Abstract
Stress
can be remembered by plants in a form of ‘stress memory’ that can alter
future phenotypes of previously stressed plants and even phenotypes of
their offspring. It was shown that DNA methylation is among the
mechanisms mediating the memory.
It
is not known for how long the memory is kept by plants. If the memory is
long lasting, it can become maladaptive in situations when
parental-offspring environment differ.
We
investigated for how long can a parental plant “remember” that it
experienced a stress and pass the memory to its clonal offspring.
We
grew parental plants of three genotypes ofTrifolium
repens for five months either in control conditions or in control
conditions that were interrupted with drought pulses applied for two
months in four different time-slots. We also treated half of the
parental plants with 5-azacytidine (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.
Such
a memory resulted in enhanced number of offspring side branches
originating from plants that experienced drought stress maximally 6
weeks before their transplantation to control environment. We did not
find any transgenerational memory in offspring of plants that
experienced drought stress later than 6 weeks before their
transplantation. 5-azaC also
reduced the effect of transgenerational memory on offspring ramets. We
confirmed that drought stress can trigger transgenerational memory inT. repens that is very likely mediated by
DNA
methylation. Most importantly,
the
memory was time limited and was gradually erased. We conclude that the
time limited memory on environmental stress can be adaptive as climate
tends to be variable and parental-offspring environmental conditions
often do not match.
Keywords Epigenetic
memory;
Memory
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 memory’ or “priming”. In some cases, the stress memory
can be passed to further generation(s) and affect thus offspring growth
and response to the stress despite no direct experience with the stress
(Cullins, 1973; Shock et al., 1998; Molinier et al., 2006; Monneveux et
al., 2013; Trewavas, 2014). This transgenerational memory 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).
One
of the intriguing questions is for how long can the str,ss memory
persist in a plant?
If
the stress memory has physiological and/or phenotypic consequences and
is maintained over long period, it could easily become maladaptive in
situations when stress events are rare or even absent. Hence, the
fundamental prerequisite for evolutionary adaptiveness of the stress
memory must be its reversibility and transiency
(Ding
et al., 2012; Virlouvet et al.,
2018). Memories 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 epigenetic memory 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).
In
the study of Shi et al. (2019) it took two years and ten clonal
generations to reset most of the environmentally induced epigenetic
memory in a clonal plant Alternanthera philoxeroides .
The
dynamic of environmental stress is, however, operating often at shorter
time scales, usually within days or months. It is thus extremely
interesting and important to focus on the stress memory from the
temporal dynamic perspective in order to improve our understanding of
stress memory in eco-evo processes in 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 than a naïve offspring (González et al.,
2017). Clonal plants usually prefer wet habitats (Klimeš et al., 1997,
van Groenendael et al., 1996; Ye et al., 2014) 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 memory that is
partly enabled by epigenetic mechanism, in this case by DNA methylation
(González et al., 2016). We have also shown that the memory can be
adaptive, i.e. offspring ramets of parents that experienced drought
responded to the drought better than naïve offspring (González et al.,
2017).
The
memory is translated into altered growth of offspring ramets in
comparison to plants without the memory (González et al., 2016, 2017).
In this study, we built on our previous studies on T. repens and
tested for how long parental plant carries the stress memory that is
detectable on clonal offspring phenotypes and whether the memory 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 memory that is time-limited and is
reset after certain period since the last drought event. (3) The drought
memory is facilitated by DNA methylation. Testing these hypotheses
should enable us to put the stress memory phenomenon into a time frame
context, which should improve our understanding of ecological and
evolutionary consequences of stress memory 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).
Each
phytomere of the plant consisting of a node, internode, subtending leaf,
axillary bud, and two nodal root initials is usually considered as a
ramet (Hay et al., 2001). Therefore, the growing stolon/branch is de
facto consisting of interconnected ramets that can develop independent
genet if connection between ramets is severed. 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 memory induction in parental generation
(further referred to as Parental generation), the second part was
designed to test for how long the parental plant carries memory on the
drought stress that affects clonal offspring generations (further
referred to as Offspring generation).
Parental
generation:Drought
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 for two weeks to allow recovery and successful rooting. The
plants were grown for five months either in control conditions (watered
when needed) or in control conditions repeatedly interrupted with
drought
pulses (watered only when leaves were wilting) that lasted for 10 weeks.
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. Plants from the
drought-stress treatments were divided in four groups in the way that
each group experienced 10-week drought period 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 as8W 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 as 6W group). In the third
group (n=8 per genotype), drought ended 4 weeks before establishment of
the Offspring generation part (further referred to as 4Wgroup). 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
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 × (watered 2 ×
more often with 4 × more water volume at each watering occasion) more
water than the drought stressed plants during the drought period. The
same level of watering as in controls was maintained in the drought
stressed plants outside the drought period. Two weeks after the last
drought event (i.e. 140th day of the experiment), we
created single standardized cutting (4 nodes with apical end) from every
plant individual. These cutting were used in the following Offspring
generation part, see later. The remaining above ground biomass of
parental plants was harvested, dried at 80°C for 48 hours and weighed.
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.
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.
Offspring
generation
On
day 140 of the experiment, we
created single standardized 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
Parental generation. We cultivated them 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 in the Parental generation and the
new,
offspring parts that have developed after transplantation (see Fig. 1b).
During the 10 weeks’ period, we recorded the length of the main stolon,
number of nodes of the main stolon and number of side branches every
week (10 times in total). This was used to calculate the growth rate of
individual plants after transplantation.
At
the end of the experiment (Ten weeks after establishment of the
Offspring generation),
we
harvested above-ground biomass separated in parental ramet planted into
Offspring generation and offspring ramets (main stolon and side
branches) that had developed after transplantation,
dried
them at 80°C for 48 hours and weighed.
Statistical analyses
We first tested the effect of
genotype (genotype A, B and C),
time
since the last drought event (2W, 4W, 6W, 8W where W means week, and
Control),
5-azaC
application and their interactions on biomass of the plant grown in
Parental generation of the experiment.
The data from continuous measurements of the plants in the Offspring
generation were used to calculated growth rate based on stolon length,
node number on main stolon and side branch
number.
To do this we fitted a growth function a × bx to the
data using a non-linear least squares (nls) function in R 3.5.1. for
each individual plant separately. The b value, representing the slope of
the growth curve, was used as a measure of growth rate (Latzel et al.
2012).
Because we had too many performance measures and they were highly
correlated to each other (r > 0.7),
we excluded some of the measures and
retained only total biomass of the offspring (i.e. grown in Offspring
generation), number of internodes of the main stolon, number of side
branches and growth rate based on node number on main stolon and side
branch number. We tested the effects of genotype, drought timing, 5-azaC
application and their interactions on these plant performance measures.
The parental biomass
(biomass
of the cutting transplanted to the Offspring generation that had
developed before transplantation) was use as a covariate to account for
potential initial size difference among transplanted ramets on the
subsequent growth. All the tests were done using generalised linear
model in R 3.5.1.
Side
branch number followed Poisson distribution. All the other variables
followed Gaussian distribution. Total biomass and growth rate of side
branch number had to be log transformed to fit the Gaussian
distribution.
Results