The effect of 5-azacytidine on Offspring generation
Application of 5-azaC on parental plants in the first phase of the study consequently increased the mean offspring biomass, reduced number of side branches in transplanted plants of the second phase (Table 1, Fig.3a, b), but did not have a main effect on the other measured variables in transplanted plants. 5-azaC significantly altered mean offspring biomass (Table 1, Fig. 4). The mean offspring biomass of parents that experienced the last drought event 2 and 8 weeks before transplantation significantly increased compared to offspring of control parents (Fig. 4). The effect of 5-azaC was strongly genotype dependent (Table 1). In A genotype, the mean offspring biomass of parents that experienced the last drought event 4 weeks before transplantation significantly increased after 5-azaC application. In genotype B, significant effect of application of 5-azaC on parental plants was detected in plants that experienced last drought event two weeks before transplantation. In genotype C, plants which experienced the last drought event 2 and 8 weeks before transplantation were significantly bigger after 5-azaC application when compared to offspring of control parents (Table 1, Fig. 5).
Discussion
Our study investigated whether drought stress in the parental generation triggers transgenerational effects in a clonal plantTrifolium repens , and if so, for how long from the last drought event the stress legacy in parental plant persists and affects the phenotype of its clonal offspring. We hypothesized that the phenotypic consequences of transgenerational effects should be gradually erased with the increasing time since the last drought event. This prediction assumes that the long-term phenotypic consequences of transgenerational effects should be not beneficial in situation when the drought stress is infrequent, time-limited or even absent for a long period (Jiang et al., 2014; Shi et al. 2019; Lukic et al., 2020).
Results of our study are not in agreement with our predictions. We found that drought stress was detectable on the number of created offspring ramets even 8 weeks after the last drought experienced by parents in all genotypes. Our results thus suggest that the legacy of drought stress in a parental plant can last for at least 8 weeks (we did not test longer period because drought events simulated in our study cannot be expected to last more than few weeks in the Central European context) and trigger transgenerational effects that are affecting offspring phenotypes ofT. repens . This contradicts our prediction that the role of transgenerational effects should be gradually erased with the increasing time since the last drought event because they could easily become maladaptive in situations when stress events are rare or even absent. On the other hand, the long-lasting transgenerational effects due to the drought resulted in increased number of offspring ramets produced by parental ramets that experienced drought. This suggests that the negative effect of the drought on parental biomass can be to some degree compensated in the offspring generation. In other words, the stress legacy can provide plants with other advantage than only better coping with future stress. Hence, even the long-lasting stress legacy may not be maladaptive as long as it provides offspring with other benefits. These findings are to some degree in line with our previous study where we showed that particular intensity of drought stress in parental generation can increase offspring growth and biomass whereas different levels of drought result in reduced biomass of offspring ramets (González et al. 2016).
Some studies showed that the environmentally induced epigenetic variation can be heritable among several (a)sexual generations in the absence of the triggering stress (Verhoeven et al., 2010; Xu et al., 2016). Shi et al. (2019) found that the environmentally induced epigenetic variation is progressively degrading over 10 clonal generations (10 offspring ramets created from the establishment of the study) in a plant Alternanthera philoxeroides when cultivated in a common environment. These studies however were focused only on molecular mechanisms and did not test the phenotypic consequences of environmentally induced epigenetic variation in plants. Despite that they provided important evidence that the environmentally induced epigenetic change can be heritable in certain cases (and species) and is carried by several (a)sexual generations. In our study, we tested the role of DNA methylation on transgenerational effects indirectly via alteration of DNA methylation of half of the plants with 5-azacytidine (5-azaC). Our results outlined that DNA methylation was likely involved in the observed transgenerational effects as the effect of parental drought on mean offspring biomass was changed in plants treated with 5-azaC in comparison to plants of the same stress history but not treated with 5-azaC. Interestingly, 5-azaC did not alter growth of control plants (see Fig. 4 and 5), which supports our conclusion that the application of 5-azaC interacted with epigenetic memory on the drought stress.
The genotype specificity of the role of 5-azaC on transgenerational effects observed in mean offspring biomass (Fig. 5) is in line with other studies demonstrating that epigenetic variation can be highly genotype dependent (Richards 2006, Bossdorf et al. 2008, Becker et al. 2011, Li et al. 2012). Alternatively, potential structural and/or morphological differences among genotypes could led to different levels of absorption of the 5-azaC and thus in different efficiency of demethylation of DNA. It should be also noted that the stress legacy can be also ascribed to other than epigenetic mechanisms such as hormonal signalling or other metabolites involved in stress signalling (Hilker and Schmülling 2019) that could be present in transplanted parental ramets.
In our study, we simulated an environment that was repeatedly desiccating during summer season, i.e. periods with sufficient water supply were interrupted by periods of water shortage. This particular setting triggered stress legacy that lasted at least for 8 weeks in the three genotypes of T. repens . Of course, it is intuitive that other scenarios with different timing and/or severity of a stress could trigger different legacy effects that can have even contrasting phenotypic consequences on the offspring generation. For instance, in our previous research on the same species, we observed that the stress legacy is established only if the drought last for a certain period. We found that the drought stress can trigger transgenerational effects if it lasted for 10 weeks but not for 4 months (González et al., 2016). This phenomenon needs to be investigated in more detail to get better idea about the role of environmental stress, its intensity and duration on induction and temporal dynamics of transgenerational effects in plants.
Previous studies investigated the role of duration or intensity of environmental stress on induction of transgenerational effects (e.g. Boyko, 2010; Verhoeven & van Gurp, 2012; Rahavi & Kovalchuk 2013a, b; González et al. 2016; Racette et al., 2019) but did not consider the temporal dynamics of the stress legacy in plants. Study by González et al. (2017) showed that the drought in parental generation can trigger adaptive transgenerational effects in T. repens , i.e. offspring performed better in drought if their parents also experienced drought in comparison to offspring of naïve parents. However, the adaptive transgenerational effects were demonstrated on offspring of parents that experienced drought period very recently before transplantation to new environment, which may be ecologically rather rare scenario. It is possible that documented patterns of transgenerational effects can be only snap shots in time, which can result in overestimation or underestimation of ecological and evolutionary aspects of transgenerational effects in plants.
Conclusion
Based on our results of the actual as well as previous studies (i.e. González et al., 2016, 2017), we argue that the next inevitable step in upcoming research should be involvement of the temporal dynamics of the stress legacy from the perspective of stress duration and the time when the stress occurred in studies on clonal transgenerational plasticity. This can help us not only better understand ecological and evolutionary aspects of the transgenerational effects in clonal plants but could also improve our predictions of plant responses to future climatic conditions. More detailed insights into molecular (epigenetic) and biochemical mechanisms involved in the stress legacy would also considerably improve our understanding of the stress legacy mechanisms in clonal plants. Although we focused on clonal generations, similar aspects of temporal dynamics of stress legacy can be expected for sexually derived individuals.