Calcium Signaling in Plant-Insect Interactions

Abstract

In plant-insect interactions, calcium (Ca²⁺) variations are among the earliest events associated with the plant perception of biotic stress. Upon herbivory, Ca²⁺ waves travel long distances to transmit and convert the local signal to a systemic defense program. Reactive oxygen species (ROS), Ca²⁺ and electrical signaling are interlinked to form a network supporting rapid signal transmission, whereas the Ca²⁺ message is decoded and relayed by Ca²⁺-binding proteins (including calmodulin, Ca²⁺-dependent protein kinases, annexins and calcineurin B-like proteins). Monitoring the generation of Ca²⁺ signals at the whole plant or cell level and their long-distance propagation during biotic interactions requires innovative imaging techniques based on sensitive sensors and using genetically encoded indicators. This review summarizes the recent advances in Ca²⁺ signaling upon herbivory and reviews the most recent Ca²⁺ imaging techniques and methods.

Keywords: CPK; MAPK; calcium imaging; calmodulin; herbivory; membrane potential; oral secretions; reactive oxygen species; signal transduction.

Figure 1

Framework of early events induced in responses to herbivory within the first minutes after infestation. Herbivory is the combination of tissue damage (wounding) and the delivery of insect oral secretions. Both herbivory and mechanical damage induce the emission of volatile organic compounds (VOCs), which depolarize the plasma membrane potential (Vm). Wounding induces variations in the water potential that affect the turgor pressure (Ψ_T), which is perceived by plasma-membrane located mechanosensitive ion channels (MSC) resulting in Vm depolarization. Type IIB auto-inhibited Ca²⁺-ATPases (ACAs) play a role in recovery of excitability after long-term herbivory by interfering with both the Vm and [Ca²⁺]_cyt. Mechanical wounding does not exert a direct effect on Vm, Ca²⁺ and K⁺ channel activity (red crosses). Herbivory delivers oral secretions that exert different effects. Upon herbivory receptors activate a plasma-membrane–localized cyclic nucleotide-gated ion channel (CNGC) which interacts with the Ca²⁺-sensor calmodulin (CaM). Oral secretions inhibit the H⁺-ATPase activity by hampering the association between 14-3-3 protein and H⁺-ATPase proteins and cause a reduced phosphohydrolitic activity of the proton pump with a reduced extrusion of H⁺ from the cytosol and an alkalinization of the apoplast (pH_APO), both concurring to the Vm depolarization. Elicitors from oral secretions like N-linolenoyl-l-glutamine (GLN18:3) induce a RBOHD and RBOHF-dependent ROS burst. Specific oligosaccharides present in the oral secretions interact with plasma membrane receptors that also trigger ROS and Ca²⁺ signaling. Extracellular DNA (eDNA), deriving from cell disruption made by herbivory, and extracellular ATP (eATP) released by wounding and herbivory increase [Ca²⁺]_cyt. In summary, oral secretions, wounding, Ψ_T reduction, pH_APO alkalinization, an increase in apoplastic amino acids (AAapo) and glutamate (Glu) and the activity of the glutamate receptor-like cation channels (GLRs) depolarize the Vm. Oral secretions and ROS/NRS activate the Ca²⁺ channel that in turn opens the inward rectifying K⁺ channel that reduces the cytosolic pH causing Vm depolarization. Connections supported by empirical studies in planta are represented by solid lines, hypothetical connections are shown in dashed lines.

Figure 2

Calcium binding proteins. The top cartoon illustrates the canonical EF-hand helix–loop–helix. EF-hand Ca²⁺ binding motif contains a 29-residue helix–loop–helix topology, much like the spread thumb and forefinger of the human hand. Calmodulin is shown with its four calcium sites (shown as green balls) occupied. Calcium binding proteins are major players in Ca²⁺ signaling. See text for more details.

Figure 3

Calcium signaling upon herbivore feeding. (A–F) false colors time-course imaging of Ca²⁺ release upon feeding with the insect Spodoptera littoralis on a rosette leaf of Arabidopsis thaliana expressing the biosensor R-GECO1. The rapid fluorescence variation (blue to green) indicates the increase in the cytosolic Ca²⁺ concentration following the insect feeding. A wave of Ca²⁺ is generated at the site of feeding and rapidly spreads through the vascular system. The acquisitions have been obtained by a fluorescence stereo microscope Nikon SMZ18 with a SHR PLAN APO 0.5X WD:71. Excitation light was produced by a fluorescent CoolLED pE-300 ultra at 580 nm. Images were collected with a Mono Camera Nikon DS-Fi3 camera. Exposure time was set to 1 sec with a resolution of 8 bit 1440 × 1024. Images were acquired every 5 sec (See also Video S1). (G,H) Confocal laser scanning micrographs showing the increased [Ca²⁺]_cyt in Lima bean (Phaseolus lunatus) leaves upon feeding by S. littoralis. The red fluorescence is associated to the chlorophyll present in the chloroplasts, whereas the green fluorescence indicates the cytosolic localization of Ca²⁺ by the indicator Calcium Orange^TM. Scale bars: G = 100 µm; H = 200 µm (Figures (G,H) by Massimo Maffei).