Periodic Reporting for period 1 - DYNAMET (Interplay of plant one-carbon metabolism and redox homeostasis in the context of dynamic DNA methylation (DYNAMET))
Periodo di rendicontazione: 2018-06-01 al 2020-05-31
Drought, heat, and other environmental stresses associated with global warming are major limiting factors for plant productivity. Concurrently, a continuous increase in crop yields is essential to keep pace with the growing world population and achieve food security for future generation - despite restricted cultivable land and other limiting natural resources. To this end, it is imperative to explore and develop new sustainable crop improvement strategies. Modern breeding methods have contributed substantially to improving agronomic traits; yet, extensive selection has resulted in losing genetic diversity in elite crop varieties. Moreover, new breeding technologies involving genetic modification face legislative barriers and consumer reservations. With regards to these challenges, natural and induced epigenetic variation constitutes an alternative source of trait modification, but its usefulness in agriculture remains unclear.
Plant acclimation to recurring stress involves epigenetic changes mediated by methylation of DNA and histones, which are the main constituents of chromatin. It is known that environmental stress causes changes in DNA and histone methylation but the underlying mechanisms and biological functions are still poorly understood. Therefore, this project focused on the molecular interactions between stress-related metabolic changes and chromatin methylation in the model plant Arabidopsis to gain new insights into plant acclimation mechanisms and how they can be used for crop production under challenging environmental conditions.
DNA and histone methylation are catalyzed by distinct methyltransferases, which require the cofactor S-adenosylmethionine (SAM) as methyl donor. During methylation, SAM is converted to S-adenosylhomocysteine (SAH), which in turn is a competitive inhibitor of SAM-dependent methyltransferases. SAM production and SAH degradation are integral parts of the methionine cycle, which depends on methyl supply by folate-mediated one-carbon (C1) metabolism. Accordingly, changes in C1 metabolism can affect DNA and histone methylation patterns and are associated with diseases and developmental defects. We have previously shown that MTHFD1, a central enzyme in folate metabolism, is required for maintaining proper DNA and histone H3K9 methylation. Here, we further investigated the molecular function of MTHFD1 to understand its role in DNA and histone methylation. Thereby, we particularly focused on the involvement of MTHFD1 in redox homeostasis.
In conclusion, we observed that MTHFD1 plays an important role in stabilizing DNA methylation patterns against environmental stress. Accordingly, mthfd1 mutants showed increased dynamics of genome-wide DNA methylation, whereas leave tissue DNA methylation patterns in wild type plants were largely robust against exposure to reactive oxygen species. In addition, we observed impaired redox homeostasis and increased levels of light- and UV-induced reactive oxygen species in leaf tissue of mthfd1 mutants, indicating that MTHFD1 is involved in antioxidant defence. Our results provide new insights into the mechanisms of DNA methylation dynamics during stress responses and contribute to our understanding of the environmental impact on epigenetic changes in plants.
Plant acclimation to recurring stress involves epigenetic changes mediated by methylation of DNA and histones, which are the main constituents of chromatin. It is known that environmental stress causes changes in DNA and histone methylation but the underlying mechanisms and biological functions are still poorly understood. Therefore, this project focused on the molecular interactions between stress-related metabolic changes and chromatin methylation in the model plant Arabidopsis to gain new insights into plant acclimation mechanisms and how they can be used for crop production under challenging environmental conditions.
DNA and histone methylation are catalyzed by distinct methyltransferases, which require the cofactor S-adenosylmethionine (SAM) as methyl donor. During methylation, SAM is converted to S-adenosylhomocysteine (SAH), which in turn is a competitive inhibitor of SAM-dependent methyltransferases. SAM production and SAH degradation are integral parts of the methionine cycle, which depends on methyl supply by folate-mediated one-carbon (C1) metabolism. Accordingly, changes in C1 metabolism can affect DNA and histone methylation patterns and are associated with diseases and developmental defects. We have previously shown that MTHFD1, a central enzyme in folate metabolism, is required for maintaining proper DNA and histone H3K9 methylation. Here, we further investigated the molecular function of MTHFD1 to understand its role in DNA and histone methylation. Thereby, we particularly focused on the involvement of MTHFD1 in redox homeostasis.
In conclusion, we observed that MTHFD1 plays an important role in stabilizing DNA methylation patterns against environmental stress. Accordingly, mthfd1 mutants showed increased dynamics of genome-wide DNA methylation, whereas leave tissue DNA methylation patterns in wild type plants were largely robust against exposure to reactive oxygen species. In addition, we observed impaired redox homeostasis and increased levels of light- and UV-induced reactive oxygen species in leaf tissue of mthfd1 mutants, indicating that MTHFD1 is involved in antioxidant defence. Our results provide new insights into the mechanisms of DNA methylation dynamics during stress responses and contribute to our understanding of the environmental impact on epigenetic changes in plants.
To determine the role of MTHFD1 in redox homeostasis, wild-type Arabidopsis plants, mthfd1 and control mutants impaired in redox homeostasis were grown under controlled environmental conditions and high-light as well as UV treatment to induce oxidative stress. Subsequently, redox status was determined by state-of-the-art metabolomics, enzymatic assays, reactive oxygen species (ROS) staining, and imaging techniques. To test, how changes in redox homeostasis affect DNA methylation, seedlings were grown on control media and media supplemented with redox-active chemicals. Subsequently, DNA methylation patterns were analyzed by whole genome bisulfite sequencing. In summary, the results showed that glutathione and other metabolite levels linked to redox homeostasis are significantly altered in mthfd1 mutants and, accordingly, mthfd1 mutants are more sensitive to oxidative stress and showed concomitant changes in genome-wide DNA methylation.
To further dissect the links between metabolism and epigenetic regulation, Arabidopsis seedlings containing a DNA methylation-sensitive GFP reporter were used to screen a small compound library for hits that alter DNA methylation in mthfd1 mutants and/or wild-type plants. Using a semi-automated imaging pipeline based on confocal microscopy of 96-well plates, we identified and confirmed candidate compounds for follow-up analyses and target identification.
Exploitation and dissemination: The results have been presented at international conferences. Manuscripts for publication of the results in peer-reviewed scientific journals are being prepared.
To further dissect the links between metabolism and epigenetic regulation, Arabidopsis seedlings containing a DNA methylation-sensitive GFP reporter were used to screen a small compound library for hits that alter DNA methylation in mthfd1 mutants and/or wild-type plants. Using a semi-automated imaging pipeline based on confocal microscopy of 96-well plates, we identified and confirmed candidate compounds for follow-up analyses and target identification.
Exploitation and dissemination: The results have been presented at international conferences. Manuscripts for publication of the results in peer-reviewed scientific journals are being prepared.
The outcome of the project sheds new light on the functions of folate metabolism in plants. According to our results, impaired MTHFD1 activity not only blocks the methionine cycle and DNA/H3K9 methylation, as previously shown, but also leads to altered glutathione redox balance and increased sensitivity to oxidative stress. In contrast to previous studies, which indicated that decreased NADPH production by MTHFD1 homologs leads to oxidative stress, the altered glutathione redox balance was not linked to decreased NADPH levels in mthfd1 mutants; instead, the redox changes are probably associated with homocysteine accumulation observed in mthfd1 mutants. Moreover, our work emphasizes the importance of folate metabolism in regulating C1 fluxes to maintain SAM-dependent methylation during cellular responses to environmental changes. Finally, the identified chemical suppressors of mthfd1 constitute a valuable resource for further investigation of the connections between folate metabolism and epigenetic regulation and to test their potential use as epigenetic drugs in plants and other organisms. In a bigger context, our results contribute to the development of new approaches for breeding and sustainable cultivation of crops with improved characteristics, such as stress resistance and nutrient or vitamin content.