Quantification of calcium signatures in roots of Arabidopsis thaliana

Calcium (Ca2+) is an essential second messenger in plant cells linking the perception of stresses at the plasma membrane to the appropriate defense response. The calcium signature theory states that for each perceived stress there is a unique calcium transient that triggers specific downstream responses. It is thought that the signaling specificity is encoded in the spatio-temporal pattern of cytosolic calcium concentration, which is in turn decoded by various intracellular calcium binding proteins. For 25 years now the calcium signature theory has not been conclusively proven, and alternative theories are now appearing. One of the problems remaining is that there is no standard method to quantify these spatio- temporal signals. The aim of this thesis was to develop a standard method to quantify calcium signatures in plants and start constructing a library of calcium signatures in response to different stresses. As a model system I used Arabidopsis thaliana roots expressing the R-GECO calcium sensor. To quantify the spatio-temporal calcium response, the calcium signature was divided into six quantifiable parameters: (a) delay of the first detected calcium signal, (b) location of the first calcium signal, (c) duration of the calcium signal, (d) distance that the calcium wave traveled along the root, (e) velocity with which the calcium wave travels towards the root tip, and (f) velocity with which the calcium wave travels towards the shoot. Principle component analysis (PCA) was used to look for similarities and analyze the data. Responses to eleven elicitors (ATP, chitin, cellobiose, cold, D-serine, elf18, flg22, glutamate, NaCl, nlp20 and PG3) were tested. The results showed that, indeed, each elicitor resulted in a unique composition of the six parameters that together form the calcium signature. Moreover, calcium signatures in response to biotic versus abiotic elicitors formed two distinct groups. While biotic stress caused delayed calcium responses specific to the elongation zone of plant roots, abiotic stresses resulted in immediate and systemic calcium signatures. Further experiments suggested that ROS play a key role in restricting calcium signatures to the elongation zone in response to biotic stress and in propagation of calcium signals through the root in response to abiotic stress, indicating that there is crosstalk between reactive oxygen species (ROS) and calcium signatures to prioritize distinct stresses

Comparing Arabidopsis receptor kinase and receptor protein-mediated immune signaling reveals BIK1-dependent differences

Pattern recognition receptors (PRRs) sense microbial patterns and activate innate immunity against attempted microbial invasions. The leucine‐rich repeat receptor kinases (LRR‐RK) FLS2 and EFR, and the LRR receptor protein (LRR‐RP) receptors RLP23 and RLP42, respectively, represent prototypical members of these two prominent and closely related PRR families. We conducted a survey of Arabidopsis thaliana immune signaling mediated by these receptors to address the question of commonalities and differences between LRR‐RK and LRR‐RP signaling. Quantitative differences in timing and amplitude were observed for several early immune responses, with RP‐mediated responses typically being slower and more prolonged than those mediated by RKs. Activation of RLP23, but not FLS2, induced the production of camalexin. Transcriptomic analysis revealed that RLP23‐regulated genes represent only a fraction of those genes differentially expressed upon FLS2 activation. Several positive and negative regulators of FLS2‐signaling play similar roles in RLP23 signaling. Intriguingly, the cytoplasmic receptor kinase BIK1, a positive regulator of RK signaling, acts as a negative regulator of RP‐type immune receptors in a manner dependent on BIK1 kinase activity. Our study unveiled unexpected differences in two closely related receptor systems and reports a new negative role of BIK1 in plant immunity