PIF-independent regulation of growth by an evening complex in the liverwort Marchantia polymorpha

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

Functional analyses of growth and development in the liverwort Marchantia polymorpha

Land plants developed from a freshwater charophycean algae about 500 million years ago. Today, they consist of two main clades, the vascular plants and the non-vascular bryophytes including hornworts, liverworts and mosses. To adapt to challenges within a terrestrial habitat, the first land plants evolved a diversity of hormonal and genetic pathways regulating growth and development. Analyses regarding these networks are mainly based on the angiosperm Arabidopsis thaliana. Genes of other land plant lineages that are inexistent in Arabidopsis are often not considered in functional studies, resulting in an incomplete picture of land plant evolution. The remarkable phylogenetic position of bryophytes makes them interesting for studies of gene function as they might carry different characteristics compared to e.g. angiosperms. In difference to vascular plants, the liverwort Marchantia polymorpha harbors a small, low genetic redundant genome containing most gene families present in Arabidopsis. Thus making it an advantageous model organism to determining specific gene function.  This thesis focuses on describing how dormancy and the circadian clock regulate growth in Marchantia. To avoid growth during unfavorable environmental, plants apply dormancy programs. Marchantia applies dormancy in gemmae, small asexual propagules produced by the shoot in a cup. Gemmae are dormant in the cup until they are dispersed by rain and subsequently germinating. I show that high levels of absisic acid (ABA), inhibits gemmae germination within the cup. Gemmae with a manipulated MpCYP707A, a gene involved in catabolism of ABA and seed dormancy regulation in Arabidopsis, showed altering dormancy suggesting that ABA homeostasis is fundamental for regulation of gemmae dormancy. Because dormant gemmae are not physically attached to the cup it has been speculated that the signal maintaining dormancy of gemmae is a gas. I found that gemmae mutated in positive and negative regulators of the ethylene signaling pathway showed decreased and increased dormancy respectively, suggesting that ethylene regulates dormancy through ABA. I also found that the circadian clock in Marchantia regulates growth of the thallus, possibly by affecting auxin levels. The circadian clock in land plants appears in structural differences between species. I showed that the gene MpDET1 has a conserved structure but harbors a different function compared to Arabidopsis. In Arabidopsis, growth is regulated by the clock through PIF genes, but in Marchantia, this pathway appears independent of PIF. Although the clock mechanism appear well conserved in land plants, its structure and function has evolved, creating diversity between land plant groups

Nyctinastic thallus movement in the liverwort Marchantia polymorpha is regulated by a circadian clock

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

PIF-independent regulation of growth by an evening complex in the liverwort Marchantia polymorpha

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.De två första författarna delar förstaförfattarskapet</p

DE‐ETIOLATED1 has a role in the circadian clock of the liverwort Marchantia polymorpha

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 co-repressor 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