An insect lands on the open leaves of a Venus flytrap plant, drawn to an appealing scent. It noses around and accidentally brushes one of the trap’s trigger hairs. An action potential shoots across the leaf blade. The insect keeps moving and bends another trigger hair, propagating a second action potential; suddenly, the leaves snap shut, trapping the insect, enveloping it in digestive juices, and absorbing the bug’s rich nutrients.
How these two light touches trigger abrupt shutting of the leaves has been hypothesized, but never proven. Now, in a new study published in Current Biology, a team of researchers knocked out two ion channels, making it harder to produce action potentials and proving the channels’ importance in leaf closing.1
“The paper is a very big technical advance,” said plant biophysicist Rainer Hedrich at the University of Wurzburg who was not involved in the study. “It is possible to knock out genes in an excitable plant and test hypotheses.”
Carl Procko holds a Venus flytrap plant in the greenhouse that stores various transgenic and wild type plants
Carnivorous plants and their quick movements have fascinated scientists for centuries. In the 1870s, Darwin and his colleagues discussed how electrical currents played a role in leaf closing.2,3,4 More recently, scientists found mechanosensitive ion channels FLYCATCHER1 (FLYC1) and FLYCATCHER2 (FLYC2) expressed in trigger hairs that may associate with touch sensitivity.5 Even though the Venus flytrap’s genome is sequenced, no targeted mutations of ion channel genes have been made to conclusively prove their roles in leaf closing.
So, plant biologists Carl Procko and Joanne Chory at the Salk Institute decided to use CRISPR-Cas9 to mutate FLYC1 and FLYC2 to investigate their functions. Scientists had hypothesized that an insect’s touch causes deformation of the trigger hair’s sensory cell membrane, which causes the opening of these ion channels and membrane depolarization and electrical signaling.
Procko grew Venus flytrap plants in tissue culture and then fired gold particles covered with plasmid DNA containing components of the CRISPR-Cas9 system into the cells. In the plasmid, the researchers also included a gene for a fluorescent protein to identify the plasmid-bearing tissue. The team propagated the genetically transformed cells and eventually grew a new plant. The plant was mosaic; it carried the plasmid DNA in some leaf arms, while others were wild type.
Procko chose leaflets that were fully transgenic (and fluorescent) and clonally separated them in tissue culture. To determine whether the leaves were single or double mutants, Procko used PCR-based Sanger sequencing and genotyping. He chose single mutants for some experiments and double mutants for others. He then planted the plants in soil and continued to grow them in a greenhouse.
Next, he triggered the double mutant plants with a touch from thin, fire-polished glass rod mounted on a micromanipulator; they closed just as often and as quickly as the wild type plants. “You get a plant that looks normal,” said Procko. “You sit there, and you scratch your head a bit.” Procko thought that perhaps the defect was smaller than could be detected using the relatively large touch of a pipette and decided to search for another more subtle quantitative assay.
Wen Mai Wong, a scientist in Sreekanth Chalasani’s lab, uses the ultrasound transducer to test a Venus flytrap leaf for its action potential.
Carl Procko and Wen Mai Wong
He collaborated with molecular neurobiologist Sreekanth Chalasani, also at the Salk Institute, who works with ultrasound. When the team tested the plants with a new, more sensitive assay using ultrasound waves to stimulate the trigger hair, the FLYC1-FLYC2 double mutants showed a significant defect: mutated plants required a greater ultrasound pressure to induce the trap closure than wild type plants. The team noted that single FLYC1 mutants stimulated with ultrasound closed just as well as the wild type plants. Procko believes that brute force mechanical stimulation with the glass rod may be so large that it could act through different mechanosensitive ion channels in the trigger hair.
“The next step now is to start looking at these other mechanosensitive channels that are within the trigger hair,” said Procko. “We can start to mutate some of these others and put them in various combinations to see exactly which mechanosensitive channels are most important or if they’re all required together to get that very exquisite touch sensitivity of the trigger hair.” Hedrich’s team is currently working to knock out a calcium channel gene hyperosmolality-gated calcium-permeable channel (OSCA).
Procko acknowledged that he didn’t know exactly how the ultrasound assay relates to touch, which limits the study. “It’s a mechanical stimulus. We like to think it’s related to touch, but it could alternatively be applying that stimulus directly to the sensory membranes and altering the membranes. So, this is still a little bit of a question mark,” said Procko.
- Procko C, et al. Mutational analysis of mechanosensitive ion channels in the carnivorous Venus flytrap plant. Curr Biol. 2023;33:3257-64.
- Darwin, C. Insectivorous Plants. 1875.
- Burdon-Sanderson, JS. Note on the electrical phenomena which accompany irritation of the leaf of Dionaea muscipula. Proc. R. Soc. Lond. 1873;21:139-147.
- Williams, SE. A salute to Sir John Burdon-Sanderson and Mr. Charles Darwin on the Centennial of the Discovery of Nerve-Like Activity in the Venus’ Flytrap. CPN. 1973; Vol. II(3) 41-43.
- Procko C, et al. Stretch-activated ion channels identified in the touch-sensitive structures of carnivorous Droseraceae plants. eLife. 2021;10:e64260.