How the Venus Flytrap Counts

The carnivorous Venus flytrap (Dionaea muscipula) can count to five: A team led by biophysicist Rainer Hedrich, professor at Julius-Maximilians-Universität (JMU) Würzburg in Bavaria, Germany, proved this in 2016. This finding received worldwide attention in science and the media.

In 2019, the JMU plant scientist was awarded the Koselleck Research Prize of the German Research Foundation (DFG) worth 1.5 million euros – and with it the opportunity to find out how the carnivorous plant counts. This is now shown by a Japanese research team, led by the developmental biologist Professor Mitsuyasu Hasebe from the University of Okazaki, and Rainer Hedrich's team in the journal Nature Plants.

If a prey touches one of the sensory hairs on the inner trap side of Dionaea, the mechanical stimulus is converted into an electrical signal. This so-called action potential spreads over the entire trap. As a reaction to this, nothing happens at first. But when within 30 seconds a second action potential electrically excites the trap, it snaps shut. If, on the other hand, the second stimulus takes longer, the first action potential is erased from the short-term memory of the Venus flytrap.

Flytrap gets calcium sensor implanted

The molecular memory of the flytrap could be based on a cellular calcium clock – this possibility was discussed by Rainer Hedrich and the Göttingen Nobel Prize winner and neurobiophysicist Erwin Neher as early as 2018 in a review article.

But how can we prove that the flytrap's memory for electric waves has something to do with the generation and storage of calcium? "By installing a calcium sensor in the plant," says Hedrich. This genetically engineered protein lights up when the cellular calcium concentration exceeds a critical level.

Such calcium sensors have already been successfully used in animals and plants to study calcium signals. This has now also worked for Dionaea. Hasebe and Hedrich had decoded the genome of the Venus flytrap and two close relatives in June 2020 and succeeded in introducing the calcium sensor GCAMP into flytrap tissue culture. Functional Venus flytraps could be regenerated from cultured cells – "this was the decisive step towards testing our hypothesis of the calcium clock," explains Hedrich.

Each action potential is accompanied by a calcium wave

Experiments with the sensor-equipped plants showed: When a sensory hair is touched, the calcium level in the cells in the foot of the sensory hair increases in a flash and spreads as a wave over the entire trap. This happens on each individual stimulus. As with every wave, however, it is a temporary phenomenon: within a few seconds after the touch, the calcium wave reaches its peak. After one minute it has largely died away.

What happens when a sensory hair is stimulated twice in a row? Then two action potentials are sent on their journey separately. The first one brings – as expected – an increase in the cellular calcium level. If the second occurs before the calcium level has fallen to its resting value, the two signals overlap. As a result, a threshold is exceeded, triggering calcium-dependent processes that in turn close the trap, Hedrich says.

"The electrical excitation of the trap cells is thus translated into an increase in the concentration of calcium. Thus the passing action potential is quasi stored in the electrically excited trap cells. If a further action potential comes, its calcium value is added to the first signal. Using this calcium clock the Venus flytrap can count the number of the touch stimulation-conditioned action potentials", explains the JMU professor.

The decisive factor is the crossing of the calcium threshold

However, if the second action potential arrives after the first calcium wave has died down, the flytrap will not close. Is it the number of action potentials that is responsible for the trap closing or the exceeding of the calcium threshold?

In the Würzburg laboratory it could be shown that after 30 seconds a second action potential does not close the trap, but a shortly following electrical excitation does. This procedure was used in the Hasebe laboratory to study the behavior of the calcium level. The result was clear: With the delayed second stimulus, the calcium level increased but remained subliminal. With the third stimulus, the threshold for triggering the trap was exceeded.

How does the Venus flytrap continue to count?

"Our findings show that short-term memory and the ability to count to two are based on the calcium clock," Hedrich concludes. But the Venus flytrap can continue to count past two. In response to subsequent action potentials, the biosynthesis of the plant hormone jasmonate is increased. From the fifth electrical excitation on, the Venus flytrap produces digestive enzymes to decompose the prey and transport proteins to aid in the assimilation of the nutrients from the meal.

In this context, the next question that the research groups will address is, whether and how the calcium clock counts to five. An important question will be to understand whether the cells that react to the first and second action potentials are different to those that respond to the action potentials three and four.

"Furthermore, we want to know how the different calcium-dependent processes are triggered after the respective calcium threshold is exceeded," says the Würzburg professor. "Our primary interest is in the calcium channels that are opened by the action potential and the process in which the calcium signal is translated into the touch hormone jasmonate.

Publication

Calcium dynamics during trap closure visualized in transgenic Venus flytrap, Nature Plants, 05 October 2020, DOI: 10.1038/s41477-020-00773-1

Contact person

Prof. Dr. Rainer Hedrich, Chair of Botany I (Plant Physiology and Biophysics), University of Würzburg, T +49 931 31-86100, hedrich@botanik.uni-wuerzburg.de