Yang Zhou

• The discovery, heterologous expression, and characterization of channelrhodopsin-2 (ChR2) – a light-sensitive cation channel found in the green alga Chlamydomonas reinhardtii – led to the success of optogenetics as a powerful technology, first in neuroscience. ChR2 was employed to induce action potentials by blue light in genetically modified nerve cells. In optogenetics, exogenous photoreceptors are expressed in cells to manipulate cellular activity. These photoreceptors were in the beginning mainly microbial opsins. During nearly two decades,The discovery, heterologous expression, and characterization of channelrhodopsin-2 (ChR2) – a light-sensitive cation channel found in the green alga Chlamydomonas reinhardtii – led to the success of optogenetics as a powerful technology, first in neuroscience. ChR2 was employed to induce action potentials by blue light in genetically modified nerve cells. In optogenetics, exogenous photoreceptors are expressed in cells to manipulate cellular activity. These photoreceptors were in the beginning mainly microbial opsins. During nearly two decades, many microbial opsins and their mutants were explored for their application in neuroscience. Until now, however, the application of optogenetics to plant studies is limited to very few reports. Several optogenetic strategies for plant research were demonstrated, in which most attempts are based on non-opsin optogenetic tools. Opsins need retinal (vitamin A) as a cofactor to generate the functional protein, the rhodopsin. As most animals have eyes that contain animal rhodopsins, they also have the enzyme - a 15, 15'-Dioxygenase - for retinal production from food-supplied provitamin A (beta-carotene). However, higher plants lack a similar enzyme, making it difficult to express functional rhodopsins successfully in plants. But plant chloroplasts contain plenty of beta-carotene. I introduced a gene, coding for a 15, 15'-Dioxygenase with a chloroplast target peptide, to tobacco plants. This enzyme converts a molecule of β-carotene into two of all-trans-retinal. After expressing this enzyme in plants, the concentration of all-trans-retinal was increased greatly. The increased retinal concentration led to increased expression of several microbial opsins, tested in model higher plants. Unfortunately, most opsins were observed intracellularly and not in the plasma membrane. To improve their localization in the plasma membrane, some reported signal peptides were fused to the N- or C-terminal end of opsins. Finally, I helped to identify three microbial opsins -- GtACR1 (a light-gated anion channel), ChR2 (a light-gated cation channel), PPR (a light-gated proton pump) which express and work well in the plasma membrane of plants. The transgene plants were grown under red light to prevent activation of the expressed opsins. Upon illumination with blue or green light, the activation of these opsins then induced the expected change of the membrane potential, dramatically changing the phenotype of plants with activated rhodopsins. This study is the first which shows the potential of microbial opsins for optogenetic research in higher plants, using the ubq10 promoter for ubiquitous expression. I expect this to be just the beginning, as many different opsins and tissue-specific promoters for selective expression now can be tested for their usefulness. It is further to be expected that the here established method will help investigators to exploit more optogenetic tools and explore the secrets, kept in the plant kingdom.…
• Die Entdeckung, heterologe Expression und Charakterisierung von Channelrhodopsin-2 (ChR2) - einem lichtempfindlichen Kationenkanal, der in der Grünalge Chlamydomonas reinhardtii vorkommt - führte zum Erfolg der Optogenetik als leistungsfähige Technologie, zunächst in den Neurowissenschaften. ChR2 wurde eingesetzt, um in genetisch veränderten Nervenzellen durch blaues Licht Aktionspotentiale zu induzieren. Bei der Optogenetik werden exogene Photorezeptoren in Zellen exprimiert, umDie Entdeckung, heterologe Expression und Charakterisierung von Channelrhodopsin-2 (ChR2) - einem lichtempfindlichen Kationenkanal, der in der Grünalge Chlamydomonas reinhardtii vorkommt - führte zum Erfolg der Optogenetik als leistungsfähige Technologie, zunächst in den Neurowissenschaften. ChR2 wurde eingesetzt, um in genetisch veränderten Nervenzellen durch blaues Licht Aktionspotentiale zu induzieren. Bei der Optogenetik werden exogene Photorezeptoren in Zellen exprimiert, um die zelluläre Aktivität zu manipulieren. Diese Photorezeptoren waren anfangs hauptsächlich mikrobielle Opsine. Im Laufe von fast zwei Jahrzehnten wurden viele mikrobielle Opsine und ihre Mutanten für ihre Anwendung in den Neurowissenschaften erforscht. Bis jetzt ist die Anwendung der Optogenetik in der Pflanzenforschung jedoch auf sehr wenige Arbeiten beschränkt. Es wurden mehrere optogenetische Strategien für die Pflanzenforschung aufgezeigt, wobei die meisten Versuche auf optogenetischen Werkzeugen, die nicht Opsine sind, beruhen. Opsine benötigen Retinal (Vitamin A) als Kofaktor, um das funktionelle Protein, das Rhodopsin, zu generieren. Da die meisten Tiere Augen haben, die tierische Rhodopsine enthalten, verfügen sie auch über das Enzym - eine 15, 15'-Dioxygenase - zur Retinalproduktion aus mit der Nahrung zugeführtem Provitamin A (Beta-Carotin). Höheren Pflanzen fehlt jedoch ein ähnliches Enzym, was es schwierig macht, funktionale Rhodopsine erfolgreich in Pflanzen zu exprimieren. Aber die Chloroplasten der Pflanzen enthalten reichlich Beta-Carotin. Ich führte ein Gen, das für eine 15, 15'-Dioxygenase mit einem Chloroplasten-Zielpeptid kodiert, in Tabakpflanzen ein. Dieses Enzym wandelt ein Molekül β-Carotin in zwei Moleküle all-trans-Retinal um. Nach Expression dieses Enzyms in Pflanzen wurde die Konzentration von all-trans-Retinal stark erhöht. ...…