Scientists have visualized how plants use volatile organic compounds (VOCs) to communicate when they are threatened. The team found that plants interpret these volatile organic compounds as danger signals, triggering a defense response. Using innovative equipment and imaging techniques, they identified specific volatile organic compounds and which cells in the plant responded first. Their research provides insights into the intricate communication mechanisms of plants and their ability to recover in the face of potential damage.
Researchers visualized plant-to-plant communication through airborne compounds, identifying specific signals and cellular responses that activate plant defenses against threats.
Plants release volatile organic compounds (VOCs) into the atmosphere when they are mechanically damaged or attacked by insects. Undamaged neighboring plants sense the released VOCs and interpret them as danger cues, activating defense responses to ward off the incoming threat (Figure 1). The airborne transmission of volatile organic compounds between plants was first documented in 1983 and has since been observed in more than 30 different plant species. However, the molecular mechanisms from sensing VOCs to inducing defense remain unclear.
Figure 1: Plants release volatile organic compounds into the atmosphere when damaged by insects. Intact neighboring plants sense VOCs and initiate a preemptive defense response against the insects. Source: MasatsuguToyota/Saitama University
Breakthrough visualization of plant conversations
The research team led by Professor Masatsugu Toyota (Saitama University, Japan) performed real-time visualization of plant-to-plant communication through VOCs and revealed how VOCs are absorbed by plants to initiate Ca2+-dependent defense responses to defend against future threats.
This breakthrough research will be published in the journal Nature Communications on October 17, 2023. Yuri Aratani and Takuya Uemura led the work as doctoral students and postdoctoral researchers, respectively, at Toyota Laboratories, collaborating with Professor Kenji Matsui of Yamaguchi University in Japan.
Video 1: Volatile organic compounds released by insect-infested plants induce Ca2+ signaling (arrow). Source: MasatsuguToyota/Saitama University
"We built a device to pump volatile organic compounds released by caterpillar-feeding plants onto undamaged neighboring plants and combined it with a real-time fluorescence imaging system in the field," Toyota said. "This innovative device allowed the observation of bursts of fluorescence in the mustard plant Arabidopsis after exposure to volatile organic compounds released by insect-infested plants (Figure 2; Video 1). The plant produced a fluorescent protein sensor for intracellular Ca2+, so changes in intracellular Ca2+ concentration could be monitored by observing changes in fluorescence."
"In addition to insect attack, volatile organic compounds released from artificially crushed leaves can also induce Ca2+ signals in undamaged neighboring plants," Toyota said (Video 2).
Figure 2: Left: Equipment for exposing intact Arabidopsis thaliana to volatile organic compounds released by insect-infested plants (dashed arrows). Right: Volatile organic compounds (dashed arrows) released by insect-infested plants induce Ca2+ signals (yellow arrows, 600 and 1200 sec). Source: MasatsuguToyota/Saitama University
Identify key volatile organic compounds and their impacts
To determine which types of VOCs induce Ca signals in plants, Toyota's team of scientists studied a variety of VOCs known to induce plant defense responses. They found that (Z)-3-hexenal (Z-3-HAL) and (E)-2-hexenal (E-2-HAL), two six-carbon aldehyde volatile organic compounds, can induce Ca2+ signals in Arabidopsis (Figure 3; Video 3). Z-3-HAL and E-2-HAL are airborne, grassy-smelling chemicals known as green leaf volatiles (GLVs) that are released from mechanically damaged and herbivore-damaged plants.
Video 2: Volatile organic compounds released from artificially crushed plants induce Ca2+ signals. Source: MasatsuguToyota/Saitama University
Exposure of Arabidopsis to Z-3-HAL and E-2-HAL resulted in upregulation of defense-related genes. To understand the relationship between Ca2+ signaling and defense responses, they treated Arabidopsis thaliana with the Ca2+ channel inhibitor LaCl3 and the Ca2+ chelator EGTA. These chemicals inhibited Ca2+ signaling and the induction of defense-related genes, thus demonstrating that Arabidopsis can sense GLV and activate defense responses in a Ca2+-dependent manner.
Figure 3: Z-3-HAL in the air (orange broken line) induces Ca2+ signals in Arabidopsis leaves (yellow arrows, 120 sec and 370 sec). Source: MasatsuguToyota/Saitama University
Guard cells: Plants’ cognitive gateways
They also determined which specific cells would generate Ca2+ signals from GLVs by engineering transgenic plants specifically expressing fluorescent protein sensors in guard cells, mesophyll cells, or epidermal cells. After exposure to Z-3-HAL, guard cells produced Ca2+ signals within approximately 1 minute, followed by mesophyll cells, while epidermal cells produced Ca2+ signals more slowly (Video 4). Guard cells are bean-shaped cells on the surface of plants that form stomata, which are small holes that connect internal tissues to the atmosphere.
Video 3: Z-3-HAL in the air (in the right tube) induces Ca2+ signals in Arabidopsis leaves. Source: MasatsuguToyota/Saitama University
"Plants don't have a 'nose,' but stomata are the plant's portals, which mediate rapid entry of GLV into leaf tissue spaces," Toyota said. In fact, they found that pretreatment with abscisic acid (ABA), a plant hormone known for closing stomata, reduced the Ca2+ response of wild-type leaves. On the other hand, in mutants with impaired ABA-induced stomatal closing function, Ca2+ signals in leaves remained normal even with ABA treatment.
"We have finally uncovered the complex story of when, where and how plants respond to airborne 'warning messages' from their threatened neighbours," he said. "Hidden from view, this communication network plays a key role in promptly protecting neighboring plants from imminent threats."
Video 4: Airborne Z-3-HAL induces Ca2+ signals in guard cells (left video), mesophyll cells (center video), and epidermal cells (right video) of Arabidopsis leaves. Source: MasatsuguToyota/Saitama University
This groundbreaking research not only deepens our understanding of the amazing world of plants, it also highlights the extraordinary ways in which nature has given plants the ability to thrive and adapt to adversity. The profound implications of these discoveries extend far beyond the boundaries of plant science, providing a glimpse into the intricate tapestry of life on Earth.