Using newly created ‘optogenetic’ tobacco plants, research teams have delved into how plants interpret external signals.
When it comes to survival, plants face a significant challenge that many other organisms do not: they cannot relocate if faced with threats from predators, diseases, or unfavorable environmental conditions.
To cope with these challenges, plants have evolved various strategies to respond to such threats. Typically, these responses are triggered by specific environmental signals. It has been established that the concentration of calcium ions within cells plays a crucial role in how these signals are processed.
In addition to fluctuations in intracellular calcium levels, variations in the cell’s membrane potential are also suspected of serving as signaling mediators. Research teams from the Departments of Neurophysiology, Pharmaceutical Biology, and Botany at the Julius-Maximilians-Universität Würzburg (JMU) have conducted a detailed examination of the relationship between calcium levels and membrane potential, with their findings published in the journal Nature.
Light-Sensitive Channels Allow Controlled Experiments
In their investigation, the researchers worked with tobacco plants equipped with ion channels that can be activated by light. Over two decades ago, Peter Hegemann, Georg Nagel, and Ernst Bamberg laid the groundwork for optogenetics by discovering and characterizing light-responsive ion channels known as channelrhodopsins. Utilizing these light-sensitive proteins derived from algae and other microorganisms, JMU scientists aimed to explore whether calcium ion influx or anion efflux-driven depolarization of the cell membrane is key to the plant’s response during specific stress events. Significant preliminary work was required before they reached this point.
Optogenetics with Rhodopsins
Channelrhodopsins possess a natural light switch based on rhodopsin, transforming neuroscience through light-controlled analysis of neuronal networks. Their application in plant studies emerged only about two decades later, due to a collaborative effort between Georg Nagel’s group at JMU’s Institute of Physiology and plant researchers from various Botany departments.
In 2021, Georg Nagel’s team, along with Dr. Kai Konrad from the JMU Chair of Prof. Rainer Hedrich Botany 1, introduced methods to optimize the use of channelrhodopsins in plants by addressing three major challenges.
Rhodopsins Depend on Vitamin A
Challenge 1: “Like all rhodopsins, including those in human eyes, channelrhodopsins need the small molecule retinal, commonly referred to as vitamin A, to absorb light. While humans obtain retinal mainly from beta-carotene, land plants contain little retinal but are rich in beta-carotene,” explains Dr. Shiqiang Gao, a co-author of the Nature publication and a ‘rhodopsin engineer’ at JMU’s Department of Neurophysiology.
In 2021, Gao achieved a significant breakthrough by combining the expression of channelrhodopsins with retinal synthesis from beta-carotene in plant cells. This advancement led to the creation of tobacco plants with elevated retinal levels and functional channelrhodopsin expression.
Dr. Markus Krischke from the Metabolomics Core Unit at JMU also confirmed the high retinal content in various transgenic tobacco plants.
Comparable transgenic tobacco plants were developed for the study recently published by Dr. Meiqi Ding, under the guidance of plant physiologist Dr. Kai Konrad from Professor Rainer Hedrich’s group at the Department of Botany I.
Tobacco Plants Require Specific Light for Growth
Challenge 2: “Most rhodopsins respond to blue or green light, which are components of white light,” points out Georg Nagel. Consequently, the tobacco plants could not thrive in greenhouses or under artificial white light typically used for growth. They were grown successfully only in specialized growth chambers with red LED light, which is photosynthetically helpful and avoids unintended rhodopsin activation. Various tests indicated, “Tobacco grows healthily under red light, similar to greenhouse conditions,” adds Dr. Kai Konrad.
Successful Channelrhodopsin Expression in Plants
Challenge 3: The expression of channelrhodopsins in tobacco cells often presents issues. In 2021, the Würzburg research team succeeded in expressing the light-activated anion channel GtACR1 in tobacco plant cells. This achievement allowed Georg Nagel’s team to create multiple channelrhodopsins tailored for efficient calcium ion permeability. Ultimately, Dr. Shiqiang Gao and Dr. Shang Yang, both part of Nagel’s group, succeeded in developing a highly efficient calcium-conducting channelrhodopsin, XXM 2.0, specifically for tobacco plants.
This marked a significant milestone: “The successful expression of varied channelrhodopsins with differing ion selectivity in plant cells allows for a simultaneous comparison of various ion signals alongside the electrical signal, known as depolarization,” notes Dr. Meiqi Ding. She employed the calcium-conducting channelrhodopsin XXM 2.0 and the light-activated anion channel GtACR1 to explore different ion signaling mechanisms within tobacco.
A New Chapter in Plant Research
The newly developed “optogenetic” tobacco plants made it possible to determine whether calcium influx or membrane depolarization is crucial for a plant’s response to a particular stress. “The findings were conclusive,” states Dr. Kai Konrad, the corresponding author. First author Dr. Meiqi Ding explains, “After activating the anion channel, the leaves wilted and exhibited classic drought responses; the plant hormone abscisic acid (ABA) was synthesized, and gene expression increased to guard against dehydration.”
“On the other hand, when the calcium channel was activated, there was no increase in ABA levels,” Dr. Ding continues. “Instead, the plants released signaling molecules and hormones to trigger defense responses against predators, evident by the white spots on their leaves,” explains Dr. Konrad.
Dr. Sönke Scherzer from Prof. Hedrich’s group demonstrated through direct measurements that reactive oxygen species (ROS) are produced during this process.
Dirk Becker and Rainer Hedrich at the Chair of Botany 1 designed an experimental framework that bolstered the findings using transcriptomic and bioinformatic analyses.
These researchers believe that their study represents merely the beginning of a new chapter in plant research, emphasizing the potential to illuminate plant signaling pathways more effectively using various rhodopsins.
