PhD Defense | Pol Laanen | The plant adaptive response to long term ionizing radiation exposure in development and reproduction: an epigenetic perspective

The plant adaptive response to long term ionizing radiation exposure in development and reproduction: an epigenetic perspective

Life is constantly exposed to some form of ionising radiation (IR) from natural sources, however, with the increase of anthropogenic activities and nuclear accidents such as Chornobyl or Fukushima this exposure can be significantly increased. Nonetheless, life persists in some of the most radiation contaminated locations thereby sparking the debate of a potential adaptation to radiation. Additionally, understanding the impact of IR on an ecosystem level is crucial for ensuring robust environmental radiation protection. Plants in particular are of interest as they are primary producers within an ecosystem while their inability to move away from danger allows for studying the impact as well as the potential for adaptation to IR. 

Adaptation can result from genetic changes such as mutations. Additionally, epigenetic changes have also been put forward as inducing faster adaptation while being sensitive to environmental cues. Previous studies have suggested that exposure to IR alters DNA methylation, one epigenetic mechanism, in plants. However, this phenomenon as well as the exact functional significance of these changes, is yet to be studied across multiple generations. To this end, we investigated the effects of IR on DNA methylation in young Arabidopsis thaliana seedlings (seven-day old) over multiple generations of exposure. As such, parent (naïve plants), generation 1, and generation 2 plants were exposed to either 30 mGy/h, 60 mGy/h, 110 mGy/h, or 430 mGy/h or to natural background radiation (control condition) over two weeks. After global methylation analysis (UPLC-MS/MS) and identification of differentially methylated regions (DMRs)(whole genome bisulphite sequencing), intra- and intergenerational comparisons of DMR-associated genes and transposable elements showed that most DNA methylation changes occurred in the CG methylation context. Furthermore, most changes were seen in the plants exposed to the lower dose rates, and especially in the second generation. Many discovered DMRs were associated with transposable elements and showed hypermethylation, likely to increase genetic stability. A significant number of genes with DMRs were found to be important in developmental as well as various (a)biotic stress responses, including DNA damage repair and RNA splicing. These findings suggest a potential role for DNA methylation in the regulation of the IR-stress response as well as a potential role in plant adaptation to IR.

In order to further study the effects of IR over multiple generations, a second experiment was set up in which Lemna minor plants were exposed for 6 weeks to either 400 µGy/h, 25 mGy/h, or 90 mGy/h or to control conditions. Additionally, a subsection of the irradiated plants were allowed to recover under control conditions, for 1 week, after 5 weeks of chronic irradiation. Phenotypic follow-up of the plants showed healthy development in all conditions. A transient hormesis effect was observed in the growth rate of the irradiated plants in the first week. An RNA-seq analysis showed a sustained stress response in the irradiated plants which remained present throughout the 6 weeks of irradiation, despite seemingly normal phenotypic growth and development. The plants that were allowed to recover showed similar growth and development to the control plants. They did, however, maintain a larger number of genes that were affected by IR similarly to the irradiated plants over multiple generations (≥2 generations), indicating a persistent transgenerational response. The recovering plants also established a new homeostasis different from the control and/or irradiated plants. The DNA methyltransferases, CMT3 and MET1, also showed time dependent and IR-induced expression changes, further indicating the importance of DNA methylation in the plant’s response to IR. Furthermore, many of the results (growth rate, endoreplication, cell division, RNA-seq) indicate the potential of adaptation to IR.

Previous research has shown that IR exposure affects the development of flowers and the timing of flower induction. Additionally, in one of the experiments of this thesis, genes associated with flowering were differentially methylated after irradiation. Consequently, we investigated the effects of chronic IR exposure on the growth, development, and flowering of a single generation. A. thaliana plants of different ages (1, 2, or 3 weeks-old) were exposed to either natural background radiation (control), 20 mGy/h, or 100 mGy/h for 14 days. After irradiation leaves of different physiological age (leaf 3, 4, 5, 6, 7, and 9) were harvested and analysed. Firstly, IR induced a growth reduction which was dependent on plant age. Secondly, an accelerated senescence was observed in the irradiated plants with increased endoreplication levels and expression of various phytohormones (IAA, ACC, JA, gibberellins). Thirdly, a stress response was observed in the plants in the form of increased levels of the stress phytohormone, JA, as well as the observed changes in endoreplication. Finally, while no significant changes were observed in flower induction after irradiation, differential expression of a selection of flowering genes was observed.

The plant microbiome, or endophyte communities, can help the plant survive various stressors including drought, pathogens, and salt stress. Additionally, endophytes have been proven to be able to be transferred vertically (from parent to offspring). Therefore, endophytes might play a role in the plant’s adaptation to stressful environments. As such, in a pilot study A. thaliana seeds were collected from 5 different locations varying in IR levels (total dose rates (internal + external) of 1.03, 46.24, 52.80, 370.69 µGy/h, and a control location (0.39 µGy/h)) in the Chornobyl exclusion zone. 16S-sequencing of plant seed endophytes showed indications of a reduction in endophyte richness resulting from increased IR exposure. Additionally, several OTUs (operational taxonomic units) were found to be limited to the control and low dose locations, potentially indicating a loss of more IR-sensitive endophyte species in the higher exposed plant seeds. Nonetheless, various OTUs were found in the higher dose rate locations with many of the linked endophyte being capable of resisting increased levels of IR exposure as well as many other stressors such as soil contamination (i.e. salt, heavy metals, arsenic). Some of these species have also been shown to be beneficial contributors to plant growth and/or stress protection. Thereby, further investigations into the potential role of the endophyte community in plant survival under IR might be advantageous.

In conclusion, our results demonstrate firstly, that IR (20-100 mGy/h) induces a stress response in naïve parental plants and over multiple generations. Secondly, the response to IR exposure is likely epigenetically (at least via DNA methylation) and hormonally regulated. Thirdly, the multigenerational IR response is likely transferred from one generation to the next (possibly via DNA methylation and endophyte communities). And lastly, despite changes on a molecular level, the plants studied in this thesis are able to grow, develop, and flower while showing IR-induced accelerated aging. Overall, the results show that long-term effects remain present at an underlying (e.g. molecular) level, over multiple generations, which is not immediately visible on a phenotypic level. Additionally, plants that are allowed to recover after irradiation seem to establish a new post-IR homeostasis which is different to non-irradiated plants. Hence, these long-term effects can lead to changes in the population, including the way the population responds to renewed IR exposure and/or other stressors, but also changes in the population (e.g. accelerated aging, changes in flower development and/or the timing of floral induction) can lead to changes in the greater ecosystem network. The findings of this thesis are therefore important in the broader scope of environmental radiation protection.

Promotor:

• Ann Cuypers (UHASSELT)

SCK CEN mentors:

• Nele Horemans

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