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Biochemical Regulation of CDPKs

The binding of calcium induces a conformational change that exposes the active site of the enzyme and induces catalytic activity as auto-phosphorylation and substrate phosphorylation (Fig. 1, left). In one topic, we address the critical calcium concentration and binding cooperativity of the different calcium binding sites, in a second, the regulatory role of auto-phosphorylation sites, and in a third, the calcium-induced changes in protein folding and structure. Conformational changes in the enzyme therefore determine the level of its activity. One can distinguish strictly calcium-dependent and catalytically highly active enzymes from isoforms that exhibit some level of basal enzyme activities that are almost calcium-independent (Fig. 2, middle).

Fig. 1 Activation model of CDPKs. Scheme of the CDPK conformation in the calcium (Ca2+)-bound and -unbound form. In the calcium-bound active conformation, the CDPK can auto-phosphorylate itself and phosphorylate substrate proteins (left panel). Measurements of the kinase activity of two CDPKs with distinct calcium-affinities over a calcium-concentrations range (middle panel). The intracellular concentration of free calcium can be visualized by using a fluorescence-based calcium-sensor. The phytohormone abscisic acid (ABA) induces calcium spiking in stomata (right panel).

Our research aims to describe this biochemical regulation in planta for selected CDPKs and to understand the biochemical regulation in the context of biological stress responses or developmental cues. To this end, we have established a FRET-based method that allows tracking the calcium-induced conformational change of several CDPKs in real time. This approach revealed a close correlation between conformational changes and activity of the enzymes. We use this method (1) to decipher specific amino acids that determine enzyme activity, (2) to study the integration of already known CPDKs in their specific signaling cascades, and (3) to identify the biological context and function of as yet poorly characterized CDPKs.


Techniques:

  • Protein chemistry and enzyme analysis
  • Phosphoproteomics
  • Fluorescence-based imaging using FRET

Plant Defense

Interactions of plants with microbial pathogens or pests lead to the activation of specific plant defense responses. These comprise of (1) a local initiation phase, which includes the recognition of the attack to the induction of intracellular signaling pathways, followed by (2) the systemic propagation of defense signals throughout the plant, and (3) the manifestation of defensive responses that establish resistance (Fig. 2, Seybold et al., New Phytol. 2015; Hake und Romeis, Plant Cell Environ. 2018).

Fig. 2: Larval feeding activates defense in Arabidopsis plants (left panel). This includes the local defense initiation phase, including recognition and induction of signaling pathways, systemic activation of defense signals and phytohormones, and the manifestation of defense responses as a prerequisite for resistance (middle panel). Local wounding induces calcium concentration changes also in distal previously unwounded leaves, which is visualized by a fluorescence-based calcium-sensor (right panel).

We have determined functions of CDPKs in all three phases of plant resistance responses and have already identified in vivo phosphorylation substrates of selected CDPKs in plant defense. These reflect multiple functions in the decoding of calcium both at the plasma membrane and in the nucleus (Dubiella et al., PNAS 2013; Liu et al., Plant Cell 2017; Guerra et al., New Phytol. 2019). In particular, CDPK function together with NADPH oxidases in signal transduction upon bacterial infection and are crucial for generation of specific pathogen-induced metabolite signals. These chemical mediators are not only a prerequisite for systemic activation of defense reactions, but also for establishing plant immune memory.

In our research, we investigate calcium-mediated molecular, biochemical, and cell biological mechanisms that underlie the spatial and temporal resolution of resistance. These include the activation of systemic acquired resistance, molecular memory of an earlier attack (so-called priming), as well as erasing this information when no further attack is anticipated. By studying CDPK-mediated decoding of calcium signals, enzyme stability and molecular interactions with other proteins, as well as changes in gene expression as a result of CDPK activation, and the pattern of specific metabolites (phytohormones), we aim to obtain a more complete picture of CDPK function in antimicrobial and antiherbivore defense.


Techniques:

  • Biological tests of bacterial growth and herbivory
  • Molecular biology and gene expression analysis
  • Fluorescence-based calcium imaging
  • Metabolomics

Biotechnological Transfer

Abiotic changes in the environment of plants, such as limited water and nutrient availability, induce biochemical and physiological mechanisms of acclimation in plants. Our research addresses the function of calcium decoding by CDPKs that underlie some of these acclimations (Liese et al., Biochim. Biophys. Acta 2013; Schulz et al., Plant Physiol. 2013; Kudla et al., New Phytol. 2018). With respect to improving drought tolerance in interplay with ABA signal transduction, we, in collaboration with other groups, are investigating the function of CDPKs in regulating the opening and closing of the guard cell induced by the phosphorylation of CDPK substrates, e.g., ion channels (Geiger et al., PNAS 2009, 2010, 2011). We also study calcium-dependent enzyme parameters, which allow differentiation of different CDPK isoforms that could regulate the same substrate. The differences in the regulation of CDPK enzyme activity provide a basis for deciding whether an enzyme is suitable for biotechnological transfer. In addition to the acclimation and adaptations of plants to drought stress, biochemical and molecular responses to osmotic stress, cold, and nutritional deficiencies are investigated. Our studies are not limited to the model plant Arabidopsis thaliana but have already been extended to tobacco (Nicotiana tabacum), barley (Hordeum vulgare) and maize (Zea mays), (Weckwerth et al., Plant Cell Environ. 2014).

Fig. 3: Workflow for biotechnology transfer in crop plants. Genes encoding signal transduction proteins are cloned into plant vectors. Changes in the DNA sequence can be inserted so that in addition to the native proteins also proteins with changes in the amino acid sequence are encoded. Upon transfer in suitable crop, transgenic plants can be assessed under various stress conditions for improved growth and yield.

Our starting point is to understand the specific biochemical mechanisms that allow maintaining plant growth under abiotic stress. We aim to characterize the protein components involved and to decipher their corresponding molecular and structural basis that collectively result in stress tolerance. The interplay of function, biochemical property, and regulation of identified signal components forms the basis for the decision for transfer to crops such as cereals or potatoes. The resulting crops are tested for altered growth, yield, and stress tolerance in various stress situations (Fig. 3).


Techniques:

  • Biological growth, yield, and tolerance tests
  • Physiological studies

This page was last modified on 13.02.2020.

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