Abstract
Stress
can be remembered by plants in a form of stress legacy that can alter
future phenotypes of previously stressed plants and even phenotypes of
their offspring. DNA methylation belongs among the mechanisms mediating
the stress legacy.
It
is however not known for how long the stress legacy is carried by
plants. If the legacy is long lasting, it can become maladaptive in
situations when parental-offspring environment do not match.
We
investigated for how long after the last exposure of a parental plant to
drought can the phenotype of its clonal offspring be altered.
We
grew parental plants of three genotypes ofTrifolium
repens for five months either in control conditions or in control
conditions that were interrupted with intense drought periods applied
for two months in four different time-slots. We also treated half of the
parental plants with a demethylating agent (5-azaC) to test for the
potential role of DNA methylation in the stress memory. Then, we
transplanted parental cuttings (ramets) individually to control
environment and allowed them to produce offspring ramets for two months.
The
drought stress experienced by parents affected phenotypes of offspring
ramets.
The
stress legacy resulted in enhanced number of offspring ramets
originating from plants that experienced drought stress even 56 days
before their transplantation to the control environment.
5-azaC altered transgenerational
effects on offspring ramets. We confirmed that drought stress can
trigger transgenerational effects in T. repens that is very
likely mediated by
DNA
methylation. Most importantly,
the
stress legacy in parental plants persisted for at least 8 weeks
suggesting that the stress legacy can persist in a clonal plantTrifolium repens for relatively long period. We suggest that the
stress legacy should be considered in future ecological studies on
clonal plants.
Keywords Epigenetic
memory;
Stress
legacy persistence; DNA methylation; 5-azacytidine
Introduction
An
increasing body of studies demonstrate that plants’ exposure to
different kinds of stresses in the past can affect their
responses
to the same and/or different stresses in the future and eventually
prepare them to respond rapidly and/or adaptively to forthcoming
stressful events
(Bruce
et al., 2007; Ding et al., 2013; Ramírez et al., 2015;
Li et al., 2014, Iwasaki &
Paszkowski, 2014, Li et al., 2019).
Such a phenomenon is commonly
called “stress legacy”, ‘stress memory’ or “priming”. In some cases,
the stress experience can be passed to further generation(s) and affect
thus offspring growth and response to the stress despite no direct
exposure to the stress (Cullins, 1973; Shock et al., 1998; Molinier et
al., 2006; Monneveux et al., 2013; Trewavas, 2014). Such
transgenerational effects can allow for rapid adaptation to
environmental condition if offspring environment resembles parental
conditions
(Mirouze
& Paszkowski, 2011; Latzel and Klimešová, 2010; Boyko & Kovalchuk,
2011; Latzel et al., 2014; González et al., 2017; Crisp et al., 2016;
González et al. 2017, Baker et al. 2019, Puy et al. 2021).
One
of the intriguing questions is for how long is the stress legacy
affecting the phenotypes of offspring?
If
the stress legacy has physiological and/or phenotypic consequences on
the offspring and is maintained over long period by the parental plant,
it could easily become maladaptive in situations when stress events are
rare or even absent. On the other hand, if the stress legacy is kept
only for a very short time it can have limited if any transgenerational
effects and thus potentially no role in transgenerational adaptation. In
other words, in order for memory to be advantageous to plants, plants
must balance between creating and keeping memory and being able to reset
the memory (Crisp et al., 2016). Information on the experienced stress
can be stored in the form of epigenetic variation
(Bruce
et al., 2007; Pascual et al., 2014;
McIntyre
& Strauss, 2014;
Richards
et al., 2017). It has been shown that environmentally induced epigenetic
variation can be transmitted to offspring generations (e.g.
Verhoeven
et al., 2010; Verhoeven &
van
Gurp, 2012; González et al., 2018) and can be gradually lost after
several sexual or asexual generations in the absence of the triggering
environmental stress (Jiang et al., 2014; Shi et al., 2019).
However,
the knowledge of temporal dynamics of the stress legacy on offspring
phenotype remains limited.
The
dynamic of environmental stress can be operating at time scales ranging
from several days to few weeks. For example, in the central European
context, common situation is when a relatively wet spring is followed by
a drier summer period that can last up to several weeks. From the
perspective of the clonal plant strategy, it only makes sense to produce
drought-ready clonal offspring when the offspring will experience
drought too. However, if the dry season is about to end it makes no
sense to keep producing drought-ready offspring. Nonetheless, we still
do not know whether such environmental dynamics is accounted for in the
stress legacy dynamics in clonal plants.
Drought is one of the main threats affecting plant growth, as water
deficit affects plants at all levels from molecular, cellular, organ to
the whole body (Li et al., 2014;
Avramova,
2015; Li & Liu, 2016;
Tombesi
et al., 2018).
Studies
have shown that plants that experienced repeated cycles of drought
stress exhibited both transcriptional and physiological responses during
a subsequent drought stress that were absent in plants without previous
drought experience (Ding et al., 2012, 2014; Virlouvet et al., 2018). It
has been also shown that the memory on drought can be passed to
(a)sexual offspring in Oryza sativa, Trifolium repens, Arabidopsis
thaliana or Zea mays(González
et al., 2016; Li et al., 2019; Ding et al., 2012, 2014; Virlouvet et
al., 2018) and can be even adaptive, i.e. offspring of stressed parents
overcome the stress better, i.e. has higher overall fitness, than a
naïve offspring (González et al., 2017). Clonal plants usually prefer
wet habitats (Klimeš et al., 1997, van Groenendael et al., 1996) making
them particularly vulnerable to drought events that should increase in
their frequency and severity in the near future
(Dai,
2012;
Sherwood
& Fu, 2014).
Clonal
plants may have greater ability to pass epigenetic information to
asexual generations than non-clonal plants to sexual generation because
of the lack of meiosis during clonal reproduction (Latzel & Klimešová,
2010; Verhoeven & Preite, 2014; Douhovnikoff & Dodd, 2015; González et
al., 2016; Paszkowski & Grossniklaus, 2011; Latzel & Münzbergová;
2018;
Münzbergová
et al., 2019). This makes clonal plants an ideal system for studying
various ecological and evolutionary aspects of transgenerational stress
memory in plants.
Our
previous studies on a clonal herb Trifolium repens have shown
that it can develop genotype specific drought stress legacy that is
partly enabled by epigenetic mechanism, in this case by DNA methylation
(González et al., 2016, 2018). We have also shown that the stress legacy
can be adaptive, i.e. offspring ramets of parents that experienced
drought responded to the drought better, produced more biomass, than
naïve offspring (González et al., 2017).
The
legacy is translated into altered growth of offspring ramets in
comparison to plants without the legacy (González et al., 2016, 2017).
Here, we built on our previous studies on T. repens and tested
for how long from the last exposure of a parental plant to the drought
can phenotype of its clonal offspring be affected and whether the
offspring phenotype alteration is co-facilitated by DNA methylation. We
tested the following
hypotheses:
(1)
Drought stress is altering growth of parental ramets. (2) This
alternation triggers drought-stress legacy that affects phenotype of
offspring ramets but is time-limited and is lost after certain period
since the last drought event. (3) The drought stress legacy is
facilitated by DNA methylation. Testing these hypotheses should enable
us to put the phenomenon of transgenerational effects into a time frame
context, which should improve our understanding of ecological and
evolutionary consequences of transgenerational effects in clonal plants.
Materials
and methods
Plant material
We
usedTrifolium
repens as the model in our study. It is a rapidly growing
polycarpic perennial herb widely
distributed in a variety of grasslands and pastures differing in soil
type, nutrient level, and soil humidity (Burdon, 1983).
In most studies, each phytomer of T. repens that consists of a
node, internode, leaf, axillary bud and two nodal root initials is
considered as a ramet (Hay et al., 2001, Goméz et al., 2007). However,
similarly to our previous studies on the species (González et al., 2016,
2017, 2018), we decided to apply more conservative approach and consideroffspring ramets only the side branches produced by elongating
main stolon, i.e. parental ramet . The monopodial growth style
of Trifolium repens means that every stolon elongates along its
main axis by producing new phytomers within which resource and
information flow is not restricted. On the other hand, the side branches
that are produced by axillary buds of the main stolon are more
independent from the main stolon because their connection to the main
stolon is limited and not permanent, which results in more limited
resources and information exchange among the main stolon and side
branches. In other words, the growth of side branches is more
independent on the physiological state of the main stolon. Such a
conservative approach provides us confidence that we can consider
potential observed environmental effects to be truly transgenerational
and ecologically relevant. See also Fig. 1 for a description of parental
and offspring clonal generations considered in our study.
We collected three cuttings taken from at least 50 meters distance from
a mesophilous meadow of the park
at
the Institute of Botany, Průhonice, Czech Republic to ensure that the
three cuttings were of different genotypes but
had similar growing conditions as
well as growing history. We vegetatively propagated them for four months
in the experimental garden prior the main experiment.
Study design
We
conducted the experiment in a greenhouse at the Institute of Botany,
Průhonice, Czech Republic with controlled temperature and light regime
from October 7, 2019 to May 4, 2020 (210 days in total). The greenhouse
had controlled temperature (23/18 °C day/night) and light regime
(12-/12-h light/night cycle). The experiment was divided in two parts.
The first consisted of stress legacy induction in parental generation,
the second was designed to test for how long the parental plant carries
legacy on the drought stress that affects clonal offspring generations.
First
phase -
d rought
stress application
We
created 120 standardized unbranched cuttings (parental ramets) from the
pre-cultivated plant material (three genotypes, 40 cuttings per
genotype) of T. repens . Each
cutting consisted of three nodes with apical end and was planted
individually into a tray 30 × 40 × 8 cm filled with standardized soil
(Trávníkový substrát, AGRO CS a.s., Rikov, Czech Republic, mixture of
sand, compost and peat, 75% mass water holding capacity). After
transplantation of parental ramets, we kept all plants in control
conditions (regular watering) for two weeks to allow recovery and
successful rooting. Afterwards, we randomly assigned plants to five
treatment combinations: control (n=8 per genotype), plants were watered
regularly to keep the soil constantly moist during the whole cultivation
period. and 4 drought-stress treatments. The plants were grown for 5
months in selected conditions. Plants assigned to drought stress
treatment experienced control conditions interrupted with drought
periods (watered only when leaves were wilting) that lasted for 10 weeks
but in different time slots (2 weeks difference among the slots, see
Fig. 1). In the first group (n=8 per genotype), the drought treatment
ended 8 weeks before establishment of the Offspring generation part
(further referred to as 8W group, see also Fig. 1). In the
second group (n=8 per genotype), drought ended 6 weeks before
establishment of the Offspring generation part (further referred to as6W group). In the third group (n=8 per genotype), drought ended
4 weeks before establishment of the Offspring generation part (further
referred to as 4W group). Finally, in the fourth group (n=8 per
genotype), drought ended 2 weeks before establishment of the Offspring
generation part (further referred to as 2W group). The drought
stress was implemented by watering a plant with 200 ml of water only
when the plant showed significant drought stress response, i.e., most
leaves
wilting.
The water volume that was determined by a pilot study
to
sufficiently moistened the soil and ensured that the next drought pulse
occurs within 4 to 7 days. During the 10-week drought period plants were
watered approximately 10 times. The control plants received 8 × more
water than the drought stressed plants during the drought period
(watered 2 × more often with 4 × more water volume at each watering
occasion) The same level of watering as in controls was maintained in
the drought stressed plants outside the drought period. The first phase
was terminated 140th day of the experiment.
5-azacytidine
application
To test for the role of DNA methylation in the stress memory induced by
drought, we applied 5-azacytidine demethylating agent on half of the
parental plants, the remaining plants were sprayed with the same volume
of pure water.
5-azacytidine
(further referred to as 5-azaC) reduces the global cytosine methylation
level of treated plants, and it has been successfully applied to
demonstrate the role of plant epigenetic memory in plant adaptation to
stress (e.g. Boyko et al., 2010; González et al., 2016). 5-azaC can be
toxic to plants and thus some growth responses of plants can be
consequences of the toxicity rather than the alteration of DNA
methylation. The unwanted side effects of 5-azaC are, however, related
almost exclusively to situations, when plants are germinated in 5-azaC
solution (Puy et al. 2018). Foliar applications of 5-azaC is bypassing
most of the negative effects on plant growth but keeps its demethylating
efficiency at comparable levels to germination plants in 5-azaC solution
(Puy et al., 2018). We
subjected
a half of the parental plants to 5-azaC treatment (4 plants per genotype
and treatment) to alter their epigenetic memory. We regularly sprayed
plants with 100 μmol solution of 5-azaC (Sigma-Aldrich, Praha, Czech
Republic) every fourth day, which resulted in 32 spraying events. The
first application was on October 21, 2019, i.e. 14 days after setting
the experiment (the day of start of the first drought treatment), and
with the last application at the time of the termination of the last
drought treatment (February 10, 2020, 126th day of the
experiment). We sprayed the plants in early morning to ensure that
plants had open stomata and the solution of 5-azaC could therefore be
easily absorbed by the leaves. We did not measure the level of
demethylation achieved by the 5-azaC treatment in this study. However,
in our previous study on the same species, by spraying plants eleven
times with 50 μmol solution of 5-azaC (i.e. half concertation and a
third of spraying events than used in this study) resulted in overall
reduction in methylation by 4.48% (González et al., 2016). Therefore,
we are confident that the application of 5-azaC was effective in this
study and resulted in reduction of overall DNA methylation level of
treated plants. However, we cannot exclude the scenario that plants
experiencing drought can react to the 5-azaC differently than plants
experiencing control conditions.
Second
phase – testing of stress legacy dynamics
On
day 140 of the experiment, we
created a single standardized
parental cutting consisting of four nodes and apical end from each
individual (40 cuttings per genotype, 120 cuttings in total) and
transplanted them individually to similar trays filled with the same
substrate as in
the
first phase. The remaining above ground biomass of parental plants
(further referred to as “parental biomass”) was harvested, dried at
80°C for 48 hours and weighed. By creating a cutting, we ensured that
the newly growing clone had no connection to the original parental plant
from the first phase. Thus, the new emerging clone could not receive any
signals from the parental plant that experienced the drought and all
phenotypic differences potentially detected on the newly emerging clone
can be ascribed to stress legacy mechanisms carried by the transplanted
cutting.
We cultivated the transplanted plants in a greenhouse under control
condition for 10 weeks (from Day 140 to Day 210 of the experiment).
We
labelled
the apical end of each transplanted
cutting to be able to identify the end of parental (transplanted) ramet
that had developed before transplantation and the
new
parts that have developed after transplantation (see Fig. 1b).
At
the end of the experiment (Ten weeks after establishment of the
Offspring generation),
we
record the number of side branches (i.e. offspring ramets) produced by
the elongating transplanted parental ramet. All clones consisted by
interconnected ramets at the end of the study. We harvested above-ground
biomass separated in parental ramet (main stolon was divided into parts
developed before and after transplantation) and offspring ramets (side
branches) that had developed after transplantation,
dried
them at 80°C for 48 hours and weighed. The mean offspring biomass was
calculated by offspring biomass divided by the number of side branches.
In a subset of randomly chosen plants we also checked the Rhizobia
colonisation of roots. We did not find any established relationship in
the 10 plants, which confirmed our previous experienced with the species
that the Rhizobia colonisation is rare under our growing conditions.
Statistical analyses
We tested the effect of genotype
(genotype A, B and C),
time
since the last drought (2W, 4W, 6W, 8W where W means week, and Control),
5-azaC
application and their interactions on parental biomass of the first
phase, mean offspring biomass developed in the second phase and number
of branches using generalised linear models with Poisson distribution
for number of branches and Gaussian distribution for the other two
variables. The significances were assessed using marginal tests, i.e.
the effect of each predictor was assessed after accounting for all the
other predictors in the model. We used duncan.test function in the
agricolae package in R to perform the post-hoc tests in case of
significant effects. The
parental
cutting biomass transplanted to the second phase of the study was used
as a covariate to account for potential initial size difference among
transplanted ramets on the subsequent growth when testing mean offspring
biomass and number of ramets. In preliminary tests, we explored whether
the effects of parental cutting size interacted with 5-azaC application,
drought treatment or genotype. As we did not detect any such significant
interaction, we did not consider these interactions in the models
presented here. To meet the assumptions of homoscedasticity and
normality, the biomass data were log transformed prior to analyses. All
analyses were done in R 3.5.1.
Results