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Application of high-sensitive and high-throughput molecular tools to disentangle the mechanisms of heavy metals accumulation and tolerance in mosses: epigenetic and transcriptomic approaches

Periodic Reporting for period 2 - BRYOMICS (Application of high-sensitive and high-throughput molecular tools to disentangle the mechanisms of heavy metals accumulation and tolerance in mosses: epigenetic and transcriptomic approaches)

Reporting period: 2018-07-01 to 2019-06-30

Anthropogenic emission of heavy metals (HM) constitutes a major environmental problem due to the high toxicity and persistence of these pollutants in different environmental compartments. Over the recent decades, Europe implemented regulatory actions to reduce the emission of HM and improve air quality. However, advancing the knowledge on the interaction between living organisms and HM pollution is also crucial. Plants constitute a very valuable biological resource to study this interaction because they are sessile and therefore unable to escape their surroundings. Our study system, terrestrial mosses, are unique among land plants because: i) they are in a key phylogenetic position between green algae and vascular plants, providing a better insight into the evolution of the mechanisms used by plants to deal with environmental contamination; and ii) mosses mostly rely on biochemical strategies to deal with HM pollution (given their low level of anatomical and morphological complexity), which adds further biotechnological value to the advance in knowledge of the mechanistic basis of their hyperaccumulator and tolerant phenotypes. BRYOMICS aims at investigating such moss specific strategies to provide key information to better understand and manage the effects of pollution in ecosystems, their components, and their functions, and to discover genes and gene products needed to develop technological tools to deal with extant contamination.
To achieve its goal, we explored the mechanisms underlying intraspecific variation in HM accumulation and tolerance in two terrestrial moss species with contrasting affinity to heavy metals, the copper (Cu) moss Scopelophila cataractae, and the cosmopolitan moss Ceratodon purpureus. We collected four populations of S. cataractae from different contamination levels within a former Cu mine, one population of C. purpureus in an urban area, and studied male and female plants of C. purpureus that were already growing in axenic conditions in the lab. We cultured all populations under control, Cd, and Cu enriched treatments and analyzed changes between control and contaminated replicates at different levels. First, we focused on functional phenotypic changes measuring Cd and Cu accumulation and plant performance (growth and oxidative damage). Second, we used epigenotyping by sequencing (epiGBS) to create methylation and genetic profiles of the samples searching for epigenetic responses. Finally, we used RNA sequencing to detect expression changes associated with Cu exposure.
For the first time, we found evidence for phenotypic differentiation for HM accumulation and tolerance among proximate populations of S. cataractae (maximum distance < 500 m), as a result of the different selective pressures experienced by these plants in the field. For C. purpureus, our results showed significant phenotypic differentiation among field and lab populations and, for the first time, sexual dimorphism for metal tolerance. We also observed population-specific gene expression profiles in response to Cu in both species that allowed us to better categorize candidate genes/pathways involved in Cu tolerance and will help to identify them once a well annotated reference genome becomes available for any of the two study species.
In addition to the scientific part, BRYOMICS developed an important outreach dimension aimed at raising general public awareness about environmental pollution, moss ability to deal with some of long-lived pollutants and how science can contribute to solve such problems.
During the grant period, we successfully accomplished the following tasks:
1. Collection of field samples of two ecologically different terrestrial moss species.
2. Implementation of a common garden experiment in the laboratory to study accumulation and tolerance to HM under controlled conditions.
3. Evaluation of the effects of HM exposure on moss performance.
4. Analysis of the total concentrations of HM in moss tissues to evaluate their accumulation capacity.
5. Implementation of the wet-lab technique for reduced representation bisulfite sequencing epiGBS to build the methylome profiles of mosses experimentally exposed to different levels of HM.
6. Implementation of the wet-lab techniques for RNA sequencing (RNAseq), to evaluate changes in gene expression and to identify candidate genes and/or pathways involved in HM tolerance in mosses.
7. Complete analysis of data generated in 3 and 4, including communication of results in scientific conferences.
8. Partial analysis of data generated in 5 and 6, including communication of results within the host institutions for discussion.
9. Substantial improvement of the applicant’s competences in bryology, microscopy, molecular genomic techniques, bioinformatics analysis of genomic data and epigenetics that have contributed to the foster some promising scientific collaborations with other researchers in the field of bryology and epigenetics.
10. Dissemination of the goals of this Marie Sklodowska-Curie Action at multiple levels and for different audiences, including the general public (by means of our project web page and Facebook page, the European Researchers’ Night, and participation in outreach activities with kids), and the scientific community (by participating in several scientific meetings and imparting several seminars, as well as the academic network ResearchGate).
Main results achieved:
1. Demonstrating, for the first time, the existence of intraspecific differences in tolerance to and accumulation of HM. In the metal specialist S. cataractae, such differences are related to the levels of contamination in their origin environments at very short spatial distances.
2. Demonstrating the existence of differences between sexes in tolerance to HM in the facultative metallophyte C. purpureus.
3. Demonstrating the existence of significant differences in gene expression in response to Cu in the two species. The magnitude of these differences varied between populations for both species, and between sexes for C. purpureus.
The results on the phenotypic response of mosses to Cd and Cu plus the total and relative concentrations of these metals in the plants’ tissues will stand alone for a publication (bullet points 1 and 2 above), currently under preparation. The results of the methylome and transcriptome profiles will be published later either in the same or in two different manuscripts depending on the final results. We recently published a book chapter entitled “Epigenetic approaches in non-model plants” describing the epiGBS method in detail within the Methods in Molecular Biology series.
BRYOMICS establishes the molecular basis of the HM accumulation and tolerance in bryophytes. Even though previous studies showed evidence of the existence of phenotypic plasticity and local adaptation to HM pollution in some mosses, yet none has looked at the possible genes and pathways involved in this phenotype. Our findings indicate that mosses previously exposed to HM have improved their ability for thriving under high levels of Cu and Cd. By comparing the underlying mechanisms driving this change in mosses with that of vascular plants BRYOMICS is also contributing to place bryophytes at the forefront of the research of the plant-environment interactions from an evolutionary perspective given their interesting phylogenetic position, between green algae and tracheophytes.
Population of Scopelophila cataractae growing in the center of the mine
Abandoned mine site where we collected populations from Scopelophila cataractae