Evolution of the Auxin Response Factors from charophyte ancestors

Author summary Plants transition from water to land was determining for the history of our planet, since it led to atmospheric and soil condition changes that promoted the appearance of other life forms. This transition initiated around 1 billion years ago from a Charophyte algae lineage that acquired features allowing it to adapt to the very different terrestrial conditions. Land plants coordinate their development with external stimuli through signalling mechanisms triggered by plant hormones. Therefore, evolution of these molecules and their signalling pathways likely played an important role in the aquatic to terrestrial move. In this manuscript we study the origin of auxin signalling, a plant hormone implicated in all plant developmental steps. Our studies suggest that out of the three families of proteins originally proposed to trigger auxin signalling in land plants, only one existed in Charophyte ancestors as a likely transcriptional repressor independent of auxin. We show that despite millions of years of evolution, this family of proteins has conserved its biochemical and structural properties that are found today in land plants. The results presented here provide an insight on how hormone signalling pathways could have evolved by co-opting a pre-existing hormone-independent transcriptional regulatory mechanism. Auxin is a major developmental regulator in plants and the acquisition of a transcriptional response to auxin likely contributed to developmental innovations at the time of water-to-land transition. Auxin Response Factors (ARFs) Transcription Factors (TFs) that mediate auxin-dependent transcriptional changes are divided into A, B and C evolutive classes in land plants. The origin and nature of the first ARF proteins in algae is still debated. Here, we identify the most 'ancient' ARF homologue to date in the early divergent charophyte algae Chlorokybus atmophyticus, CaARF. Structural modelling combined with biochemical studies showed that CaARF already shares many features with modern ARFs: it is capable of oligomerization, interacts with the TOPLESS co-repressor and specifically binds Auxin Response Elements as dimer. In addition, CaARF possesses a DNA-binding specificity that differs from class A and B ARFs and that was maintained in class C ARF along plants evolution. Phylogenetic evidence together with CaARF biochemical properties indicate that the different classes of ARFs likely arose from an ancestral proto-ARF protein with class C-like features. The foundation of auxin signalling would have thus happened from a pre-existing hormone-independent transcriptional regulation together with the emergence of a functional hormone perception complex.