Periodic Reporting for period 1 - 3D-Plant2Cells (Exploring the Impact of Pesticide on the 3D Metabolome and the Microbiota from the Whole Plant to the Cell Scale)
Okres sprawozdawczy: 2017-01-01 do 2018-12-31
Conventional agriculture relies on the use of inorganic fertilizers and synthetic pesticides, whereas organic agriculture uses sustainable alternatives in order to exert a positive role on the cultivation environment. The influence of the agronomic practices of crops cultivated for human consumption have been subject to numerous studies, and showed that vegetal foods produced organically tends to have a higher content of metabolites associated with health benefices, such as phenolic derivatives with antioxidant activity and vitamins. On the other hand, fruits and vegetables obtained from crops grown conventionally contain more nitrates and pesticide residues. In addition to severely impacting natural ecosystems, many of these residues are involved or suspected to be involved in numerous diseases, such as autoimmunodisease or cancers, which has an important cost on the European society. Furthermore, organic farming promote plant microbial diversity, and fruits produced by organic and conventional agricultural practices have differences in their associated microbial communities. These plant-associated micro-organisms (plant microbiota) are key factors in the assimilation of nutriments but also in the defense against various pathogens. Plant-associated microbes produce specialized metabolites that facilitate nutrients absorptions, or that possessed antibiotic activity, which influence the plant microbial communities composition. Furthermore, micro-organisms present in crops for human consumption influence directly the human gut microbiome, and relationships had been observed between gut microbiota and numerous chronic diseases in humans, such as obesity or cardiovascular disorders. With the development of culture-independent methods based on high-throughput microbial DNA profiling (microbiome analysis), it is possible to study the real diversity of micro-organisms in their environment. The microbiome revolution revealed that only a minority of plant- associated micro-organism are cultivable with conventional methods, and were so far inaccessible. Thus, the chemistry and biological activities of their microbially-derived metabolites remain uncharacterized because most of the current scientific knowledge is derived from cultured micro- organism. To understand the role of microbial and host metabolites (metabolome) in situ, mass spectrometry-based metabolomics and mass spectrometry imaging have emerged as a potent tools to investigate the entire pool of metabolites in their ecosystem.
The project 3D-Plant2Cells proposes to explore the impact of the agricultural mode on crops grown for human consumption. We aimed at developing and applying innovative interdisciplinary approaches to study the metabolome and the microbiota of plants in three-dimensions (3D) at three different scales: the whole plant, the fruit, and the cellular scales. The objective of this study is to get a new insight on the metabolome and microbiota response to a pesticide treatment. Our hypothesis is that the molecular profiles and the microbiome can change upon the use of conventional cultivation managements. To explore this hypothesis, two groups of plants will be cultivated in growth chamber and one will received pesticide treatment(s). Then, we will apply the developed method to explore the host and microbial metabolome and the plant- associated commensal microbiota on the whole plant surface. Samples will be collected on various location of the plant, and the metabolites present will be profiled by high-resolution tandem-mass spectrometry, while the plant-associated microbial communities will be studied by targeted amplicon sequencing analysis. Results of these both methods will be uploaded into a bioinformatic model representing a 3D topological map of the metabolome/microbiome over the entire organism surface. In a second part of the research project, we will study fruits of the cultivated specimens by 3D mass spectrometry imaging. The goals of this study are to visualize, in a spatially-resolved way, the metabolome/microbiota response to pesticide treatments along with the pesticide distribution in 3D. The entire project can be an important contribution to assess the impact of pesticide on plant metabolome/microbiota and food safety, but also for the development of new methodological approaches to study the relationships between plant microbial communities and their host metabolome.
The project 3D-Plant2Cells proposes to explore the impact of the agricultural mode on crops grown for human consumption. We aimed at developing and applying innovative interdisciplinary approaches to study the metabolome and the microbiota of plants in three-dimensions (3D) at three different scales: the whole plant, the fruit, and the cellular scales. The objective of this study is to get a new insight on the metabolome and microbiota response to a pesticide treatment. Our hypothesis is that the molecular profiles and the microbiome can change upon the use of conventional cultivation managements. To explore this hypothesis, two groups of plants will be cultivated in growth chamber and one will received pesticide treatment(s). Then, we will apply the developed method to explore the host and microbial metabolome and the plant- associated commensal microbiota on the whole plant surface. Samples will be collected on various location of the plant, and the metabolites present will be profiled by high-resolution tandem-mass spectrometry, while the plant-associated microbial communities will be studied by targeted amplicon sequencing analysis. Results of these both methods will be uploaded into a bioinformatic model representing a 3D topological map of the metabolome/microbiome over the entire organism surface. In a second part of the research project, we will study fruits of the cultivated specimens by 3D mass spectrometry imaging. The goals of this study are to visualize, in a spatially-resolved way, the metabolome/microbiota response to pesticide treatments along with the pesticide distribution in 3D. The entire project can be an important contribution to assess the impact of pesticide on plant metabolome/microbiota and food safety, but also for the development of new methodological approaches to study the relationships between plant microbial communities and their host metabolome.
During this project, the largest tandem mass spectral (MS/MS) library of pesticides (729 spectra from 204 pesticides) was generated and made publicly available on the GNPS website [https://gnps.ucsd.edu/ProteoSAFe/libraries.jsp].
Moreover, a protocol for mass spectrometry (MS)-based 3D molecular cartography of pesticides residues and other metabolite on fruit surfaces was developed. Cotton swabs were used to sample various location of common fruits (tomato, apple, bell pepper, peach, mango, cucumber). The corresponding samples were then extracted and analysed by liquid phase chromatography (LC) coupled to tandem MS (MS/MS) in data dependent mode. The obtained data were processed with MassFuser [https://github.com/alexandrovteam/MassFuser] a new software that combines the mass spectral data from two complementary LC-MS/MS runs to achieve an optimal downstream analysis.
The MS data were mapped on the 3D model of the corresponding fruit using the ‘ili web-based software [https://ili.embl.de/ and https://www.nature.com/articles/nprot.2017.122]. The results of the 3D molecular cartography can be accessed at the following website [https://github.com/lfnothias/3d-plant2cells]. Our results showed that our protocol enables the detection of pesticides residues, and other metabolites. Annotations were performed with the GNPS web-platform (http://gnps.ucsd.edu) and the Sirius software (https://bio.informatik.uni-jena.de/software/sirius/). The mass spectrometry data are available on the MassIVE repository https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=13e66f51afc04712bfce317bb74a3d37 .
Note that the project was terminated one year after its initiation by the host beneficiary (January 2018), following the departure of the main researcher for an other research institution.
Moreover, a protocol for mass spectrometry (MS)-based 3D molecular cartography of pesticides residues and other metabolite on fruit surfaces was developed. Cotton swabs were used to sample various location of common fruits (tomato, apple, bell pepper, peach, mango, cucumber). The corresponding samples were then extracted and analysed by liquid phase chromatography (LC) coupled to tandem MS (MS/MS) in data dependent mode. The obtained data were processed with MassFuser [https://github.com/alexandrovteam/MassFuser] a new software that combines the mass spectral data from two complementary LC-MS/MS runs to achieve an optimal downstream analysis.
The MS data were mapped on the 3D model of the corresponding fruit using the ‘ili web-based software [https://ili.embl.de/ and https://www.nature.com/articles/nprot.2017.122]. The results of the 3D molecular cartography can be accessed at the following website [https://github.com/lfnothias/3d-plant2cells]. Our results showed that our protocol enables the detection of pesticides residues, and other metabolites. Annotations were performed with the GNPS web-platform (http://gnps.ucsd.edu) and the Sirius software (https://bio.informatik.uni-jena.de/software/sirius/). The mass spectrometry data are available on the MassIVE repository https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=13e66f51afc04712bfce317bb74a3d37 .
Note that the project was terminated one year after its initiation by the host beneficiary (January 2018), following the departure of the main researcher for an other research institution.
This study showed that swab-based 3D molecular cartography and liquid chromatography coupled to tandem mass spectrometry can be used to study pesticides residues and other metabolites on the surface of plants and fruits.