The effect of 5-azacytidine on Offspring generation
Application of 5-azaC on parental plants reduced number of side branches in offspring plants (Table 1, Control:10.45 ± 0.37, 5-azaC: 8.33 ± 0.37), but did not have a main effect on the other measured variables in offspring. The effect of 5-azaC interacted with time since last drought for node number and its growth rate and number of branches (Table 1, Fig. 2a, b). Drought stress in parental generation generally increased the number of nodes of the main stolon in Offspring generation, the 5-azaC significantly reduced number of the nodes in offspring of parents that experienced last drought event two weeks before transplantation (Fig. 2a). The application of 5-azaC in parents did not affect number of side branches produced by control parents (Fig. 2b), but reduced number of side branches in offspring of parents that experienced the last drought event 4 and 6 weeks before transplantation (Fig. 2b). Growth rate of the main stolon node number was interactively affected by the time since last drought and 5azaC. When 5-azaC was applied to parental ramets, the growth rate of offspring of parents that experienced the last drought event 2 and 8 weeks before transplantation significantly declined compared to offspring of control parents (Fig. 4; Fig S3).
Discussion
Our study investigated whether a clonal plantTrifolium repens can remember drought stress, and if so, for how long. We hypothesized that the memory should be gradually erased with the increasing time since the last drought experience. This prediction assumes that the long-term phenotypic consequences of the memory should be not beneficial in situation when the drought stress is infrequent or absent for a long period (Jiang et al., 2014; Shi et al. 2019; Lukic et al., 2020).
Results of our study are mostly in agreement with the predictions. We found that growth of offspring ramets was affected by the drought experienced by parents, which can be considered as an evidence of transgenerational stress memory. Such a memory was detected for offspring of parental ramets that experienced the last drought event 4 or 6 weeks before they were transplanted into the control environment in most of measured parameters but offspring biomass. Offspring biomass was affected even for offspring of parental ramets that experienced the last drought event 2 weeks before transplantation to control environment. Interestingly, some of the memory effects on the growth of offspring were absent in offspring of parents that were treated by 5-azaC. Our results thus suggest that the drought in parental generation can trigger transgenerational memory that can be carried maximally six weeks by parents of T. repens . The drought memory was in majority of cases erased by application of 5-azaC indicating that DNA methylation was involved in the memory. Our results thus indirectly supported the mounting evidence that epigenetic processes are involved in stress memory in plants (e.g. Molinier et al., 2006; Boyko et al., 2007; Whittle et al., 2009; Verhoeven et al., 2010; Xu et al., 2016; Nakamura & Hennig, 2017).
Some studies showed that the environmentally induced epigenetic change can be heritable among several sexual generations in the absence of the triggering stress (Verhoeven et al., 2010; Xu et al., 2016). Shi et al. (2019) showed that the environmentally induced epigenetic variation is 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 did not test the phenotypic consequences of epigenetic memory in plants but demonstrated that the environmentally induced epigenetic change can be heritable in certain cases (and species) and is carried by several (a)sexual generations. This is very intriguing phenomenon suggesting that some environments trigger memory that is fixed over longer period (and generations) than others and that the memory can be species specific. In our study, we simulated an environment that is repeatedly desiccating during summer season, i.e. periods with sufficient water supply were interrupted by periods of water shortage. This particular setting triggered memory that lasted for 6 weeks in the three genotypes of T. repens . Other scenarios with different timing and/or severity of a stress could trigger different memory effects that can have different phenotypic consequences on offspring generation. For instance, in our previous research on the same species, we observed that the stress memory is established only if the drought last for a certain period. We found that the drought stress can trigger transgenerational effects if it last for 10 weeks but not for 4 months (Rendina 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 stress memory in plants.
Previous studies investigated the role 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 memory. For instance, study by González et al. (2017) showed that the drought in parental generation can trigger adaptive stress memory in T. repens , i.e. offspring performed better in drought if their parents also experienced drought in comparison to offspring of naïve parents. However, they demonstrated it on offspring of parents that experienced drought period only recently, which may be ecologically rather rare scenario. It is possible that documented patterns of plant memory effects on transgenerational plasticity can be only snap shots in time, which can result in overestimation or underestimation of ecological and evolutionary aspects of memory in plants.
Our results along with other studies (Verhoeven & van Gurp, 2012; González, 2016; Münzbergová et al. 2019, Racette et al., 2019) point also on the genotype specificity of the stress memory. The length and number of nodes of the main stolon (parental ramet) were significantly or marginally significantly affected by the drought timing, genotype and 5-azaC application (Table 1; Fig. S2). The inherent differences among genotypes support the assumption that the existence and mechanisms of parental stress memory vary between genotypes. Growth rate of parental stolon length was significantly affected by interaction between time since last drought and methylation (Table 1). Our results suggest a possibility that DNA methylation is associated with, and may partially regulate, growth of the main stolon under drought stress, through a dynamic alteration of methylation patterns.
Other potential mechanisms involved in observed patterns of stress memory
The more vigorous growth of offspring of parents that experienced the last drought event 2 to 6 weeks indicates that the stress memory cannot be ascribed to negative consequences of physiological damage of the parents but rather to other mechanisms including DNA methylation. The mostly comparable growth of offspring of parents that experienced the last drought event 2 weeks prior their transplantation to control environment with controls implies that the stress memory effect was likely to some degree downregulated by remaining negative physiological consequences of the not so long-ago experienced drought (e.g. Kannenberg et al., 2020). On the other hand, the comparable growth of offspring of parents that experienced last drought 8 weeks and controls can be best explained by the loss of the drought memory. Similar biomass produced by parents from drought treatments applied in different time slots indicates that the stress intensity was similar across the parental treatments despite parents experienced the stress in their slightly different developmental stages. This is a good indicator that the differing stress memory observed in offspring was not a consequence of different stress intensity in parents.
Conclusion
The term stress memory has been already well established in plant ecology and became commonly accepted by experts. Based on our results of the actual as well as previous studies (e.g. Rendina González et al., 2016), we argue that the next inevitable step in upcoming research should be involvement of the temporal dynamics of the stress memory from the perspective of stress duration and the time when the stress occurred. This can help us not only better understand ecological and evolutionary aspects of the memory in 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 memory would also considerably improve our understanding of the memory mechanisms in our study. Although we focused on clonal generations, similar aspects of temporal dynamics of stress memory could be relevant for sexually derived individuals too.