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.