Calcium signaling in Arabidopsis and potato

Abstract

This doctoral thesis presents an investigation into stress-related Ca2+ signaling in the model plant Arabidopsis thaliana and the crop Solanum tuberosum (potato). Chapter one describes the generation and characterization of Solanum tuberosum lines expressing the genetically encoded Ca2+ biosensor apoaequorin. We measured Ca2+ transients in response to different biotic and abiotic stimuli in comparison with the response observed in an established Arabidopsis apoaequorin line. We observed dose-dependent calcium signatures in response to a series of abiotic and biotic stress stimuli, including H2O2, NaCl, mannitol and the pathogen-associated molecular patterns (PAMPs), flg22 and Pep-13 with stimuli-specific kinetics. Direct comparison with Arabidopsis revealed differences in the kinetics and amplitude of Ca2+ transients between both species, implying species-specific sensitivity for different stress conditions. Furthermore, an additional potato sensor line expressing the redox-sensitive Grx1-roGFP2 probe was introduced. This system enabled the analysis of cytosolic redox dynamics in S. tuberosum and facilitated comparative studies with an existing redox-sensitive Arabidopsis sensor line. We observed that potato has a higher basal oxidative state compared to Arabidopsis, which may explain the differences in their Ca2+ signature in response to H2O2.<br /> Chapter two shows a very different approach towards the elucidation of Ca2+ signaling. Here we performed a full proteome analysis, investigating the effect of Ca2+ transients induced by an oxidative stress stimulus (H2O2) on protein regulation after 10 and 30 minutes of stress treatment. To differentiate between Ca2+-dependent and Ca2+-independent protein responses to oxidative stress, a subset of samples was treated with the Ca2+ channel inhibitor LaCl3, thereby suppressing the transient Ca2+ signal. Comparative analysis of proteomic data between H2O2 and LaCl3 + H2O2 treated samples vs control samples provided insights into the distinct regulatory mechanisms associated with oxidative stress. We identified a high number of differentially abundant proteins (DAPs) after the combined treatment with LaCl3 + H2O2 that did not change upon treatment with H2O2 alone, indicating a strong attenuating effect of Ca2+ signaling on the oxidative stress response. In addition, we identified H2O2 responsive proteins after 10 and 30 minutes of stress treatment, resulting in two distinct subsets, indicating that the duration of the stress exposure significantly shapes proteome-wide adaptations. These H2O2 responsive proteins were further categorized as strictly Ca2+-dependent, partially Ca2+-dependent or Ca2+-independent. This categorisation revealed Ca2+-dependent shifts in three proteins between the two time points, suggesting a dynamic role of Ca2+ in regulating proteomic changes. Interestingly, strictly Ca2+-dependent proteins predominantly showed reduced abundance, implying a role for Ca2+ in protein degradation, while Ca2+-independent proteins generally exhibited increased abundance, suggesting potential upregulation, possibly through transcription.<br /> Chapter three extends the findings of chapter two by employing a novel analytical framework, the Stress Knowledge Map (SKM), to further analyse the data. This computational tool compilates existing knowledge of plant stress response mechanisms extracted from published datasets, facilitating a systems-level analysis of stress signaling networks. The proteomic dataset obtained and analysed in Chapter 2 provided one of two case studies demonstration the application of SKM in complex analyses. It focussed on a subset of H2O2-responsive proteins and revealed that they could be linked to a source set of proteins related to Ca2+-signaling, either directly or through pathways of up to four steps. Integrating all identified shortest paths into a single network highlighted major network hubs connected to multiple known Ca2+ signaling genes, suggesting their potential role in regulating multiple targets.