The Arabidopsis plant cells glow green after exposure to VOCs, signifying plant-to-plant communication. (Image Credit: Aratani et al. Nature Communications, 2023)
When a plant is damaged by insects or other means, it releases volatile organic compounds (VOCs) in the air. Undamaged nearby plants detect those VOCs, which works like a warning system, alerting them of danger or potential harm. The plants then respond with Ca2+ defenses to keep themselves protected. Two scientists at Saitama University, Yuri Aratani and Takuya Uemura, used imaging technology to record plants communicating with each other in this manner.
The team started by genetically modifying the plants’ guard, mesophyll, and epidermal cells with a biosensor so that it would illuminate green after detecting calcium ions. Afterward, they made a setup containing an air pump, two flow meters, plastic bottles, and a plastic dish. This system constantly pumps VOCs from tomato leaves, which are being consumed by caterpillars, to the unharmed Arabidopsis plant.
They also used an upright confocal laser scanning microscope to capture GCaMP3 signals for recording Ca2+ imaging at the cellular level. According to the team, the Arabidopsis plant’s guard cells produced calcium signals within one minute after exposure to the Z-3-HAL compounds. Mesophyll cells then detected those compounds. Exposing Z-3-HAL and E-2-HAL boosted the plant’s defense genes. Plus, the scientists treated the plant with LaCl3, the Ca2+ channel inhibitor, and EGTA, the Ca2+ chelating agent. As a result, these suppressed the Ca2+ signals and defense-related genes, proving that this plant senses green leaf volatiles (GLVs) and activates defense responses.
Furthermore, calcium signaling decreases when the stomata, small pores that allow plants to take in carbon dioxide, are closed with a phytohormone. The researchers think this stomata work like “plant nostrils.” This means they found where, when, and how plants respond to warnings from neighbor plants.
The VOCs (sourced from the plastic bottle with caterpillars) are pumped to the Arabidopsis plant, which picks up those compounds. (Image Credit: Aratani et al. Nature Communications, 2023)
A 488-nm laser/488-nm dichroic mirror excited the GCaMP3, and the microscope’s GaAsP detector detected the fluorescent signals. Then, the scientists used NIS-Elements imaging software for GCaMP3 signal analysis over time at varying areas. When they analyzed the compounds, the team discovered the Z-3-HAL and E-2 HAL compounds activated the Arabidopsis’ calcium signals.
In addition, the scientists used an operational amplifier, head stage amplifier, digitizer, and electrophysiology data acquisition software to measure surface potential changes. All data points were extracted at 0.5 Hz to compare the signal change with Ca2+ increases.
By using this wide-field real-time imaging technique with the intact Arabidopsis plant, the researchers were able to see the details of Ca2+ signals due to GLVs. This method made it possible to observe spatial and temporal aspects of GLV perception pathways at the cellular level. Also, integrating real-time imaging with other approaches allowed the team to understand the “comprehensive orchestration of GLV responses, including Ca2+ signals.” The team believes their approach can be improved to study VOC signaling networks “across plant taxa using mutants that are defective in the putative elements of VOC responses.”
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