Periodic Reporting for period 1 - REOX (Reconstructing the origin of the auxin response system)
Okres sprawozdawczy: 2022-01-10 do 2024-01-09
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.
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.
Due to technical problems based mainly on the lack of functionality in the algal transformation protocol, the objectives proposed in the project have been approached and changed in different ways as described below.
WP1's objective was to determine proto-ARF proteins' intrinsic activity. We have followed what was initially proposed and systematically performed Mparf mutant complementation using multiple protoARFs, both full length and only certain domains. We focused on arf1 mutant but we also implemented arf3 complementation to further understand the functionality of these proteins. Task 2 has only been partially done for reconstructing a DNA binding domain, as the rest of the protein was difficult to predict in a statistically robust manner. Task 3 has been performed as expected, which has opened new lines regarding the evolution of transcriptional activation.
Our main findings suggest that algal A/B-class ARFs do not retain A nor B-class function in land plants as they cannot complement the simple mutants in Marchantia. This indicates that A and B divergence was accompanied by a full neofunctionalization of their respective functions. Interestingly, major DNA binding properties are likely conserved between A/B ARFs and both A and B classes, suggesting that these functions are linked to either their transcriptional activities or interacting ability. In turn, algal C-class ARFs seem to fully complement land plant C-ARFs, indicating a deep conservation of C-class function, both in terms of DNA binding and biochemical function.
WP2 objective was to establish P. margaritaceum as a model and study PmARFs physiological relevance. However, after several attempts, we could not make the published Agrobacterium-mediated transformation protocol work. Instead, after two years of trying. we have been able to adapt an electroporation protocol for direct protein delivery.
For WP3 we followed our contingency plan, using a heterologous system, yeast, to reconstruct the auxin pathway. Yeast has proven very valuable for many other functional assays, specially interaction between multiple components. With this, we have shown that algal ARFs from both A/B and C classes act as repressors, and can oligomerize. We have also found that Aux/IAA can interact with protoARFs, this indicates that ARFs were pre-adapted to the recruitment by the auxin nuclear response system, but also suggests that an Aux/IAA-ARF co-repressor module likely exists in algae, hinting at the plausible existence of a yet-to-discover mechanism for Aux/IAA regulation unrelated to the auxin-sensing TIR1-mediated signalling.
The results produced to date are limited to the electroporation of algal cells protocol. This has been published in an special issue of Physiologia Plantarum in open access form (doi: 10.1111/ppl.14121) and is available through our university repository. Future results will follow the same path, but have been already discussed in different congresses.
WP1's objective was to determine proto-ARF proteins' intrinsic activity. We have followed what was initially proposed and systematically performed Mparf mutant complementation using multiple protoARFs, both full length and only certain domains. We focused on arf1 mutant but we also implemented arf3 complementation to further understand the functionality of these proteins. Task 2 has only been partially done for reconstructing a DNA binding domain, as the rest of the protein was difficult to predict in a statistically robust manner. Task 3 has been performed as expected, which has opened new lines regarding the evolution of transcriptional activation.
Our main findings suggest that algal A/B-class ARFs do not retain A nor B-class function in land plants as they cannot complement the simple mutants in Marchantia. This indicates that A and B divergence was accompanied by a full neofunctionalization of their respective functions. Interestingly, major DNA binding properties are likely conserved between A/B ARFs and both A and B classes, suggesting that these functions are linked to either their transcriptional activities or interacting ability. In turn, algal C-class ARFs seem to fully complement land plant C-ARFs, indicating a deep conservation of C-class function, both in terms of DNA binding and biochemical function.
WP2 objective was to establish P. margaritaceum as a model and study PmARFs physiological relevance. However, after several attempts, we could not make the published Agrobacterium-mediated transformation protocol work. Instead, after two years of trying. we have been able to adapt an electroporation protocol for direct protein delivery.
For WP3 we followed our contingency plan, using a heterologous system, yeast, to reconstruct the auxin pathway. Yeast has proven very valuable for many other functional assays, specially interaction between multiple components. With this, we have shown that algal ARFs from both A/B and C classes act as repressors, and can oligomerize. We have also found that Aux/IAA can interact with protoARFs, this indicates that ARFs were pre-adapted to the recruitment by the auxin nuclear response system, but also suggests that an Aux/IAA-ARF co-repressor module likely exists in algae, hinting at the plausible existence of a yet-to-discover mechanism for Aux/IAA regulation unrelated to the auxin-sensing TIR1-mediated signalling.
The results produced to date are limited to the electroporation of algal cells protocol. This has been published in an special issue of Physiologia Plantarum in open access form (doi: 10.1111/ppl.14121) and is available through our university repository. Future results will follow the same path, but have been already discussed in different congresses.
The progress has been covered in the previous section. As explained above, most of the problems were linked to algal transformation. With new tools as the electroporation method we have developed, it would be possible to do so but time forbids it, and is almost impossible to predict what kind of resources and timeframes would be necessary. However, the outcome of the remaining tasks has been very successful and we will be able to publish at least two additional manuscripts. The method published itself has been already well received by the community and we expect it to be adopted.
Following WP1, we expect results linked to the evolution and divergence of the transcriptional activity of ARF proteins which will likely lead to high impact publications.
Following WP1, we expect results linked to the evolution and divergence of the transcriptional activity of ARF proteins which will likely lead to high impact publications.