Mosses and phytochromes

To most people, mosses are boring because they are so small and simple, but that is exactly why they are useful as model plants for basic research. One of the most intriguing things about mosses is how they steer their filaments to grow towards a source of light. Of course, it makes sense that they do this because they need light to carry out photosynthesis, but what is interesting is how they manage to "see" from which direction the light is coming and orientate their tip growth accordingly.  In contrast to higher plants whose "phototropic" growth is controlled by the blue-light receptor phototropin, moss filaments use phytochrome. Phytochromes are  red/far-red photochromic photoreceptors used by all plants to adjust their development according to their light environment. What makes the moss's physiology so interesting is that everyone believes that phytochromes act by regulating gene expression in the nucleus. Ok, so if that's the whole story, how can the phytochrome in the moss cell tell in which direction it should grow? In fact, it is impossible for this information to be transmitted via gene activation! So we got interested in this little problem in the hope of helping to understand how phytochrome really works (and even today, that's only partly known).

Homologous recombination and the Physcomitrella genome

Then at the FU in Berlin, a lot of this work was done together with Mathias Zeidler (a PhD student of mine who joined us in Giessen in 2002 after a spell at the Rockefeller in NYC), Franz Mittmann (a PhD student who later came to Giessen as a postdoc for several years too), and Tilman Lamparter (now at Uni Karlsruhe). We found that, both spectrally and biochemically, phytochromes from higher and lower plants are very similar. Just at that time Didier Schäfer in Lausanne discovered that mosses - unlike any other group of plants - show high recombination rates with homologous foreign DNA, thereby allowing efficient chromosomal gene targeting or even replacement. This is an important tool in studying gene function in prokaryotes, yeast and even mammals - but unfortunately it doesn't work in higher plants. Thus mosses provided plant biology with an extremely useful molecular genetic tool. In fact, gene targeting in Physcomitrella is so useful that BASF spent millions of Euros to knock out many thousands  of genes and thereby find out what their functions are (at least that was the idea...).

We began work using homologous recombination to find out how lower plants use phytochrome to sense light direction. Franz Mittmann cloned four Physcomitrella phytochrome genes de novo, then targetted each of them, knocking them out one after another. We found that one of these mutants showed a weaker response to light direction - so we expected that this gene product in particular seems to be responsible for steering the phototropic response (Mittmann et al. (2004) PNAS). With the help of a substantial DFG grant, we tried to find out how - but things became more complicated than we had expected.... 

Neochrome

In the mean time, together with Masamitsu Wada's group (NIBB, Japan) we discovered the photoreceptor probably responsible for steering chloroplast rotation in the alga Mougeotia. This phenomenon is described lovingly in most plant physiology textbooks together with a wonderful theory that phytochrome molecules are attached coherently at the plasma membrane and that they flip over when they are photoconverted from Pr to Pfr. Unfortunately, however, this very cool explanation contradicts almost everything currently known about phytochrome biochemistry and cell biology! We found, however, that the photoreceptor involved in Mougeotia is a neochrome, a hybrid between a phytochrome and a phototropin. This largely solves the paradox of a membrane-associated phytochrome, because phototropins are invariably attached to the plasma membrane in plant cells. This work (Suetsugu et al., (2005) PNAS) was even featured on the PNAS front cover. 

Phytochrome and Phototropin

So what about phototropism in the moss? First the bad news: mosses don't have neochromes! Now the good news: we think we've found the answer, and it's rather nice. To our great surprise, we discovered that a subpopulation of phytochrome molecules hitch a ride on phototropins at the plasma membrane (Jaedicke et al. (2012) PNAS). We think that the same thing happens in higher plants too, and of course the story fits beautifully with that of neochrome in Mougeotia, because in that case the phytochrome and the phototropin are fused together. The people who write the textbooks should include this new aspect of the story. Most of this work was done by Kathi Mailliet (née Jaedicke) - for which she won the University Prize for the best PhD in Sciences in 2013.

Seven phytochromes and CRISPR/Cas9

In the mean time, though, the Physcomitrella genome had been sequenced. We didn't do any of the dirty work to generate the sequence, but I spent a lot of time searching for interesting genes and annotating them, so I was a co-author on the paper (Rensig et al. (2008) Science). A rather disturbing surprise for us was that the genome contained not 4 but 7 phytochrome genes, more than in any other plant species known to date. That of course complicated our work on phytochrome function in Physcomitrella. But help was at hand....

Although homologous recombination in Physcomitrella is nice, CRISPR/Cas9-based genome editing is revolutionary - and works in most organisms. But CRISPR exploits the same machinery that allows homologous recombination, so we wondered if perhaps CRISPR might work especially well in Physcomitrella. Silvia Trogu from Sardinia joined the lab as a PhD student to exploit this possibility - and it worked even better than we had hoped! In fact, we currently hold the world record for multplex mutagenesis by knocking out all 7 Physcomitrella phytochrome genes in a single experiment (Trogu et al. 2020). In fact, the phototropic phenotypes she has found are quite remarkable - so we hope that we can now explain this paradoxical phenomenon.

My contribution to the Annual Review of Plant Biology 2013 "Phytochrome cytoplasmic signalling" focuses on this whole theme.

(All the papers cited above are in the publications list)