Johanna Knab (Universität Erlangen-Nürnberg)
Confocal optical sections through a Physcomitrium patens protonemal cell accumulating a ChR-2-XXL-GFP fusion protein in the plasma membrane (green). Chloroplasts display red autofluorescence. Scale bar: 15 μm. Image: Johanna Knab
Johanna Knab's Master thesis was awarded with the Prize for the Best Plant Science Master Thesis, which was carried out at Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) in the year 2020.
Title: Analysis of CNGCs in Physcomitrium patens and generation of optogenetic lines and a pH marker for live-cell imaging
Local Ca2+-import trough CNGC channels appears to modulate tip growth in moss (Physcomitrium patens) protonemata and can be investigated using a newly established optogenetic system.
In plants, "cyclic nucleotide-gated channels" (CNGCs) regulate numerous biological processes ranging from development to tip growth and immune responses. Eight CNGCs have been identified in the moss Physcomitrium patens, whose functions are largely unexplored. By contrast, in the model plant Arabidopsis thaliana essential functions of different CNGCs in the tip growth of root hairs and pollen tubes have already been established. In order to investigate the role of CNGCs in tip growth in moss, P. patens CNGC knock-out lines were generated using CRISPR/Cas methodology. Four different cngc single KO lines, two cngc double KO lines and one cngc triple KO line were established. Despite extensive efforts, it was not possible to generate KO lines disrupted in the expression of the remaining four CNGCs, indicating essential functions of these channels in moss. Investigation of the established cngc KO lines has shown that three P. patens CNGCs in fact modulate tip growth in protonemata. cngc-b, cngc-c, and cngc-h single KO lines, as well as two of the cngc double KO lines, showed significantly increased elongation of protonemal cells. This effect was particularly pronounced in the cngc-b / cngc-c double knockout line, which indicates additive functional interactions between CNGCc and CNGCb.
Furthermore, experiments were performed to establish an optogenetic system in P. patens. Optogenetics is a cell biological method, which enables the control of processes in living cells by light pulses using light-sensitive proteins. In cooperation with Prof. Dr. Nagel from the University of Würzburg, transgenic P. patens lines were generated expressing a recombinant channel rhodopsin tagged with a green fluorescent protein (ChR-2-XXL::GFP). Similar channel rhodopsins have already been used successfully in neurobiology to generate light-induced action potentials. Channel rhodopsin-2 is a light-controlled cation channel from the unicellular green alga Chlamydomonas reinhardtii. On the basis of this protein, Prof. Dr. Nagel developed the ChR-2-XXL channel, which allows blue-light-controlled local stimulation of Ca2+ import through the plasma membrane. This channel was successfully expressed for the first time in P. patens, and was found to be targeted to plasma membrane of protonemata cells, an important prerequisite for its function in Ca2+ import. In addition, a dioxygenase fused to a chloroplast targeting sequence was demonstrated to be imported into chloroplasts, where this enzyme can synthesize retinal using ß-carotene as a precursor. Retinal is essential for the function of the ChR-2-XXL channel. With this system it should now be possible to examine effects on tip growth of local cell depolarization triggered by blue light stimulation.
In the “Cell Biology Divison” at FAU, the role of Rac/Rop-dependent signal cascades in the control of tip growth in plants has been investigated for many years. The work described here represents an excellent basis for the future investigation of functional interactions between Rac/Rop- and Ca2+-dependent signal cascades in the moss P. patens.
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Johanna Knab conducted this work in the FAU Division of Cell Biology under the supervision of Dr. Maria Ntefidou in the team of Prof. Dr. Benedikt Kost.