Diffusion and bulk flow of amino acids mediate calcium waves in plants.

In plants, a variety of stimuli trigger long-range calcium signals that travel rapidly along the vasculature to distal tissues via poorly understood mechanisms. Here, we use quantitative imaging and analysis to demonstrate that traveling calcium waves are mediated by diffusion and bulk flow of amino acid chemical messengers. We propose that wounding triggers release of amino acids that diffuse locally through the apoplast, activating the calcium-permeable channel GLUTAMATE RECEPTOR-LIKE 3.3 as they pass. Over long distances through the vasculature, the wound-triggered dynamics of a fluorescent tracer show that calcium waves are likely driven by bulk flow of a channel-activating chemical. We observed that multiple stimuli trigger calcium waves with similar dynamics, but calcium waves alone cannot initiate all systemic defense responses, suggesting that mobile chemical messengers are a core component of complex systemic signaling in plants.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor.

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Interplay of Plasma Membrane and Vacuolar Ion Channels, Together with BAK1, Elicits Rapid Cytosolic Calcium Elevations in Arabidopsis during Aphid Feeding.

A transient rise in cytosolic calcium ion concentration is one of the main signals used by plants in perception of their environment. The role of calcium in the detection of abiotic stress is well documented; however, its role during biotic interactions remains unclear. Here, we use a fluorescent calcium biosensor (GCaMP3) in combination with the green peach aphid (Myzus persicae) as a tool to study Arabidopsis thaliana calcium dynamics in vivo and in real time during a live biotic interaction. We demonstrate rapid and highly localized plant calcium elevations around the feeding sites of M. persicae, and by monitoring aphid feeding behavior electrophysiologically, we demonstrate that these elevations correlate with aphid probing of epidermal and mesophyll cells. Furthermore, we dissect the molecular mechanisms involved, showing that interplay between the plant defense coreceptor BRASSINOSTEROID INSENSITIVE-ASSOCIATED KINASE1 (BAK1), the plasma membrane ion channels GLUTAMATE RECEPTOR-LIKE 3.3 and 3.6 (GLR3.3 and GLR3.6), and the vacuolar ion channel TWO-PORE CHANNEL1 (TPC1) mediate these calcium elevations. Consequently, we identify a link between plant perception of biotic threats by BAK1, cellular calcium entry mediated by GLRs, and intracellular calcium release by TPC1 during a biologically relevant interaction.