding of Ca2+-dependent signaling networks in plants. Among the range of topics covered by the RU questions towards the contribution of different sources of calcium entry, the molecular pathways for calcium transport, the decoding elements of calcium signaling and their targets as well as their role within certain pathways have been addressed. Complementary methods including Ca2+ imaging, proteomics, genetics, biophysical characterization of transport protein function as well as in vitro and in vivo protein interaction analyses have been assembled in a multidisciplinary team allowing a qualitative and quantitative analysis of selected physiological processes such as control of water-use efficiency, adaptation to nutrient shortage or interaction of plant cells with microbes. The formation of this RU has allowed to bundle the efforts for investigating Ca2+-mediated regulation and information processes in an integrative approach and has enabled researchers to jointly explore key aspects of Ca2+ signaling that they would not have been able to address in individual projects. The synergistic efforts of this RU have advanced our understanding of the role of phosphorylation mediated processes in Ca2+ signaling to a point that facilitates to approach a scientifically most interesting and innovative strategic goal that arose from the results of collaborative research during the last funding period:

The in vivo and in vitro analysis and reconstitution of Ca2+-regulated signaling pathways

In order to achieve this goal we will further pursue and advance several complementary approaches:

  1. We will use our advanced Ca2+ reporter protein systems for investigation of Ca2+ dynamics in several model systems at high tissue-specific and sub-cellular resolution and will further extend our Ca2+ reporter protein toolkit. Here, one focus will be the nodulation response in Lotus japonicus. In Arabidopsis we will exemplarily investigate Ca2+ signatures in response to cold and salt stress as well after ABA application. Our preliminary data have already provided evidence that these responses trigger tissue-specific Ca2+ elevations and, therefore, this so far unknown aspect of Ca2+ signaling should be addresses in depth. Moreover, our already available specifically sub-cellular targeted Ca2+ reporter proteins will enable the investigations in so far non-preceded sub-cellular resolution. In addtion, these tools will allow completely new designs of experimental setups. For example, for stimuli like hyper-polarization, that induce simultaneous Ca2+ signatures in the cytosol and in the nucleus, the NLS-YC3.6 reporter can be used to mirror the cytoplasmic Ca2+ signatures. By combination of the NLS-YC3.6 reporter with organelle targeted (e.g. chloroplast targeted) or membrane targeted (e.g. plasma membrane targeted) Ca2+ sensors in the same plant it will become feasible to simultaneously investigate the temporal order of Ca2+ dynamics, for example, at the plasma membrane (or the chloroplast) with changes in cytoplasmic Ca2+ dynamics (that will be mirrored by NLS-YC3.6). Moreover, during the proposed funding period we will further extend this toolkit for analyzing Ca2+ dynamics in plants by generating Ca2+ reporter proteins that are targeted to additional compartments (e.g. ER, Golgi). Finally, by performing analyses of Ca2+ dynamics in mutant backgrounds of Ca2+ responders or relays we can uncover the function of these proteins in shaping Ca2+ signatures.

  2. We will further dissect the role of phosphorylation of the CCaMK target CYCLOPS for organogenesis and symbiotic infection. The surprising discovery that CYCLOPS double D is leading to spontaneous nodulation has revealed an unpredicted key role of CYCLOPS in the regulation of organogenesis. However CYCLOPS also has an essential function in symbiotic infection (Yano et al., 2008). We hypothesize that additional phosphorylation sites are involved in this process, since CYCLOPS double D did not complement infection in a ccamk mutant background. We will subject the CCaMK/CYCLOPS complex to a detailed biochemical analysis and will attempt a structural characterization. Moreover, we will investigate candidate components involved in the crosstalk between nodulation and ABA signaling

  3. A central scientific question addressed by several groups of the RU will be the modulation and interconnection of hormone perception by Ca2+-regulated kinases/phosphatases. Here, the ABA signaling system will represent a prominent model system but also the interconnection to jasmonate signaling will be addressed. We will pursue several complementary approaches that will encompass mutant analyses, interaction studies, biochemical and electrophysiological analyses as well as cell biological studies. A most promising approach is provided by the simultaneous reconstitution of complete ABA signaling pathways and their interacting Ca2+ signaling components for example in yeast, Xenopus oocytes and in vitro. Central to these investigations will be the components of the proposed supra-molecular complex or network represented by RCARs, PP2Cs, SnRKs2, CDPKs, CIPKs and WNKs as well as their target proteins. The surprising finding by the group of D. Becker that ABI2 and its interacting CBL/CIPK complexes represent central components of jasmonate signaling and GORK regulation, will allow to investigate whether and how JA feeds back on the regulation of potassium channel activity and will allow to highlight novel interconnections in hormone and Ca2+ signaling, like a role of ABI2 as a merging point of ABA and JA signaling pathways. Furthermore, this research approach may be complemented by the group of Maik Böhmer (see below, depending on success of his funding application), who also addresses JA responses in the context of ABA signaling.

  4. The functional characterization of Ca2+-regulated ion transport processes and transcriptional responses. Here, in addition to investigate the interplay of phosphorylation/dephosphorylation in regulation of target proteins, a most interesting novel aspect will be the interconnection of CDPK and CIPK mediated target phosphorylation. Based on the results of the last funding period this research will focus on channels like TPC1, GORK, TPK1 and CNGCs. These studies will be extended by analyses of the functional assembly of R-type anion channel subunits (ALMTs) and their interaction/regulation by Ca2+-dependent kinases. This research will be further extended to investigating the interconnection of Ca2+ signaling and transcriptional responses as exemplified by the regulation of the ABA-responsive transcription factor HB6 by CIPK15/ABI1.

  5. We will perform mass-spectrometry based phosphorylation site mapping on kinases and kinase substrates to enable the functional characterization of the role of these phosphorylations and will perform non-biased kinase target identification by comparative phospho-proteomics analyses. For comparative purposes differential 14N/15N labeling will be employed using assays with 14N/15N-labeled recombinant protein kinases and their respective differentially labeled substrates. From these assays the efficiency and amount of specific site phosphorylation events will be enumerated.

Funding by:


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