The ability to use multiple molecules as signals to communicate within and between cells is instrumental for the coordination of biological processes. Extant living beings contain different arrays of signalling molecules, many known as hormones, to regulate physiological and developmental processes. Intriguingly, a single hormone can trigger very different responses depending on the context they are acting. The presence of multiple forms of signal transduction proteins is a likely cause for the variation in the response, as most hormone signalling pathways have multiple isoforms of receptors, effectors, and other regulators. The majority of our knowledge in hormone signalling comes from studies in the so-called “higher” organisms, i.e: multicellular organisms with multiple tissues. But the ancestral functions of hormone pathways before complexification, or response diversification, are unknown. To understand what these functions were, it is necessary to study organisms with reduced complexity within their hormonal response systems, or with states representing the situation before the establishment of complete response systems.
To approach this question we propose the use of a key and multifunctional plant hormone: auxin, and its nuclear signalling pathway, and the addition of a Charophycean algal model that contains an incomplete set of auxin response components. We aim to understand the function of the Auxin Response System (ARS) elements in such organisms, lacking a full canonical auxin response, and to reconstruct the emergence of this system to uncover the characteristics required to assemble a hormonal response in plants. The study of these mechanisms and functions will reveal the origin and ancestral functioning of a major hormone response system in plants. The approaches for this project include the development of algal genetic tools, contributing to establishing models for the study of plant evolution in general. My long-term goal is to understand the molecular basis of how response systems emerge and diversify to generate multiple outputs.
Given that no species is representative of a simpler, ancestral state from which land plant lineages diverged, we propose to use Charophycean algae and bryophytes to reconstruct the evolutionary trajectory that allowed the emergence of the canonical auxin response pathway by focusing on the closest orthologs of the transcriptional regulatory proteins, the ARFs, and to recreate their regulation by auxin. The project specific objectives are:
1) To characterize the properties of algal proto-ARF in terms of biochemistry and activity. With the current knowledge on phylogenies and sequences available, we will resurrect the closest ancestral proteins to analyse their functionalities. We hypothesize that these proteins will also act as transcriptional regulators, representing the ancestral ARF functional state.
2) To analyse proto-ARF functional relevance in Penium margaritaceum cells. For this we aim to adapt genetic tools amenable for functional analysis of algal biology. Given the accessibility to a proper genome assembly and previous success in genetic transformation, we expect to be able to characterize proto-ARF activity in algal cells. We will also analyse the regulatory complex that fine-tunes the activity of proto-ARFs using interactomic approaches.
3) To reconstruct the auxin response system in a Charophyte. Using the tools and knowledge gathered, we will resurrect the ancestral proteins involved in auxin response and introduce the minimal protein set to constitute an auxin response module. This will help us understand the steps auxin response system needed to be functional and create a chassis to work on to understand complexity.
