Periodic Reporting for period 1 - PlantSeeFe (Plants seeking iron: unraveling the molecular mechanisms involved in iron-mobilizing coumarins distribution and trafficking)
Berichtszeitraum: 2021-09-01 bis 2023-08-31
Fe is an essential metal for a wide range of physiological events in many organisms, since it serves as a central component of various types of metalloproteins. A deficiency of Fe nutrition often causes developmental and growth issues for both animals and plants. This project, by studying the Fe acquisition by plants might help improving Fe content in crops and thus provide a sustainable wat to provide Fe to people who are suffering from Fe-dependent anaemia all over the world (about one billion persons).
Fe is the 4th most abundant metal on earth; however, the majority of Fe existing in natural soils is non-bioavailable as it is present in the form insoluble oxyhydroxides, in particular under aerobic and/or alkaline conditions. To cope with these harsh environments, plants have evolved sophisticated mechanisms to efficiently absorb Fe. Recent studies reported coumarin secretion from roots to play an important role in this process in non-gramineous plants, like Brassica and legume species. In particular, Fe-mobilizing coumarins (FMCs), mainly fraxetin and sideretin, are major compounds involved in Fe acquisition because of their chelation activity against trivalent metals such as ferric Fe (Fe3+). Interestingly, fraxetin was recently revealed to be accumulated in specific cell types within roots (i.e. epidermis, cortex and endodermis cell layers) using the model plant Arabidopsis thaliana. This suggested that the formation of uneven distribution of coumarins within roots should be essential for optimizing their secretion and Fe acquisition. However, the molecular mechanisms underlying FMCs transport remained largely unknown.
The overall objective of this project was to decipher how coumarin distribution and secretion are controlled in Arabidopsis roots in order to acquire Fe from soils. The Fellow recently isolated from Arabidopsis, by analysing genome-wide gene expression of Arabidopsis roots exposed to Fe deficient conditions, Coumarin Import Transporter (CIT) genes. Within the frame of this project, the Fellow aimed at answering the following three questions
(1) Which coumarins (aglycones or glycosides) are the transport substrates of CITs?
(2) Where (tissue, cell or organelle) do CITs mediate coumarin transport activity?
(3) How are CIT transport activities modulated?
Fe is the 4th most abundant metal on earth; however, the majority of Fe existing in natural soils is non-bioavailable as it is present in the form insoluble oxyhydroxides, in particular under aerobic and/or alkaline conditions. To cope with these harsh environments, plants have evolved sophisticated mechanisms to efficiently absorb Fe. Recent studies reported coumarin secretion from roots to play an important role in this process in non-gramineous plants, like Brassica and legume species. In particular, Fe-mobilizing coumarins (FMCs), mainly fraxetin and sideretin, are major compounds involved in Fe acquisition because of their chelation activity against trivalent metals such as ferric Fe (Fe3+). Interestingly, fraxetin was recently revealed to be accumulated in specific cell types within roots (i.e. epidermis, cortex and endodermis cell layers) using the model plant Arabidopsis thaliana. This suggested that the formation of uneven distribution of coumarins within roots should be essential for optimizing their secretion and Fe acquisition. However, the molecular mechanisms underlying FMCs transport remained largely unknown.
The overall objective of this project was to decipher how coumarin distribution and secretion are controlled in Arabidopsis roots in order to acquire Fe from soils. The Fellow recently isolated from Arabidopsis, by analysing genome-wide gene expression of Arabidopsis roots exposed to Fe deficient conditions, Coumarin Import Transporter (CIT) genes. Within the frame of this project, the Fellow aimed at answering the following three questions
(1) Which coumarins (aglycones or glycosides) are the transport substrates of CITs?
(2) Where (tissue, cell or organelle) do CITs mediate coumarin transport activity?
(3) How are CIT transport activities modulated?
The PlantSeeFe project is composed of four work packages. WP1, WP2, and WP3 were focused on scientific aspects to answer the open questions on CIT functions in coumarin trafficking within Arabidopsis roots while WP4 was entirely dedicated to the coordination and management of the project toward achieving the goal.
In WP1, two different approaches were exploited to identify transport substrates of CIT and reveal its physiological function: (i) by evaluating transport activity of heterologously expressed CIT in yeast cells and (ii) by phenotyping Arabidopsis mutants defective in CIT gene in Fe-deficient/-limiting conditions. With regard to (i), uptake transport activities of CIT were tested against several types of coumarin compounds using transgenic cells of Saccharomyces cerevisiae expressing CIT proteins. Levels of fraxetin and scopoletin, a major FMC and its precursor, respectively, were significantly higher in transgenic cells expressing CIT than that of control. These results suggest that fraxetin and scopoletin would be major transport substrates of CIT in Arabidopsis. Concerning (ii), mutants defective in CIT (cit) were tested for morphological alteration under Fe-limiting conditions. The cit showed severe growth retardation with lower fresh weight and chlorophyll levels and shorter primary root length under this condition if compared to wild-type. In accordance with this, coumarin secretion seemed to be lower in the cit than that of wild-type. On the other hand, these symptoms were drastically rescued either by fraxetin supplementation or by complementation with CIT-GPF fusion proteins driven by the CIT promoter (ProCIT). Taken together with the uptake transport activities, CIT is involved in enhancing the coumarin secretion in response to Fe shortage by mediating cellular uptake of scopoletin and fraxetin, leading to the formation of a bioavailable Fe in the rhizosphere.
In WP2, spatiotemporal expression of CIT within roots was analyzed using promoter-reporter systems to identify cell types in which CIT mediates cellular uptakes of coumarin in Arabidopsis roots. ProCIT-driven GUS reporter showed that CIT transcription was drastically increased by Fe deficiency and GUS activities were strongly expressed in cortex and epidermis cells of primary roots of plants grown under this condition. Microscopic observation using the ProCIT:gCIT-GFP fusion construct revealed that localization of CIT protein accumulation seemed to be equivalent to that of the GUS staining patterns. CIT-GFP proteins were dominantly detected in plasma membranes in these cell layers. The results obtained from the expression and localization analyses suggest that CIT proteins are inductively accumulated in cortex and epidermis cells of roots when plants encounter Fe-deficient conditions.
In WP3, a co-immunoprecipitation (Co-IP) experiment using GFP as an epitope tag was employed to identify CIT protein partners. Different GFP fusion constructs were assayed in Arabidopsis roots and ProCIT:gCIT-GFP was selected, since the accumulation of CIT-GFP fusion proteins was detected in roots under Fe deficiency and was sufficient to rescue the phenotypes of the cit mutant as above-described (WP1 and WP2). Experiments were conducted but no clear CIT partner were identified maybe because the transgenic lines used were not strongly enough expressing the CIT-GFP protein. Further investigation by Co-IP will be necessary to progress toward the identification of CIT interactors and thus to better characterize the regulatory mechanisms controlling CIT transport activities.
In WP1, two different approaches were exploited to identify transport substrates of CIT and reveal its physiological function: (i) by evaluating transport activity of heterologously expressed CIT in yeast cells and (ii) by phenotyping Arabidopsis mutants defective in CIT gene in Fe-deficient/-limiting conditions. With regard to (i), uptake transport activities of CIT were tested against several types of coumarin compounds using transgenic cells of Saccharomyces cerevisiae expressing CIT proteins. Levels of fraxetin and scopoletin, a major FMC and its precursor, respectively, were significantly higher in transgenic cells expressing CIT than that of control. These results suggest that fraxetin and scopoletin would be major transport substrates of CIT in Arabidopsis. Concerning (ii), mutants defective in CIT (cit) were tested for morphological alteration under Fe-limiting conditions. The cit showed severe growth retardation with lower fresh weight and chlorophyll levels and shorter primary root length under this condition if compared to wild-type. In accordance with this, coumarin secretion seemed to be lower in the cit than that of wild-type. On the other hand, these symptoms were drastically rescued either by fraxetin supplementation or by complementation with CIT-GPF fusion proteins driven by the CIT promoter (ProCIT). Taken together with the uptake transport activities, CIT is involved in enhancing the coumarin secretion in response to Fe shortage by mediating cellular uptake of scopoletin and fraxetin, leading to the formation of a bioavailable Fe in the rhizosphere.
In WP2, spatiotemporal expression of CIT within roots was analyzed using promoter-reporter systems to identify cell types in which CIT mediates cellular uptakes of coumarin in Arabidopsis roots. ProCIT-driven GUS reporter showed that CIT transcription was drastically increased by Fe deficiency and GUS activities were strongly expressed in cortex and epidermis cells of primary roots of plants grown under this condition. Microscopic observation using the ProCIT:gCIT-GFP fusion construct revealed that localization of CIT protein accumulation seemed to be equivalent to that of the GUS staining patterns. CIT-GFP proteins were dominantly detected in plasma membranes in these cell layers. The results obtained from the expression and localization analyses suggest that CIT proteins are inductively accumulated in cortex and epidermis cells of roots when plants encounter Fe-deficient conditions.
In WP3, a co-immunoprecipitation (Co-IP) experiment using GFP as an epitope tag was employed to identify CIT protein partners. Different GFP fusion constructs were assayed in Arabidopsis roots and ProCIT:gCIT-GFP was selected, since the accumulation of CIT-GFP fusion proteins was detected in roots under Fe deficiency and was sufficient to rescue the phenotypes of the cit mutant as above-described (WP1 and WP2). Experiments were conducted but no clear CIT partner were identified maybe because the transgenic lines used were not strongly enough expressing the CIT-GFP protein. Further investigation by Co-IP will be necessary to progress toward the identification of CIT interactors and thus to better characterize the regulatory mechanisms controlling CIT transport activities.
The PlantSeeFe project yielded excellent scientific results, leading to a better understanding of the molecular mechanism of coumarin trafficking within roots. A remarkable achievement of the project is the identification of the transporter mediating cellular uptake of coumarin, which is a completely novel piece in the elaborate mechanism of the coumarin secretion-based Fe acquisition strategy of plants. The up-to-date definition of plant coumarin secretion integrating CIT, provided by the project, will further support sustainable developments of human beings as well as plants because human beings Fe diet rely on plants. Therefore, the findings obtained from this project will benefit both quantitative and qualitative improvement of agricultural production. From the instructive point of view of the Fellow’s career, the implementation of the project also provided important opportunities for the Fellow to enhance his experimental techniques and skills for project management, global collaborative activities, presentation of findings and writing up scientific articles. These experiences will allow him to lead the global research team as a principal investigator and achieve multidisciplinary, innovative, and international collaborative research in near future.