The circadian clock coordinates an organism's growth, development and physiology with environmental factors. One illuminating example is the rhythmic growth of hypocotyls and cotyledons in Arabidopsis thaliana. Such daily oscillations in leaf position are often referred to as sleep movements or nyctinasty. Here, we report that plantlets of the liverwort Marchantia polymorpha show analogous rhythmic movements of thallus lobes, and that the circadian clock controls this rhythm, with auxin a likely output pathway affecting these movements. The mechanisms of this circadian clock are partly conserved as compared to angiosperms, with homologs to the core clock genes PRR, RVE and TOC1 forming a core transcriptional feedback loop also in M. polymorpha. Rhythmic movements of plant organs were documented already several centuries BC, but the first known experiments searching for the origin of such rhythms were conducted by the French astronomer de Mairan. Working with a sensitive plant (likely Mimosa pudica), he could show that leaves moving in day/night conditions continued to move in constant darkness. During the following centuries, experiments with what Linnaeus later termed "sleep movements" resulted in both the concept of the circadian clock and that of osmotic motors 1,2. These so called nyctinastic movements often occur in non-growing tissue and are reversible as in several legumes. The reversible movements involve osmotic motors in the pulvinus organ 3 , but rhythmic leaf movements can also be growth associated and thus non-reversible. Such rhythms are evident in the movement of leaves in tobacco and cotyledons in Arabidopsis thaliana 4,5. The irreversibility of this process is probably due to deposition of new cell wall material and decreased wall extensibility, but tissue expansion likely results from mechanisms in common with those in pulvinus tissue 6. Since the introduction of the concept of a circadian or endogenous biological clock great progress has been achieved in understanding the mechanisms behind such internal rhythms. In plants most of this work has been performed in the flowering plant Arabidopsis 7. A working model of the plant circadian clock comprises a self-sustaining oscillator with an approximately 24-hour rhythm resulting mainly from transcriptional and translational feedback loops 8. In short, the main components in such models are a set of single MYB domain transcription factors, a family of PSEUDO-RESPONSE REGULATORs (PRRs), and a few plant specific genes with unknown biochemical function. The early morning phased genes CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) encode two MYB-like transcription factors that function mainly as repressors of day-and evening-phased genes 9-13. A second sub-family of related MYB-like transcription factors including REVEILLE4 (RVE4), RVE6 and RVE8 has an opposite function, enhancing clock pace through the activation of several core clock genes 14,15. The family of PRR genes comprise five members in Arabidopsis: PRR1, PRR3, PRR5, PRR...

14The circadian clock coordinates an organism's growth, development and physiology with 15 environmental factors. One illuminating example is the rhythmic growth of hypocotyls and 16 cotyledons in Arabidopsis thaliana. Such daily oscillations in leaf position are often referred to as 17 sleep movements or nyctinasty. Here, we report that plantlets of the liverwort Marchantia 18 polymorpha show analogous rhythmic movements of thallus lobes, and that the circadian clock 19 controls this rhythm, with auxin a likely meditator. The mechanisms of this circadian clock are 20 partly conserved as compared to angiosperms, with homologs to the core clock genes PRR, RVE 21 and TOC1 forming a core transcriptional feedback loop also in M. polymorpha. 22 23 24 25 26

Previous studies of plant circadian clock evolution have often relied on clock models and genes defined in Arabidopsis. These studies identified homologues with seemingly conserved function, as well as frequent gene loss. In the present study, we aimed to identify candidate clock genes in the liverwort Marchantia polymorpha using a more unbiased approach.To identify genes with circadian rhythm we sequenced the transcriptomes of gemmalings in a time series in constant light conditions. Subsequently, we performed functional studies using loss-of-function mutants and gene expression reporters.Among the genes displaying circadian rhythm, a homologue to the transcriptional corepressor Arabidopsis DE-ETIOLATED1 showed high amplitude and morning phase. Because AtDET1 is arrhythmic and associated with the morning gene function of AtCCA1/LHY, that lack a homologue in liverworts, we functionally studied DET1 in M. polymorpha.We found that the circadian rhythm of MpDET1 expression is disrupted in loss-of-function mutants of core clock genes and putative evening-complex genes. MpDET1 knock-down in turn results in altered circadian rhythm of nyctinastic thallus movement and clock gene expression. We could not detect any effect of MpDET1 knock-down on circadian response to light, suggesting that MpDET1 has a yet unknown function in the M. polymorpha circadian clock.

Previous studies in the liverwort Marchantia polymorpha have shown that the putative evening complex (EC) genes LUX ARRHYTHMO (LUX) and ELF4-LIKE (EFL) have a function in the liverwort circadian clock. Here, we studied the growth phenotypes of MpLUX and MpEFL loss-of-function mutants, to establish if PHYTOCHROME-INTERACTING FACTOR (PIF) and auxin act downstream of the M. polymorpha EC in a growth-related pathway similar to the one described for the flowering plant Arabidopsis. We examined growth rates and cell properties of loss-of-function mutants, analyzed protein-protein interactions and performed gene expression studies using reporter genes. Obtained data indicate that an EC can form in M. polymorpha and that this EC regulates growth of the thallus. Altered auxin levels in Mplux mutants could explain some of the phenotypes related to an increased thallus surface area. However, because MpPIF is not regulated by the EC, and because Mppif mutants do not show reduced growth, the growth phenotype of EC-mutants is likely not mediated via MpPIF. In Arabidopsis, the circadian clock regulates elongation growth via PIF and auxin, but this is likely not an evolutionarily conserved growth mechanism in land plants. Previous inventories of orthologs to Arabidopsis clock genes in various plant lineages showed that there is high levels of structural differences between clocks of different plant lineages. Here, we conclude that there is also variation in the output pathways used by the different plant clocks to control growth and development.