Calcium signalling in the chloroplast and in the regulation of nuclear gene expression

Calcium is a universal second messenger involved in nearly every aspect of plant physiology and development. In response to a variety of biotic or abiotic stresses, calcium rapidly and transiently increases in the cytosol and in this way triggers the appropriate downstream response. To date, most of the research on calcium has focused on cytosolic calcium signalling, however recent advances have demonstrated that in the chloroplast Ca2+ concentrations are also controlled, and that chloroplast calcium signalling is involved in regulating the plant cell physiology. This thesis describes work investigating both cytosolic and chloroplast calcium signalling, In the first case, I examined how cytosolic Ca2+ increases with different kinetics (Ca2+-signatures) can encode specific information, and how this can be translated into appropriate changes in transcript expression. To this aim, a dynamic mathematical model of the SA-mediated pathogen network was developed. Calcium is responsible for activating this defence pathway by a complex regulation of the components of this network. This model was able to predict fold-changes and kinetics of gene expression in response to any given calcium signature, hence it was able to accurately describe how specificity is encoded in plant cells. The properties emerging from this model provided insights into the mechanistic basis of calcium signature decoding. Work on chloroplast calcium signalling focused on two different aspects. Firstly, the hypothesis that chloroplast calcium might regulate chloroplast gene expression was tested, and it was found to not be the case. Secondly, a new chloroplast-specific calcium response was discovered, in response to heat. Properties of this response were investigated, as well as its possible physiological functions. Finally, by using this calcium response as a readout, I addressed the question of heat-sensing in plants. Using this approach I discovered that there is a prominent role for membrane fluidity in controlling this heat-induced calcium increase

Design principles for decoding calcium signals to generate specific gene expression via transcription

The second messenger calcium plays a key role in conveying specificity of signalling pathways in plant cells. Specific calcium signatures are decoded to generate correct gene expression responses and amplification of calcium signatures is vital to this process. It is not known: (1) if this amplification is an intrinsic property of all calcium-regulated gene expression responses and whether all calcium signatures have the potential to be amplified, and (2) how does a given calcium signature maintain specificity in cells containing a great number of transcription factors (TFs) and other proteins with the potential to be calcium-regulated? The work presented here uncovers the design principle by which it is possible to decode calcium signals into specific changes in gene transcription in plant cells. Regarding the first question, we found that the binding mechanism between protein components possesses an intrinsic property that will nonlinearly amplify any calcium signal. This nonlinear amplification allows plant cells to effectively distinguish the kinetics of different calcium signatures to produce specific and appropriate changes in gene expression. Regarding the second question, we found that the large number of calmodulin (CaM)-binding transcription factors (TFs) or proteins in plant cells form a buffering system such that the concentration of an active CaM-binding TF is insensitive to the concentration of any other CaM-binding protein, thus maintaining specificity. The design principle revealed by this work can be used to explain how any CaM-binding TF decodes calcium signals to generate specific gene expression responses in plant cells via transcription

ACA pumps maintain leaf excitability during herbivore onslaught

Recurrent damage by lepidopteran folivores triggers repeated leaf-to-leaf electrical signaling. We found that the ability to propagate electrical signals—called slow wave potentials—was unexpectedly robust and was maintained in plants that had experienced severe damage. We sought genes that maintain tissue excitability during group insect attack. When Arabidopsis thaliana P-Type II Ca2+-ATPase mutants were mechanically wounded, all mutants tested displayed leaf-to-leaf electrical signals. However, when the auto-inhibited Ca2+-ATPase double-mutant aca10 aca12 was attacked by Spodoptera littoralis caterpillars, electrical signaling failed catastrophically, and the insects consumed these plants rapidly. The attacked double mutant displayed petiole base deformation and chlorosis, which spread acropetally into laminas and led to senescence. A phloem-feeding aphid recapitulated these effects, implicating the vasculature in electrical signaling failure. Consistent with this, ACA10 expressed in phloem companion cells in an aca10 aca12 background rescued electrical signaling and defense during protracted S. littoralis attack. When expressed in xylem contact cells, ACA10 partially rescued these phenotypes. Extending our analyses, we found that prolonged darkness also caused wound-response electrical signaling failure in aca10 aca12 mutants. Our results lead to a model in which the plant vasculature acts as a capacitor that discharges temporarily when leaves are subjected to energy-depleting stresses. Under these conditions, ACA10 and ACA12 function allows the restoration of vein cell membrane potentials. In the absence of these gene functions, vascular cell excitability can no longer be restored efficiently. Additionally, this work demonstrates that non-invasive electrophysiology is a powerful tool for probing early events underlying senescence.ISSN:0960-9822ISSN:1879-044

Wound- and mechanostimulated electrical signals control hormone responses.

Plants in nature are constantly exposed to organisms that touch them and wound them. A highly conserved response to these stimuli is a rapid collapse of membrane potential (i.e. a decrease of electrical field strength across membranes). This can be coupled to the production and/or action of jasmonate or ethylene. Here, the various types of electrical signals in plants are discussed in the context of hormone responses. Genetic approaches are revealing genes involved in wound-induced electrical signalling. These include clade 3 GLUTAMATE RECEPTOR-LIKE (GLR) genes, Arabidopsis H <sup>+</sup> -ATPases (AHAs), RESPIRATORY BURST OXIDASE HOMOLOGUEs (RBOHs), and genes that determine cell wall properties. We briefly review touch- and wound-induced increases in cytosolic Ca <sup>2+</sup> concentrations and their temporal relationship to electrical activities. We then look at the questions that need addressing to link mechanostimulation and wound-induced electrical activity to hormone responses. Utilizing recently published results, we also present a hypothesis for wound-response leaf-to-leaf electrical signalling. This model is based on rapid electro-osmotic coupling between the phloem and xylem. The model suggests that the depolarization of membranes within the vascular matrix triggered by physical stimuli and/or chemical elicitors is linked to changes in phloem turgor and that this plays vital roles in leaf-to-leaf electrical signal propagation