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.