Veesam Prathyusha

Elucidating the molecular mechanisms
underlying thermopriming mediated

acquired thermotolerance in
Arabidopsis thaliana
Master's Thesis
submitted to
Indian Institute of Science Education and Research Tirupati
in partial fulfilment of the requirements for the
BS-MS Dual Degree Programme
by
Prathyusha Veesam
Roll Number: 201701094
Supervisor: Dr. Annapurna Devi Allu

IISER TIRUPATI
April 2022
© Prathyusha Veesam (2022)
All rights reserved
1
Certificate
This is to certify that the MS Thesis entitled “Elucidating the molecular mechanisms
underlying thermopriming mediated acquired thermotolerance in Arabidopsis
thaliana” submitted towards the partial fulfilment of the BS-MS dual degree programme
at the Indian Institute of Science Education and Research Tirupati, represents the study
/ work carried out by Ms. Prathyusha Veesam at Indian Institute of Science Education
and Research, Tirupati under the supervision of Dr. Annapurna Devi Allu, Department
of Biology, during the academic year 2021-2022 and the same has not been submitted
for any other degree or diploma of any other university or institute.
Name of student: Prathyusha Veesam
Signature of student:
Date:10/04/2022
Name of Supervisor: Dr. Annapurna Devi Allu Name of Co-Supervisor:
Signature of Supervisor: Signature of Co-Supervisor:

Date: 10.04.2022 Date:
2
Declaration
I declare that the matter presented in the MS thesis entitled “Elucidating the
molecular mechanisms underlying thermopriming mediated acquired
thermotolerance in Arabidopsis thaliana” are the results of the work carried out by me
at the Department of Biology, Indian Institute of Science Education and Research Tirupati
under the supervision of Dr. Annapurna Devi Allu
The contents are expressed in my own words and where other’s ideas have been
included, I have adequately cited and referenced the original sources. I also declare that
I have adhered to all principles of academic ethics and integrity and have not fabricated
or falsified any idea / data / fact / image source in my submission.
I understand that violation of the above will lead to disciplinary action by the Institute
and can also evoke penal action from the sources which have thus not been properly
cited or from whom proper permission has not been obtained.
Name of student: Prathyusha Veesam
Signature of student:
Date: 10/04/2022
Endorsed by
Name of Supervisor: Dr. Annapurna Devi Allu Name of Co-Supervisor:
Signature of Supervisor: Signature of Co-Supervisor:
Date:10.04.2022 Date:
3
Acknowledgements
I would like to thank Dr. Annapurna Devi Allu for her constant guidance and support
throughout the project.
I also extend my sincere gratitude to my TAC member Dr. Sreenivas Chavali for his
suggestions.
I sincerely thank Devidutta Samantaray, Ph.D. scholar in our group for supporting me
through the important experiments. I also thank Durga Prasad Naik, previous MS-thesis
student for laying the foundation for this project and for his valuable suggestions which
helped me in improving my work. I thank Akshay U Nair, Noble Louis, Sai Thejas, Lohitha
Cheenepalli and the rest of the plant stress biology lab members for their continuous
cooperation and support.
I'd like to thank my family members, particularly my mother, Mrs. Subhadra Cheemala,
for her unwavering support, love and encouragement as well as my close friends Kedar
Prasanna N.D, Bhavya Pasupuleti for their unconditional support through my highs and
lows.
Lastly, I would like to thank IISER TIRUPATI for providing the platform for carrying out
my research.
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Contents
TITLE PAGE. NO
Certificate….............................................…......... 2
Declaration…...........................................…......... 3
Acknowledgements…..............................…......... 4
Abbreviations…........................................…......... 6
Abstract…................................................…......... 7
Chapter 1 Introduction…..........................…......... 8-14
Chapter 2 Materials and methods…........…......... 15-17
Chapter 3 Results and discussion……..…........... 18-41
Conclusion and Future perspectives………......... 42-43
References…....................................................… 44-48
5
Abbreviations
C Control
CMT3 Chromomethyltransferase 3
CM-H2DCFDA Chloromethyl derivative of H2DCFDA
DAT Days after Triggering
DMSO Dimethyl sulfoxide
HDA6 Histone deacetylase 6
HSPs Heat shock proteins
HSFs Heat shock factors
HDACs Histone deacetylases
KYP/SUVH4 KRYPTONITE/SU(VAR)3-9 homolog
KO Knockout
LAT Long-term acquired thermotolerance
MET1 Methyltransferase 1
PS Priming stimulus
PC Primed-control
P Priming
R Recovery
ROS Reactive oxygen species
RdDM RNA directed DNA methylation
SAT Short-term acquired thermotolerance
TS Triggering stimulus
TFs Transcription factors
TSA Trichostatin-A
UP Unprimed
WT Wild-type
6
Abstract
High temperature is a serious threat to plant growth and crop yield. Plants possess innate
capacity to cope with the changing climatic conditions. However, they exhibit a
remarkable ability to mount a modified, and robust stress response, due to pre-exposure
to a stress (termed as priming). Thermopriming is one such phenomenon that aids the
plant to cope with high temperatures, effectively. However, the molecular mechanisms
underlying priming-induced plant stress responses are not well understood and are
emerging. One such mechanism is the RdDM pathway which plays an important role in
maintaining the plant growth & development, genomic stability, and stress responses.
We analysed some of the RdDM pathway genes for their basal and priming-induced heat
stress response (acquired thermotolerance) using T-DNA insertion mutants. Our results
suggest that HDA6, KYP, and CMT3 act as negative regulators of acquired
thermotolerance. Further analysis revealed that HDA6 may not be involved in regulating
the duration of stress memory. Further, our data suggests that HDAC activity during the
recovery phase may play an important role in regulating the acquired thermotolerance.
Determination of ROS in wild-type and hda6 mutant seedlings under heat stress (with or
without priming) revealed higher ROS accumulation in the root meristematic region. To
study if KYP and CMT3 cooperatively act with HDA6 in the regulation of acquired
thermotolerance and thermomemory, the double ko mutants were generated. Further
analysis of these mutants will allow us to gain more insights into the involvement of the
HDA6-KYP6-CMT3 loop in regulating thermomemory.
7
Chapter 1
Introduction
Plants being sessile, are often exposed to unfavourable environmental conditions that
affect their growth and development. Both biotic and abiotic factors are known to pose
significant threats to crop development and productivity at multiple levels (Pandey et al.,
2017). More importantly, the recent changes in climatic conditions led to an increase in
the intensity and frequency of abiotic stresses. High temperature (referred to as ‘Heat or
heat stress’) and water deficit (referred to as ‘drought’) are the most devastating abiotic
stresses affecting crop productivity (Lamaoui et al., 2018). Notably, the recent report on
climate change predicted a steady rise in temperature in the near future (IPCC, 2021),
which is detrimental to the crop yield. Every 1°C rise in temperature has been shown to
reduce the crop yield by about 10% (Zhao et al., 2017). However, the effect of heat stress
on the growth, yield and even survival of the plants is prominent at almost all the
developmental stages of the plant (Prasad et al., 2008). To overcome the adverse climatic
conditions, plants have developed several strategies. Extensive research in the recent
past has substantially contributed to our current understanding of the plant heat stress
responses, which involve mechanisms at morphological, physio-biochemical, and
molecular levels. Molecular regulation includes modulation at transcriptional, post-
transcriptional, and epigenetic levels (Boyko et al., 2008).
1.1 Priming and stress memory

Recent evidence suggests that plants can adapt to the ever-changing environmental
conditions by remembering their previous experience of stress (which is referred to as
priming). ‘Priming’ refers to the pre-exposure of plants to mild stress, where the plant
remembers such event (referred to as ‘stress memory’), which enhances the plant’s ability
to respond to future stress(es) (Conrath et al., 2007; Lämke et al., 2016; Nair et al., 2022).
Interestingly, apart from the biotic and abiotic factors, several signaling molecules such
as hydrogen peroxide or reactive oxygen species (ROS) and phytohormones can also
induce a ‘primed’ state (Das et al., 2014; Hasanuzzaman et al., 2020;). Notably, the
duration of stress memory ranges from hours to days and weeks (somatic memory: short-
or long-term stress tolerance) and even to subsequent generations (transgenerational
memory) (Lamke et al., 2017).
Interestingly, basal and acquired stress tolerance involves common mechanisms/factors
such as efficient redox homeostasis, several transcription factors (TFs), histone and DNA
8
modifications, and signalling molecules (Ohama et al., 2016; Nair et al., 2022). Priming
alters gene expression dynamics, thereby aiding in mounting an efficient response. Rapid
recruitment of several regulatory factors renders the plants to mount timely responses by
activating multiple defence mechanisms to overcome future stress(es) (Kara et al., 2019).
These features of ‘priming’ attracted significant attention from plant science researchers
in the recent past. In particular, priming-induced heat stress tolerance is one of the
emerging mechanisms to combat abiotic stresses that requires extensive investigation.
1.2 Basal and acquired thermotolerance

As discussed above, the inherent ability of plants to respond under a higher temperature
than the optimal temperature for their growth is referred to as basal thermotolerance (Fig.
1). Pre-exposure to a mild temperature (referred to as ‘thermopriming’) tremendously
enhances the plant heat stress tolerance, referred to as ‘priming-induced or acquired
thermotolerance’ (Fig. 1) (Nair et al., 2022). Acquired thermotolerance is one of the most
studied priming-induced stress tolerance mechanisms at molecular level. However, more
studies are essential to gain a deeper understanding of the multi-layered regulatory
mechanisms. Apart from the model plant, Arabidopsis thaliana, thermopriming-induced
stress tolerance has been reported in several crop plants such as rice, wheat, and maize
(Liu et al., 2021). Notably, thermopriming-induced heat stress memory may last for
several hours (short-term acquired thermotolerance, SAT) to days (long-term acquired
thermotolerance, LAT) or even generations (transgenerational) (Baurle et al., 2016).
Heat stress Response strength
Unprimed plant Basal thermotolerance
Stress responses
Naïve plant
Short term-acquired
thermotolerance (SAT)
Priming stimulus
Acquired thermotolerance Long term-acquired
Primed plant thermotolerance (LAT)
9
Figure 1. Schema representing basal- and priming-induced acquired thermotolerance in
plants (Modified from: Nair et al., 2022).
1.3 Molecular mechanisms underlying plant stress memory

Priming-mediated thermotolerance and the associated stress memory is regulated at
multiple levels. The study conducted by Conrath et al. (2006) suggests two potential
mechanisms to be involved in imprinting and maintenance of the stress memory. One is
through the accumulation of signaling molecules (proteins) or TFs, and the second is
through epigenetic modifications (Fig. 2).
STRESS (pre-exposure)
Priming - for biotic stress
Hardening – for abiotic stress
Accumulation of proteins Epigenetic change

Or TFs (Mechanism 2)
(Mechanism 1)
Stress imprint
Stress exposure Enhanced physiological

response
Figure 2. Figure showing the mechanism of priming-induced stress tolerance and

formation of stress-imprint (modified and redrawn from Bruce et al., 2007).
Upon exposure to several stresses, the signaling molecules may accumulate in inactive
configurations that get activated upon exposure to subsequent stresses with the help of
protein kinases (get activated by changes in calcium levels) (Bruce et al., 2007). Several
heat shock proteins (HSPs) and heat shock factors (HSFs) that regulate the stress-
induced gene expression are activated in response to heat stress and also are involved
in the establishment and maintenance of the acquired thermotolerance (Schoffl et al.,
1998). Notably, accumulation of TFs in primed plants enhances defence gene
transcription after stress recognition (Conrath et al., 2006). It has been shown that there
are several stress-induced TFs identified in Arabidopsis thaliana under cold, drought, and
salinity stresses (Msanne et al., 2011; Pandey et al., 2013). HIGH EXPRESSION OF
OSMOTICALLY RESPONSIVE GENES 1 (HOS10) encodes R2P3 type MYB TF,
10
essential for cold acclimation, and appears to affect dehydration stress tolerance in plants
by controlling stress-induced ABA biosynthesis (Zhu et al., 2005; Chinnusamy et al.,
2010).
Other mechanism involved in imprinting stress memory include epigenetic modifications,
including DNA modifications (methylation of cytosines) and histone modifications
(acetylation and deacetylation, SUMOylation, ubiquitination, phosphorylation, etc.)
(Madlung et al., 2004; Loidle et al., 2004; Dowen et al., 2012; Mozgova et al., 2019).
Chromatin modifications regulate the activation or repression of gene expression. In
Arabidopsis thaliana various epigenetic modifications maintain vernalization through the
FLOWERING LOCUS C (FLC) gene (Song et al., 2004; Xiao et al., 2013). Epigenetic
modifications play a role in the growth and development of plants and their stress
responses (Li et al., 2000; Yamamuro et al., 2016; Rehman et al., 2020).
1.4 HDACs and their role in plants

In plants, epigenetic mechanisms such as histone modifications, nucleosome positioning,
and miRNA accumulation aid in maintaining the thermomemory by altering memory-
related gene expression levels (Yamaguchi et al., 2021). Several epigenetic regulators
such as methyltransferases, acetyltransferases, deacetylases, and demethylases
regulate the methylation and acetylation patterns of several heat-responsive TFs and
heat-responsive genes during the heat stress responses (Lämke & Bäurle et al., 2017;
Xu et al., 2017). Histone deacetylases (HDACs) are one of the key chromatin modifiers
involved in the regulation of various developmental processes and stress responses
(Yruela et al., 2021). In plants, 3 different classes of HDACs are found including
Rpd3/Hda1 (reduced potassium-dependence 3/histone deacetylase 1; Zn2+-dependent
hydrolases), SIR2 (silent information regulator 2; NAD+ -dependent enzymes) and HD2
(HD-tuins) class of HDACs. According to sequence similarity, the Rpd3/Hda1, Zn2+-
dependent HDACs are further sub-divided into class I, III, and IV HDACs (Yruela et al.,
2021). Among the different classes of HDACs, class I HDACs (HDA6, HDA7, HDA9,
HDA19) were identified to be more associated with the plant stress responses (Yruela et
al., 2021). HDA6 belongs to class 1 RPD3- like HDACs, required for cytosine methylation,
transposable elements, and rRNA gene silencing (Early et al., 2010). HDA6 is responsible
for the deacetylation of lysine residues in the N-terminal part of core histones which helps
in epigenetic repression and plays a crucial role in transcriptional regulation, cell cycle
progression, and plant developmental events as well as stress responses. Mutations in
hda6 induce high acetylation at H4 lysine residues, increase the methylation of H3 at the
lysine 4 position, and affect the transgene-silencing and regulation of rRNA genes
(Aufsatz et al., 2002). Wu et al., 2008 reported that HDA6 is required for jasmonic acid
(JA) response, senescence, and flowering in Arabidopsis. Also, it has been shown that
HDACs are directly involved in ABA and abiotic stress responses (Song et al., 2005).
Although several previous studies identified differential histone acetylation/methylation to
11
be associated with imprinting or maintenance of plant stress memory, functional validation
of the histone modifiers is at a nascent state. Previous studies in our group identified a
histone deacetylase, HDA6 acts as a potent negative regulator of thermopriming-
mediated acquired thermotolerance (Unpublished data). Further investigations are
essential to understand the HDA6-driven mechanisms that govern acquired
thermotolerance and the heat stress memory.
1.5 RdDM pathway involved in the regulation of heat stress responses

RNA directed DNA methylation (RdDM) pathway is one of the chromatin-modifying
signaling pathways extensively involved in various stress responses. It involves the
degradation of transposable elements (TEs) through 24 short nucleotide RNAs and the
expression of adjacent stress-responsive genes (Pikaard et al., 2008; Popova et al.,
2013). In Arabidopsis, about 15 homologs of SU(VAR)39 histone methyltransferases,
SET domain proteins, associated with RdDM pathway were reported to be involved in
heat stress responses (Xiao et al., 2019). Among them (KYP/SUVH4) is the major histone
methyltransferase that methylates H3K9 at repeated sequences and SUVH5 and SUVH6
maintain epigenetic transcriptional repression (Ebbs et al., 2006; Underwood et al., 2018).
Mutations in this KYP/SUVH4 cause increased seed dormancy and its expression is
regulated by abscisic acid (ABA) and gibberellic acid (GA) (Zheng et al., 2012). There is
growing evidence that KYP/SUVH5/6 directly interacts with HDA6 to regulate the TEs
expression (Yu et al., 2017; Hung et al., 2022). In most cases, DNA methylation acts as
a repressive mark in transcription. De novo methylation of cytosine residues in CG, CHG,
and CHH (H= A, T, G) are methylated by Methyltransferase 1 (MET1) with cooperation of
DRM and Chromethylase3 (CMT3) in plants (Cao et al., 2003). Maintenance of CHG
methylation involves the combined action of CMT3 and several SET domain H3K9
methyltransferases (Cao et al., 2002). Several studies have shown that in Arabidopsis,
histone H3K9 dimethylation and CHG methylation are reciprocally maintained by a loop
between CMT3 and KYP (Cao et al., 2003; Sun et al., 2015). Popova et al., 2013 had
shown that the RdDM pathway is essential for basal thermotolerance by studying DNA
methylation deficient mutants. Transcriptional gene silencing through the RdDM pathway
depends on DNA methylation levels and histone modifications (Sake et al., 2012). TE
silencing modulated by DNA methylation, histone acetylation, and histone methylation is
further supported by Liu et al., 2012, where HDA6 and MET1 interact directly to silence
TEs. Several previous studies revealed that CMT3 predominantly methylates at the H3K9
position (Johnson et al., 2002; Lindorth et al., 2004), suggesting that HDA6 indirectly
maintains methylation on CHG and CHH by regulating H3K9me2 of TEs (Liu et al., 2012).
There is growing evidence that epigenetic pathways play a role in maintaining plant
responses under stressful conditions but not much information is known about heat stress
memory. To obtain detailed insights into, mutants of the RdDM pathway viz. cmt3 and
kyp6 have been studied for their thermomemory. Ultimately, our research aims at
12
understanding the role of class-I HDACs and the RdDM pathway genes (such as KYP
and CMT3) in the regulation of long-term acquired thermotolerance (LAT) and sustenance
of heat stress memory.
1.6 Hypocotyl elongation dynamics

Heat stress mediates in inhibition of the cell elongation. Hypocotyl of Arabidopsis thaliana
is widely used as a model to study the effect of light and other growth factors on cell
elongation (Howel et al., 1995). The extent of hypocotyl elongation depends on the
presence and absence of light. In the absence of light (skotomorphogenesis), the
development of etiolated seedlings occurs with closed cotyledons, and the formation of
apical hooks. Conversely, in the presence of light (photomorphogenesis) de-etiolated
seedlings with open and green expanded hypocotyls are observed (Wiebosch et al.,
1973; Wei et al., 1994; Josse et al., 2008). Other than dark and light conditions, several
growth hormones such as auxin, gibberellic acid, brassinosteroid (BR), cytokinins (CK),
ABA, and ethylene (ET) regulate hypocotyl growth. Auxin, GA, and BR aid in the growth
of hypocotyl, whereas CK, ET, and ABA decrease the growth of hypocotyl (Clouse et al.,
1996). By using hypocotyl elongation as a model, Hong et al., 2000 developed a
screening method to identify the mutants which are defective in acquired thermotolerance
and identified four separate genetic loci involved in the process of acquired
thermotolerance. This hypocotyl elongation assay can be used for testing the effect of
temperature on seedling growth (Charng et al., 2006). For instance, analysing hypocotyl
growth in HSP21 transgenic plants showed that the HSP21 overexpression and wild-type
seedlings retained their ability to elongate hypocotyl after thermopriming treatment,
(Sedaghatmehr et al., 2016). In the case of hsp21-amiRNA seedlings, the hypocotyl
elongation ceased under the thermopriming conditions, proving that HSP21 is crucial for
extending thermomemory. It is previously shown that thermopriming shows its effect on
cell elongation we are interested to see if this is mediated through hda6 i.e. if the hda6
mutant shows any difference in the hypocotyl elongation under dark treatment, with or
without priming in response to heat stress.
1.7 The interplay between ROS generation and epigenetic regulation

Upon heat stress Reactive oxygen species (ROS) are produced in plants. ROS are
recognized as toxic by-products of aerobic metabolism, a crucial player in plant stress
signalling (Baxter et al., 2013). Several studies have shown the importance of ROS in
signalling pathways, controlling plant growth and development. Its primary role in
response to abiotic and biotic stresses is well studied (Liu et al., 2016). ROS is majorly
localized in the chloroplast, mitochondria, and peroxisomes (Das et al., 2014) and
participates in several plant stress responses by acting as signalling molecules. However,
at elevated concentrations, ROS can cause irreversible cellular damage leading to
13
alterations in plant morphological, biochemical, and physiological homeostasis (Wahid et
al., 2007; Bose et al., 2014).
ROS has an impact on epigenetic mechanisms regulating gene expression. Simazu et
al., 2013 showed that HDACs in mammals are involved in epigenetic regulation and
oxidative stress, which can also change their localization under oxidative stress
conditions (Doyle and Fitzpatrick et al., 2010). Several histone modifications such as
H3K4me2/3, H3K9me2, H3K27me3, and H3K79me3 increase due to increase in ROS
levels and lead to inhibition of histone demethylation (Zhou et al., 2008; Niu et al., 2015;
Chen et al., 2016). There is also growing evidence that there exists a close link between
ROS metabolism and epigenetic regulation during plant growth and environmental
acclimation (Kohli et al., 2019; Hasanuzzaman et al., 2020).
In rice, overexpression of OsSRT1, which is a SILENT INFORMATION REGULATOR 2
related HDAC gene showed enhanced tolerance to oxidative stress, while the RNAi line
of the same gene induces H2O2 levels, DNA fragmentation, and cell death (Huang et al.,
2007). Studies conducted on HDA19 and HDA9 showed that the activity of both the genes
reduces by cellular oxidation thereby enhancing histone acetylation and transcription of
stress-responsive genes (Liu et al., 2015).We are interested in understanding the cross-
talk between ROS production and epigenetic regulation during the thermopriming in hda6
mutant and the wild-type, which can aid in gaining insights into the signalling
pathways/mechanisms being differentially regulated upon thermopriming mediated
acquired thermotolerance.
14
Chapter 2
Materials And Methods
2.1 Plant materials and growth conditions
2.1.1 Basal and acquired thermotolerance
Seeds of Arabidopsis thaliana wild-type (Col-0) and other mutants were procured from
the Arabidopsis Biological Resource Centre (ABRC). Arabidopsis thaliana (Col-0 and
other mutants) seeds were surface sterilized by washing with 80% ethanol and 1%
sodium hypochlorite solution followed by washing with sterile double distilled water.
Sterilized seeds were semi-dried on autoclaved filter papers and were sown on 1⁄2 MS
media plates (Murashige and Skoog, 1962) and kept for stratification (2 days at 4℃ in
dark condition). After stratification, plates were transferred to optimal growth conditions
(light: 16 h, 22℃; dark: 8 h, 22℃: relative humidity, 50-60 % throughout the light-dark
cycle). Four-day-old seedlings were used for thermopriming and HDAC inhibition assays.
The heat priming treatments were provided to Arabidopsis seedlings as described in the
Fig. 3A. Briefly, one group of seedlings (16 seedlings/plate) were subjected to a double
priming cycle of priming stimulus 1 (PS1) at 37°C for 90 minutes (min) followed by a
recovery period of 90 min at optimal growth conditions and a second cycle at 40°C for 45
min termed as primed-control plants. These set of primed-control plants do not get
exposed to triggering heat stress. One set of seedlings were kept at 22°C (optimal
conditions) and served as control. Another set of plants were exposed to triggering
stimulus (TS) at 45°C for 90 min after two cycles of PS followed by a recovery phase (3,
4 or 5 days) termed as primed plants. The fourth set of plants were directly exposed to
the TS at 45°C for 90 min termed as unprimed plants. The basal and acquired
thermotolerance of the plants were quantified in terms of loss of chlorophyll (chlorosis),
death and shoot regeneration.
For determining the effect of 1°C decrease in priming and triggering on plant phenotype,
following treatment conditions were used. For basal and acquired thermotolerance
seedlings were subjected to PS2 of 44°C for 45 min and TS of 44℃ for 90 min in case of
primed and unprimed plants, respectively (Sedaghatmehr et al., 2016). Plant response to
the heat stress was quantified in terms of loss of chlorophyll (chlorosis), death and shoot
regeneration.
2.1.2 Plant growth parameters

To determine the effect of stress treatment on seedlings, fresh and dry weights were
measured at the end of the treatments. After treatment, entire rosettes of the primed
plants were collected, excessive water was removed using tissue paper and measured
the total shoot fresh weight (FW). After noting the fresh weight, the rosettes were wrapped
15
in aluminium foil, oven dried (70°C for 72-hour, h) and weighed for total shoot dry weight
(DW) using a highly sensitive digital balance (Sartorius).
2.2 Generation of double T-DNA insertional mutants

The double knockout (KO) mutants of hda6╳cmt3, hda6╳kyp6, and cmt3╳kyp6 were
generated by reciprocal crossing of the respective homozygous single KO mutants (hda6,
kyp6, and cmt3), and the F1 plants were allowed to self-pollinate. For the confirmation of
double KO mutants, leaves of F2 plants were used for RNA isolation, cDNA preparation
followed by genotyping PCR using respective gene-specific primers.
2.3 ROS staining

Arabidopsis seedlings were collected at different time points (3h after PS2, before
triggering, 3h after TS and 2 days after TS) in 2ml centrifuge tubes and immediately
processed for ROS staining using ROS specific dye. For detection of ROS (H2O2),
chloromethyl derivative of H2DCFDA (CM-H2DCFDA) was used for staining. Harvested
seedlings at required time points were incubated in 10µM H2DCFDA and vacuum
infiltrated for 5 minutes. The infiltrated seedlings were washed 2-3 times with double
distilled water and used for microscopic imaging using OLYMPUS 1X83 Epifluorescence
microscope.
2.4 Dark hypocotyl assay

Seeds (wild-type and hda6) were surface sterilized as described in the above section and
sown to position them vertically on 1⁄2 MS media plates (Murashige and Skoog, 1962) and
kept for stratification (2 days at 4℃ in dark condition). Plates were completely covered
with aluminium foil to induce dark conditions to the seedlings. After stratification, plates
were transferred to wooden racks to maintain the vertical position and grown under
optimal growth conditions (light: 16 h, 22℃; dark: 8 h, 22℃: relative humidity, 50-60 %
throughout the light-dark cycle). For checking acquired thermotolerance in the dark
condition, 4-day-old vertically grown seedlings, covered with foil were subjected to double
priming cycle, PS1 and PS2 (as mentioned in (Fig. 4A) followed by a recovery period of
2 and 3 days and TS of 45°C for 90 min (primed plants). Unprimed plants were directly
exposed to the TS at 45°C for 90 min. Control plants were grown in optimal growth
conditions without heat stimulus. After 3 days of TS, the seedlings were analysed for
hypocotyl length.
2.5 HDAC inhibition assay

Surface sterilized wild-type seeds were sown in 1⁄2 MS media plates followed by 2 days
of stratification and then transferred to the growth chamber (22°C; 16h/8h day/night
conditions). Four-day-old seedlings were used to determine the effect of HDAC activity
inhibition on the priming-induced thermomemory. Trichostatin-A (TSA), a general HDAC
16
inhibitor was used to inhibit HDAC activity with a concentration of 50 μg/μl, dissolved in
solvent Dimethyl sulfoxide (DMSO) by directly adding it to the MS media plates. Priming
and triggering stimuli were the same as described in (Fig. 6A) with 3 days of recovery at
optimal growth conditions. Plants were transferred to TSA containing MS media plates at
multiple stages viz. during Priming cycles (PS1 & PS2) alone (referred as priming TSA),
3-day recovery period alone (referred as Recovery TSA) and at all the stages such as
PS1, PS2, recovery and during TS (All TSA). DMSO, the solvent for TSA, was used as a
reference control for the respective treatments. The basal and acquired thermotolerance
of the plants was quantified in terms of loss of chlorophyll (chlorosis), death and shoot
regeneration.
2.6 HDAC activity assay

To determine the HDAC activity during priming-mediated acquired thermotolerance, 4-
day-old wild-type and hda6 seedlings were subjected to priming and triggering cycles as
mentioned in (Fig. 7A). The control, primed, primed control (seedlings exposed to only
priming stimulus, PS1 and PS2 and then kept under optimal control conditions), and
unprimed were harvested at several time points viz. after priming, during recovery and
after triggering as mentioned in (Fig. 7A). Seedlings treated with TSA were also harvested
to determine the HDAC activity at the same time points as mentioned above. All the
samples were stored in -80°C until further analysis. The samples were weighed and
processed for total nuclei isolation using the protocol described in Kaufmann et al. (2010).
The isolated nuclei pellets were stored at -80℃ for further processing. The nuclei pellets
were used for nuclear extract preparation using protocol from EpiQuik™ Nuclear
Extraction Kit I. The Epigenase™ HDAC Activity/Inhibition Direct Assay Kit with a
complete set of optimized buffers and reagents was used for measuring the activity or
inhibition of total HDAC enzymes in nuclear extracts from the samples harvested at
different time periods.
17
Chapter 3
Results and discussion
3.1 HDA6 is a negative regulator of acquired thermotolerance but may

not modulate the duration of stress memory
Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are involved in
transcriptional activation/repression through reversible acetylation of histone tails(Liu et
al., 2014). Among the stress-responsive class-I HDACs, our previous study identified
HDA6 to be involved in the regulation of thermopriming-mediated acquired
thermotolerance (Unpublished data). HDA6 has also been reported to be involved in
regulating ABA and salt stress responses (Sridha et al., 2006). Further, hda6 mutant
shows higher H3K9/K14Ac levels compared to wild-type (Chen et al., 2010). In the current
study, we aim to gain more insights into HDA6 regulation of acquired thermotolerance
and heat stress memory.
Firstly, we re-analysed the response of wild-type (Col-0, WT) and the hda6 KO mutant’s
basal, and acquired thermotolerance by exposing them to triggering heat stress with or
without thermopriming. Upon thermopriming, hda6 mutant displayed a slower rate of
chlorophyll loss and faster, and higher regeneration rates in response to heat stress
compared to WT. With a 3 days of recovery period between the priming and triggering
stress exposure, the hda6 mutant showed a faster and slightly higher regeneration rate
compared to wild-type seedlings (Fig. 3B). Further, to understand the role of HDA6 in
regulating the duration of thermomemory, the hda6 mutant and wild-type seedlings were
subjected to triggering heat stress with 3, 4, and 5 days of recovery after exposure to
priming stimulus. The seedlings were monitored regularly for chlorosis symptoms,
survival rate, and shoot regeneration. The primed seedlings tend to lose chlorophyll
content in the cotyledons, retaining hypocotyl alone (Fig. 3A), which aids in the shoot
regeneration in a few days after exposure to triggering stress. Seedlings of wild-type and
hda6 showed better regeneration with 3 days of recovery (Fig. 3B), whereas all the
seedlings were dead under 4 and 5 days of the recovery period (Fig. 3C-D). These
observations clearly indicate that the duration of thermopriming-induced stress memory
in Arabidopsis seedlings is a maximum of three days. Further, it suggests that HDA6 may
not be involved in modulating the duration of stress memory. However, optimization of
the priming duration/intensity vs the recovery time may result in longer stress memory,
which still needs to be explored. Nevertheless, at 6 days after treatment, wild-type
seedlings showed a faster rate of chlorosis (Fig. 3E), more dead and pale seedlings
compared to hda6 mutant. In contrast, hda6 plates had more greener seedlings than wild-
type (Fig. 3F). The number of seedlings regenerated was also slightly more in hda6
mutants than in WT (Fig. 3F, 3H). As the difference in the acquired thermotolerance of
hda6 and wild-type was visibly evident, fresh weight of both the sets of seedlings was
18
determined as an end-point observation for the shoot regeneration. Primed seedlings of
hda6 displayed more fresh weight than the primed seedlings of wild-type (Fig. 3I),
suggesting that hda6 mutants showed better growth than the WT upon exposure to heat
stress. hda6 seedlings showed faster regeneration than wild-type seedlings (Fig. 3J).
19
3E.
Control Primed
WT
hda6
3F.
Control Primed
WT
hda6
3G. 3H.
20
20
Average no. of
15
Average no. of
Dead
regenerated
15
seedlings
seedlings
10
Weak 10
5 5
Green
0 0
WT hda6
Dead Weak Green
hda6 WT
3I. 3J.
0.14
100 WT hda6
0.12 90 **
Average fresh weight of
**
80
% Regeneration
0.1 70
seedlings(g)
60
0.08
50
0.06 40
30
0.04 20
10
0.02 0
0 Days 1 2 3 4 5 6 7 8 9 10 11 12 13
WT hda6
20
Figure 3. Acquired thermotolerance of Arabidopsis wild-type and hda6 seedlings.
Long-term acquired thermotolerance phenotype of wild-type and hda6 seedlings with
3days (3A-3B), 4 d (3C) and 5 days (3D) of recovery period. Phenotype images of wild-
type and hda6 at 6 (3A,3E) 13 (3B) and 14 DAT (3F). Bar plots representing the
phenotypic classes - dead, weak, green of wild-type and hda6 seedlings (3G). Bar plots
representing the shoot regeneration capacity of wild-type and hda6 (3H). Fresh and dry
weights of wild-type, hda6 seedlings (3I) (n=48,16 seedlings/each). Line graph
representing the % regeneration of wild-type and hda6 seedlings(3J). DAT: Days after
Triggering, Wild-type (Col-0), hda6: Histone deacetylase 6 KO, PS: Priming stimulus, TS:
Triggering stimulus, R: Recovery.
3.2 HDA6 may not be involved in the regulation of cell elongation during
heat stress acclimation
Hypocotyl elongation assay is used as a tool to understand the role of heat acclimation in
lethal heat stress response (Kim et al., 2017). To delineate the role of HDA6 in regulating
the acquired thermotolerance, we analysed hypocotyl elongation of the hda6 mutant
along with the wild-type(Fig. 4A). For this, seedlings of both wild-type and hda6 mutant
were treated with a standard priming protocol (with 3 days of the recovery period) in dark
conditions. Under unprimed condition, wild-type and hda6 displayed inhibition of
hypocotyl elongation compared to their respective controls, which is expected due to the
effect of lethal heat stress on cell elongation. Although unprimed hda6 hypocotyl length
is slightly higher than wild-type unprimed, the difference is not statistically significant.
Thermopriming had a positive effect on hypocotyl elongation under lethal heat stress in
both wild-type and hda6, as the primed hypocotyls were longer compared to the
unprimed. Both wild-type and hda6 displayed no significant difference in hypocotyl length
in the primed condition compared to their respective controls. This suggests that the
thermopriming alleviated the effect of lethal heat stress on hypocotyl elongation, but may
not completely abolish the heat stress-induced effects. Further, under control or primed
condition, the difference between hda6 and wild-type was not significant. Interestingly,
the wild-type displayed significant difference in hypocotyl length in the primed compared
to unprimed, while such difference was not observed in the hda6 mutant. Notably, the
same conditions had a positive influence on plant survival and shoot regeneration in both
wild-type and hda6, when analysed at seedling stage, where hda6 had faster and higher
growth rates. Nevertheless, to delineate the effect of the recovery period and the
thermopriming intensity and duration on thermopriming-induced response to heat stress,
seedlings of both wild-type and hda6 were treated with standard priming protocol with 2
days of recovery period between priming and triggering stimulus, with a slight change in
priming and triggering temperatures (with a single priming cycle). Hypocotyl length of both
wild-type and hda6 mutant seedlings was significantly less in unprimed condition
compared to their respective controls, indicating their basal response level to heat stress
21
(Fig. 4A). Even at 2 days of recovery, the primed seedlings of both wild-type and hda6
mutant did not show a significant difference in their hypocotyl length compared to their
respective control (Fig. 4B). Taken together, these results suggest that HDA6 may not be
crucial for the hypocotyl elongation under heat stress or thermopriming-mediated
acclimation responses.
4A.
Control Primed Unprimed
3
∗∗∗ ∗∗∗
Average hypocotyl length(cm)

2.5 ∗∗∗ ∗∗∗
WT
1.5
1
hda6
0.5
0 hda6
WT
4B.
∗∗∗ ∗∗∗
2.5
∗∗∗ ∗∗∗
WT
2
Average hypocotyl
∗∗
length(cm)
1.5
1
hda6
0.5
0
WT hda6
Figure 4. Hypocotyl elongation assay to elucidate the role of heat acclimation in

heat stress response in wild-type and hda6 seedlings. Figure showing the phenotype
of control, primed and unprimed hypocotyls of both WT and hda6 seedlings with 3 (4A)
or 2 days of recovery (4B). Bar plot representing the average hypocotyl length (cm) of WT
and hda6 of control, primed, unprimed (n=15 seedlings/per condition) given 3 (4A) and 2
22
days of recovery (4B). The hypocotyl length was analysed using ImageJ software.
Student's t-test was used to analyse significance. *** indicate a p-value ≤ 0.0001.
3.3 Quantification of ROS in wild-type and hda6 seedlings

Heat stress is known to induce cellular reactive oxygen species (ROS) levels. ROS at low
concentration can act as a signaling molecule, but can be detrimental to the plant survival
at higher concentrations (Mittler et al., 2017). Notably, an interplay between ROS
production and epigenetic modifications has been reported (Huang et al., 2019). As
thermopriming induces heat stress tolerance, which was relatively efficient in the hda6
mutant compared to WT, we were interested in understanding if HDA6 is involved in the
regulation of ROS homeostasis. To this end, ROS levels were analysed in the hda6 and
WT seedlings during the basal and acquired thermotolerance conditions using a ROS
specific dye, H2DCFDA (CM-H2DCFDA). Untreated seedlings served as control. As the
visualization of the signal is more efficient in root than shoot tissue due to the chlorophyll
autofluorescence, all the results presented here are from root tissues. Both WT and hda6
seedlings displayed minimal ROS signal in control and 3 hours post exposure to mild
priming stimulus (Fig. 5A). However, slight increase in ROS signal was observed just
before triggering heat stress, which is during the 3 days of recovery period (Fig. 5B). In
response to lethal heat stress (3h post TS), ROS visualized as bright green signal was
observed more in the meristematic region of roots in both primed and unprimed conditions
of WT and hda6 compared to their respective controls showing significant difference
against the controls (Fig. 5C). Two days post triggering stress, both WT and hda6
seedlings displayed elevated ROS signal at all the conditions tested which are
significantly different when compared with the control wild-type, also the control hda6 and
primed hda6 showed significant difference in ROS levels (Fig. 5D). To quantify the ROS
production, fluorescence intensity was quantified using image j software.
Results suggest that ROS production is seen in the later stages of heat stress. As the
results were from one experiment, repetition of the experiment is needed to consolidate
our findings.
23
5A.
Control Primed
WT
3hr after PS
fluorescence unit
30
hda6
Relative
20
10
0
WT hda6
100µM
Control Primed
5B.
Control Primed
Before TS
WT
Relative fluorescence
60
50
40 *
unit
30
20
hda6
10
0 WT hda6
Control Primed
100µM
5C.
WT
hda6
100µM
24
3hr after TS
fluorescence unit
80 * ** *
*
Relative
60
40
20
0
WT hda6
5D.
WT
hda6
100µM
2 DAT
fluorescence unit
100
Relative
*
80 * *
*
60
40
20
0
WT hda6
Figure 5. Quantifications of ROS in wild-type and hda6 seedlings. Microscopic

images of root showing GFP fluorescence which represents the amount of ROS present
in the particular region (meristematic region) during 3 hrs. after PS, before TS, 3hrs after
TS, and 2 days after TS (Fig. 5A-5D). Bar plots representing the relative fluorescence of
ROS detected in the meristematic region of roots measured through Image J software.
Student's t-test was used to analyse significance, (*) indicate a p-value ≤ 0.05, ** ≤ 0.005.
3.4 Delineating the role of HDA6 in regulating the priming-mediated

acquired thermotolerance
As aforementioned, HDA6 has been identified as a negative regulator of thermomemory.
However, HDA6 transcript levels were only marginally altered in the primed compared to
25
unprimed seedlings at several time points after exposure to triggering heat stress
(Unpublished data). In order to delineate the time-window at which HDA6 might be
regulating the acquired thermotolerance, we employed (i) HDAC inhibitor assay along
with (ii) total HDAC activity assay during the course of priming/recovery/triggering.
Trichostatin-A, a general HDAC inhibitor, inhibits HDAC catalysis by chelating zinc ions
in the enzyme's active site pocket via hydroxamic acid, thereby inhibiting HDAC activity
(Furami et al., 2000). TSA has been shown to inhibit HDAC activity in roots, causing root
regeneration to be delayed and primary root growth to be inhibited (Ma et al., 2016). In
the current study, TSA was used as a potential inhibitor at different time points to
understand the time-window at which HDAC activity may be involved in regulating
acquired thermotolerance. The seedlings were treated with TSA or DMSO (solvent for
TSA; as an internal control) at multiple time points such as during exposure to PS,
recovery alone or during all stages i.e., PS, recovery and TS. Seedlings were given three
days of recovery period between the priming and TS as shown in (Fig. 6A). Post exposure
to TS, seedlings were returned to control conditions and were monitored for the
phenotypic characteristics such as chlorosis, seedling survival and regeneration. By 6
days after treatment, most of the seedlings turned pale yellow, having green hypocotyl
regions retained (Fig. 6B). Seedlings treated with TSA at all the stages tend to retain
chlorophyll content longer compared to DMSO treated control seedlings under heat
stress, further supporting our previous results and hypothesis. Total number of dead
seedlings were more in R (DMSO) and All (TSA) than the other conditions, whereas the
number of green seedlings were more in PS (TSA), R (TSA) and ALL (DMSO) (Fig. 6D).
Notably, all the seedlings started to regenerate after 8 days of triggering. TSA treated
seedlings during recovery displayed more regeneration (with enhanced growth)
compared to DMSO treated seedlings (Fig. 6C). Number of dead seedlings were more in
R (DMSO) and ALL (TSA), whereas the number of green seedlings were more in PS
(TSA), R (TSA) and they are significantly different compared to All (TSA) seedlings. The
PS (TSA) seedlings and R (TSA) seedlings showed better regeneration than its control,
while ALL DMSO seedlings displayed more regeneration than ALL TSA seedlings. R
(TSA) showed better regeneration which is significantly different form its control R
(DMSO) (Fig. 6E). Notably, HDAC inhibitor alone did not alter the basal thermotolerance
of the seedlings (data not shown here). Taken together, these results suggest that the
HDAC activity during recovery alone or during priming and recovery may play an
important role in modulating the thermopriming-induced thermotolerance.
26
27
6C.
PS(TSA)
PS(DMSO)
Control
R(TSA)
Control(DMSO)
R(DMSO)
PS(DMSO)
PS,R&T(DMSO)
ALL(DMSO)
ALL(TSA)
6D. 6E.
18
Average no. of seedlings
* 16
16 *
**
* 14
14 *
**
12 12
10 10
* Dead
8 8
** 6
6 Weak
4 4
2 2
Green
0 0
Dead Weak Green
SO
A
SO
A
SO
A
TS
TS
TS
PS-TSA PS-DMSO R-TSA

M
DM
DM
L-
R-
-
D
PS
AL
L-
R-
-
PS
AL
R-DMSO ALL-TSA ALL-DMSO
Figure 6. Plant response to heat stress with or without inhibition of HDAC activity.
The experimental design for the HDAC inhibition assay (6A). Phenotype of seedlings after
6 days of TS in different experimental conditions (6B). Regeneration phenotype of
seedlings in different conditions after 15 days of triggering (6C). Bar plots representing
the phenotypic classes - dead, weak, green under different conditions (6D). Bar plots
showing total number of regenerated seedlings in different experimental conditions (6E).
Student's t test is done to analyse the significant difference between the conditions. * If p
≤ 0.05, *** if p ≤ 0.001.
28
(ii) To determine the HDAC activity during the course of priming-mediated acquired
thermotolerance, wild-type seedlings were harvested at several time points as shown in
(Fig. 7A). Further, to understand if hda6 mutant still displays any basal HDAC activity
during acquired thermotolerance, we harvested the hda6 mutant at similar time points as
wild-type (Fig. 7A). The TSA treated seedlings were also harvested at the same time
points to evaluate the HDAC activity. 4day-old seedlings of both wild-type and hda6 were
categorized into different sets viz. control, primed-control, primed, unprimed and
harvested at several time points after priming/triggering (Fig. 7A). Wild-type samples at
different timepoints were collected, stored in -80°C and processed for total nuclei
isolation. Isolated nuclei pellets were used for nuclei extract preparation. By using the
Epigenase HDAC Activity/Inhibition kit the total HDAC activity of the samples were
measured. Results suggest that the amount of deacetylated products/HDAC activity is
more in control than primed-control samples immediately after priming and 3 hours after
priming (Fig. 7B-7C), suggesting activation of gene expression upon priming stimulus. In
contrast, during the recovery phase the HDAC activity is more in primed-control than the
control samples at both 1 and 2 days. This suggests that during recovery after heat stress,
there could be active resetting of the transcriptional programs that is reflected by the
induced HDAC activity (Fig. 7D-7E). Such induced HDAC activity continued for 2 days
that correspond to after triggering time points (Fig. 7F-7G), which reduced at 2 days after
triggering time point (Fig. 7H). However, upon triggering heat stress exposure, primed
seedlings displayed lower HDAC activity at 0min and 2 days after TS, while the activity
was slightly high 1 day after TS, compared to control and unprimed (Fig. 7F-H). Notably,
the unprimed seedlings displayed induced HDAC activity at the initial time point (0 min
TS, Fig. 7F), while it remained similar to control at 1 day after TS (Fig. 7G). However
unprimed displayed lower HDAC activity only at 2 days after TS compared to control (Fig.
7H), suggesting a delayed activation of transcriptional programs. The above observations
suggest that HDAC activity suggests differential modulation of transcriptional programs
after exposure to a mild stress (high immediately after PS), during recovery period (reset
during the course of recovery, as observed in primed-control samples) and induced upon
exposure to TS after PS (as observed in primed samples). However, the time-window for
HDAC activity still needs to be re-confirmed along with hda6 mutant samples to nullify the
possibility of background HDAC activity. Further, the control seedlings display differential
HDAC activity, potentially could be due to the heterogeneity in the seedlings or the time
of the day during harvest, which needs to be carefully evaluated in the future experiments.
29
7A. 7B.
0 min after PS
3.5
HDAC activity(ng/min/mg)
3
2.5
1.5
0.5
0
Control Primed-control
7C. 7D. 1.2

1d after PS
3 hrs after PS
3 1
2.5 0.8
2
0.6
1.5
0.4
1
0.2
0.5
0
0 Control Primed-control
Control Primed-control
7E.
3
2d after PS 0 min after TS
0.45 2.5
0.4
0.35 2
0.3
0.25 1.5
0.2
1
0.15
0.1 0.5
0.05
0 0
Control Primed-control Control Primed-control Primed Unprimed
7G. 7H.
1d after TS 1.6 2d after TS

1.4
3.5
1.2
3
1
2.5
0.8
2
1.5 0.6
1 0.4
0.5 0.2
0 0
Control Primed-control Primed Unprimed Control Primed- Primed Unprimed
control
30
Figure 7. Figure showing the experimental plan and HDAC activity at different time
points during the course of priming-mediated acquired thermotolerance. Schematic
representation for work plan for harvesting samples for HDAC activity (7A). Bar plots
represent the activity of HDAC during the respective time points and plant growth
conditions (7B-7H). PS: Priming stimulus, TS: Triggering stimulus, R: Recovery.
3.5 KYP6 is involved in the establishment of thermomemory

KYP/SUVH4 is an SU(VAR)3-9 homolog that encodes a H3K9 specific methyltransferase
that aids in maintenance of DNA methylation. Luna et al., 2014 demonstrated that KYP
and NPR1 play a role in the maintenance of β-aminobutyric acid-induced long-term
resistance, with KYP suppressing gene transcription via methylation of H3K9 and DNA
methylation at CpHpG residues, implying that KYP regulates expression of long-term
defence gene during priming by silencing suppressor genes for SA/NPR1-dependent
genes. We aim to study the involvement of KYP in regulating thermomemory during
priming-mediated acquired thermotolerance.
Towards that, we reanalysed the response of wild-type and kyp6 KO mutant for their basal
and acquired thermotolerance (previously the phenotype was identified in our group).
Upon thermopriming, kyp6 mutant seedlings did not show significant symptoms of
chlorosis compared to wild-type. Both kyp6 and wild-type did not differ much in response
to basal heat stress as all the seedlings failed to survive by 7 days of TS (Fig. 8A). With
3 days of recovery period before TS, seedlings of kyp6 showed higher regeneration than
wild-type seedlings (Fig. 8B), suggesting that kyp6 plays a crucial role in maintaining
thermopriming-mediated acquired thermotolerance. The same is re-confirmed by the
number of dead seedlings that were more in the wild-type than the kyp6, whereas the
number of green seedlings were more in mutant than the wild-type (Fig. 8C). kyp6
seedlings showed better regeneration than the wild-type seedlings (Fig. 8D). kyp6
showed faster regeneration than wild-type seedlings (Fig. 8E). To understand the role of
KYP in regulating the duration of thermomemory, kyp6 and wild-type seedlings were
subjected to PS and TS with a recovery period of 4 days. As previously seen, the kyp6
mutant and wild-type did not differ in basal thermotolerance (Fig. 8F) and primed
seedlings showed maximum dead seedlings (Fig. 8G-8H). It suggests that kyp6 is
involved in maintaining thermomemory without modulating the duration of stress memory.
31
8A.
WT
kyp6
8B. 8C.
Control Primed
20
15
WT Dead
10
Weak
5
0 Green
Dead Weak Green 15 days
kyp6 WT kyp6
8D. 8E.
14 * 80 * WT
12 WT kyp6 70 kyp6
Average no. of
60
% Regeneration
10
seedlings
50
8
40
6
30
4
20
2 10
0 0
Days 7 9 10
Regeneration
15 days
32
8F.
WT
kyp6
8G.
Control Primed
WT
kyp6
8H.
20 WT kyp6
15
Average no. of
Dead
seedlings
10
Weak
5
0 Green
Dead Weak Green
33
Figure 8. Basal and Acquired thermotolerance of wild-type and kyp6 mutant
seedlings. Long-term acquired thermotolerance phenotype of wild-type and kyp6
seedlings with 3 days of recovery (8A-8B). Bar plots representing the phenotypic classes
- dead, weak, green of WT and kyp6 seedlings after 15 DAT(8C). Bar plots representing
the shoot regeneration capacity of WT and kyp6 seedlings given 3 days of recovery after
15 DAT (8D). Line graph showing the % regeneration of wild-type and kyp6
seedlings(8E). Phenotype images of WT and kyp6 at 7 (8F) and 13 DAT(8G). Bar plots
representing the phenotypic classes - dead, weak, green of WT and kyp6 seedlings (8H).
DAT: Days after Triggering, WT: wild-type, kyp6: KRYPTONITE/SU (VAR) HOMOLOG 4.
Student's t test was used for checking the significance (*) if p ≤ 0.05.
3.6 CMT3 is involved in the regulation of thermomemory

DNA methylation plays an important role in transposon silencing and maintenance of the
integrity of the genome. CMT3 which is CHROMO METHYLTRANSFERASE 3 is a plant
specific DNA methyltransferase that helps in maintaining DNA methylation.
The wild-type and cmt3 KO mutants were re-analysed (Phenotype was previously
identified in our group) for their acquired thermotolerance by exposing them to triggering
stimulus. Upon thermopriming, cmt3 seedlings displayed a similar rate of chlorosis
compared to wild-type (Fig. 9A). With 3 days of recovery period between priming and
triggering stimulus cmt3 seedlings showed better regeneration than wild-type seedlings
with 3 days of recovery (Fig. 9B), The cmt3 seedlings showed more green seedlings and
less dead seedlings, whereas wild-type seedlings showed opposite trend at 3 days of
recovery(Fig. 9C). On the other hand, cmt3 showed more shoot regeneration than wild-
type seedlings with no significant difference(Fig. 9D). As the difference in number of
seedlings regenerated was more in cmt3 than the wild-type which was visibly evident,
fresh weight of both cmt3 and wild-type was determined as an end-point observation for
the shoot regeneration. Result has shown that the cmt3 seedlings displayed more fresh
weight than primed seedlings of wild-type (Fig. 9E). cmt3 showed faster regeneration
than wild-type seedlings (Fig.9F). To check whether cmt3 modulate the duration of stress
memory seedlings were treated with priming and triggering stimulus with 4 days of
recovery (Fig. 9G) . cmt3 and wild-type seedlings with 4 days of recovery showed more
dead phenotype and less green seedlings (Fig. 9H). The wild-type seedlings showed
similar regeneration as cmt3 seedlings (Fig. 9I). Providing evidence that cmt3 is involved
in maintaining thermomemory without modulating the duration of stress memory.
34
9A.
Control Primed
WT
cmt3
9B.
Control Primed
WT
cmt3
9C. 9E. 0.1 WT cmt3

Average fresh weight
15
of seedlings(g)
* 0.095
Average no. of
Dead
seedlings
10
0.09
Weak
5
0.085
0 Green
Dead Weak Green
0.08
WT cmt3
9D. 9F.
100
WT cmt3
% Regeneration
16.5 80
16
60
15.5
15 40
14.5
20
14
13.5 0
WT cmt3 Days 6 8 10 13
Regeneration
35
9G.
Control Control Primed

Primed
WT
cmt3
9H. 9I.
20 WT cmt3 4 WT cmt3
Average no. of
Average no. of
15 Dead 3
seedlings
10 seedlings 2
Weak
5 1
Green
0 0
Dead Weak Green
Regeneration
Figure 9. Acquired thermotolerance of cmt3 and wild-type. Figure showing the

phenotype of wild-type and cmt3 after 7, 15, and 16 days after triggering stress (9A-9B).
Bar plots representing the phenotypic classes of WT and cmt3 seedlings of 3-day
recovery (n=16 seedlings/plate) (9C). Bar plots representing the shoot apical meristem
regeneration of WT, cmt3 seedlings (n=16 seedlings/plate) (9D). Bar plots representing
the fresh weights of the WT and cmt3 seedlings (n=48 seedlings,16 seedlings/plate) (9E).
Line graph representing the % regeneration graph of cmt3 and wild-type seedlings(9F).
Figure showing the phenotype of wild-type and cmt3 after 7, 15, and 16 days after
triggering stress(9G). Bar plots representing the phenotypic classes of WT, cmt3
seedlings of 4d recovery and SAM regeneration capacity (9H) of WT, cmt3 seedlings
(n=16 seedlings/plate) (9I). DAT: Days after Triggering, WT: Wild-type, cmt3: Chromo
methyltransferase3.
3.7 Alteration in priming and triggering stimulus by decrease in 1°C ,

increases the amplitude of thermotolerance
Previous experiments on wild-type, hda6, and kyp6 mutants with a recovery period of 4
days resulted in the death of maximum seedlings of both wild-type and mutants with less
36
regeneration ability. To check the effect of 1°C of temperature decrease in PS2 and TS
treatments, 5-day old seedlings of wild-type, hda6 and kyp6 were exposed to PS1- 37°C,
PS2 & TS-44°C. When subjected to 44°C TS for 90 min wild-type, kyp6 and hda6
seedlings showed no significant difference in basal thermotolerance (Fig. 10A, bottom
panel). Whereas, when primed seedlings were exposed to PS1- 37°C, PS2 & TS - 44°C
with 4 days of recovery period kyp6 mutant showed a slow rate of chlorosis than wild-
type and hda6, hda6 mutants showed chlorosis similar to wild-type (Fig. 10A-right panel).
After 15 DAT kyp6 and hda6 seedlings showed better regeneration than wild-type
seedlings (Fig. 10B). The number of green seedlings were more in kyp6 than in hda6 and
wild-type and also hda6 showed more green seedlings than wild-type (Fig. 10C), whereas
wild-type showed more dead seedlings than hda6 and kyp6 (Fig. 10D-C). kyp6 seedlings
showed better regeneration than hda6 and wild-type seedlings (Fig. 10D). kyp6 showed
faster regeneration than wild-type seedlings (Fig. 10E). These results suggest that with a
given 4-day recovery period and with a 1°C change in TS temperature of 45°C
temperature, mutant seedlings of both hda6 and kyp6 were able to retain memory and
hence showed better regeneration than wild-type. However, kyp6 displayed significant
difference in maintaining the memory of stress than both hda6 and wild-type. This result
also gives us the idea that as the seedlings were already primed for 44°C they were able
to retain the memory of the exposed temperature and were able to survive after they were
exposed to triggering stimuli of 44°C and survived better than wild-type.
37
10A. 10B.
Control Control
WT hda6 kyp6 WT hda6 kyp6
Primed Primed
WT hda6 kyp6 WT hda6 kyp6
Unprimed
10C.
20
WT hda6 kyp6
Average no. of
*
15 Dead
seedlings
10
Weak
5 *
Green
0
Dead Weak Green
WT hda6 kyp6
38
10D. 10E.
20 100 WT hda6 kyp6 *
% Regeneration
*
WT hda6 kyp6 * 80
Average no. of
15
seedlings
60
10 40
20
5
0
0 Days 7 9 11 13 15
Regeneration
Figure 10. Acquired thermotolerance of wild-type, hda6, kyp6 with 1°C change in
temperature. LAT phenotype of wild type, kyp6, hda6 after 7 days of triggering (10A).
Phenotype images wild-type, kyp6, hda6 at 15 DAT (10B). Bar plots representing the
phenotypic classes- dead, weak, green of WT, kyp6, hda6 (10C). Bar plots represent the
shoot regeneration capacity of WT, kyp6, hda6. Line graph representing the %
regeneration graph of kyp6, hda6, wild-type seedlings(10E). LAT: Long-term acquired
thermotolerance, DAT: Days after triggering, WT: Wild-type, PS: Priming stimulus, R:
Recovery, TS: Triggering stimulus. The student’s t-test was used to check for significance
against control. (*) if p value is ≤ 0.05
3.8 Does the HDA6-CMT3-KYP regulate priming-mediated acquired

thermotolerance?
Johnson et al., 2002 and Lindroth et al., 2004 showed that methylation maintained by
CMT3 depends on H3K9Me2 and HDA6 indirectly maintains CHG, CHH methylation by
regulating TEs of H3K9Me2. A previous study in the group has identified genes
associated with the RdDM pathway such as HDA6, KYP/SUVH4 and CMT3 to be involved
in regulating priming-induced thermomemory/acquired thermotolerance. To understand if
the HDA6, CMT3, and KYP axis co-operatively regulate the thermomemory, it is important
to study the acquired thermotolerance of double/triple mutants of these genes. For this,
the reciprocal crossing has been performed for the three mutants (Table 1). Flowers from
the maternal mutants were emasculated and allowed to be pollinated by the pollens from
paternal mutant plants. Siliques developed after pollination were carefully bagged and
seeds were collected.
F1 seeds of kyp6(♀)×hda6(♂) & cmt3(♀) ×kyp6(♂) double mutants were sown and leaves
were harvested for checking the expression of the genes of the double mutants (Fig. 11A,
11B, 11C). To get homozygous double KO lines, F1 plants were allowed to self-pollinate
and F2 seeds were collected, seeds were sown and leaves will be harvested and they
will be analyzed for their homozygosity for both the genes. Once confirmed, the
39
homozygous double mutants will be analyzed for their response to thermopriming-
mediated heat stress responses.
Table 1. Table showing the combination of parents involved in the reciprocal crossing for
the generation of double mutants. Yes/No indicates the status of the formation of siliques
in the double mutant plants. ♀: female parent, ♂: male parent.
Double mutants Formation of siliques
hda6♀ ×kyp6♂ No
cmt3♀ × kyp6♂ Yes
kyp6♀ × hda6♂ Yes
cmt3♀ × hda6♂ No
kyp6♀ × cmt3♂ No
hda6♀ × cmt3♂ Yes
40
Figure 11. Genotyping PCR of double mutant kyp6(♀) ×hda6(♂) & cmt3(♀) ×kyp6(♂)
Gene specific primers of HDA6 were used to check the presence of its transcript, and
bands demonstrate the presence of HDA6 transcript in WT and double mutants (11A).
Gene specific primers of KYP6 were used to check the presence of its transcripts; bands
in gel demonstrate the presence of KYP6 transcript in both wild type and double mutants
(11B,11C). Actin is used as a reference gene. WT: Wild-type (Col-0) is used as positive
control.
41
Conclusion and future perspectives
The current global environmental changes are posing a great threat to crop productivity
and demands for a crop developmental approach in a sustainable manner.
The gradual increase in global temperature as a consequence of global warming is a
devastating abiotic factor that minimizes plant growth and development to a greater
extent. Our major interest is to understand the mechanism of heat stress responses of
plants at multiple levels (physiological, biochemical, and molecular) and identify
regulators of these responses at the molecular level. Some of the epigenetic regulators
such as HDACs, methylases (DNA and histone), and demethylases are reported to be
involved in the regulation of abiotic stress responses and can be used as key targets for
the improvement of plant stress tolerance.
Recent studies have identified the inherent ability of the plants to use the memory from a
previous stress exposure (referred to as priming) to mount a robust response aiding in
enhancing tolerance to subsequent stresses (referred to as acquired tolerance).
Our major interest is in identifying the potential regulators of thermomemory and
unravelling the mechanisms involved in long-term acquired thermotolerance. Previous
studies in our group have identified some potential epigenetic regulators involved in the
establishment and maintenance of thermomemory during acquired-thermotolerance. Two
of the RdDM pathway regulators genes and a HDAC were analyzed in the current study
to understand their role in regulating thermomemory and acquired thermotolerance.
HDA6, one of the class I HDACs was identified to act as a potential negative regulator of
thermomemory during priming-induced acquired thermotolerance. Epigenetic regulators
involved in RdDM pathways CMT3 and KYP were also identified to mediate the
establishment of thermomemory. However, the observations suggested that these
regulators do not modulate the duration of thermomemory. However, modulation of
priming intensity suggested their (HDA6 and KYP) involvement in the regulation of the
duration of the stress memory, which needs to be further analyzed.
The observations from the growth parameters and phenotypic data revealed that HDA6,
CMT3, and KYP potentially regulate the heat stress responses of plants upon thermo
priming as the mutants (hda6, cmt3, and kyp6) showed better survival and growth than
the wild-type plants during acquired thermotolerance, while there is not much difference
in basal thermotolerance of mutants as well as the wild-type plants.
Eventhough, HDA6 is involved in the establishment of thermomemory upon
thermopriming, the phenotypic responses of plants suggest that it may not involve cell
elongation as suggested by hypocotyl experiment. ROS production and homeostasis
plays a significant role in initiating and determining the plant stress responses. Our
observations of ROS production upon heat stresses (primed and unprimed plants)
suggested that HDA6 may play a role in maintaining the ROS homeostasis as the mutant
42
plants displayed lesser ROS production than the wild-type plants. Nevertheless, these
observations need to be re-confirmed.
Experiments for understanding the time-window for HDAC activity (HDAC inhibition assay
and total HDAC deacetylation/inhibition assay at different time points) indicated the
recovery phase as the probable time-window for HDAC activity in re-setting thermo
memory upon priming-induced acquired thermotolerance.
We also aim to understand the mechanism of action and co-relation between these
epigenetic regulators. HDA6 was reported to act upstream to pol IV (for its recruitment)
and also interact with MET1 to establish epigenetic memory through RdDM pathway
(Blevins et al., 2014). HDA6 and KYP act co-operatively during the regulation of various
plant developmental processes and stress responses in Arabidopsis (Gu et al., 2019). It
is also reported that HDA6 mediated deacetylation acts as a pre-requisite for the
recruitment of DNA methylases like MET1 and CMT3 to enhance CG and CNG
methylation (Aufsatz et al., 2002). These reports suggest the involvement of the HDA6-
KYP-CMT3 axis in regulating the thermomemory establishment and maintenance. To test
this, we have generated double mutants of these genes by reciprocal crossing method,
As F1 generation of two of the double mutants (kyp6(♀) ×hda6(♂) & cmt3(♀) ×kyp6(♂)
showed heterozygosity, F2 generation seeds were collected and need to be screened for
the homozygosity of both genes. Once confirmed the double mutants can be analyzed
for their response to thermopriming-mediated heat stress response. Further studies
include global histone acetylation assays, based on outcomes of the acetylation assays,
western blot analysis for selective histone acetylation marks can be analyzed.
Collectively, these observations suggests that the epigenetic regulators play a significant
role in priming-mediated stress tolerance mechanisms in plants, but their detailed mode
of action and targets are still undetermined. We aim to gain deeper understanding of the
mechanism of action of these epigenetic regulators, the time-window of their activity, and
to get the potential target genes for crop improvement programs mediated by thermo
priming.
43
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Veesam Prathyusha

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Elucidating the molecular mechanisms

underlying thermopriming mediated

Indian Institute of Science Education and Research Tirupati

in partial fulfilment of the requirements for the

BS-MS Dual Degree Programme

Supervisor: Dr. Annapurna Devi Allu

Name of student: Prathyusha Veesam

Roll Number: 201701094

Name of Supervisor: Dr. Annapurna Devi Allu Name of Co-Supervisor:

Signature of Supervisor: Signature of Co-Supervisor:

Name of student: Prathyusha Veesam

Roll Number: 201701094

Name of Supervisor: Dr. Annapurna Devi Allu Name of Co-Supervisor:

Signature of Supervisor: Signature of Co-Supervisor:

Chapter 1 Introduction…..........................…......... 8-14

Chapter 2 Materials and methods…........…......... 15-17

Chapter 3 Results and discussion……..…........... 18-41

Conclusion and Future perspectives………......... 42-43

1.1 Priming and stress memory

1.2 Basal and acquired thermotolerance

Heat stress Response strength

Unprimed plant Basal thermotolerance

1.3 Molecular mechanisms underlying plant stress memory

Accumulation of proteins Epigenetic change

Stress exposure Enhanced physiological

Figure 2. Figure showing the mechanism of priming-induced stress tolerance and

1.4 HDACs and their role in plants

1.5 RdDM pathway involved in the regulation of heat stress responses

1.6 Hypocotyl elongation dynamics

1.7 The interplay between ROS generation and epigenetic regulation

2.1.2 Plant growth parameters

2.2 Generation of double T-DNA insertional mutants

2.3 ROS staining

2.4 Dark hypocotyl assay

2.5 HDAC inhibition assay

2.6 HDAC activity assay

3.1 HDA6 is a negative regulator of acquired thermotolerance but may

Average hypocotyl length(cm)

Control Primed Unprimed

Figure 4. Hypocotyl elongation assay to elucidate the role of heat acclimation in

3.3 Quantification of ROS in wild-type and hda6 seedlings

Control Primed Unprimed

Figure 5. Quantifications of ROS in wild-type and hda6 seedlings. Microscopic

3.4 Delineating the role of HDA6 in regulating the priming-mediated

PS-TSA PS-DMSO R-TSA

R-DMSO ALL-TSA ALL-DMSO

7C. 7D. 1.2

1d after TS 1.6 2d after TS

3.5 KYP6 is involved in the establishment of thermomemory

3.6 CMT3 is involved in the regulation of thermomemory

9C. 9E. 0.1 WT cmt3

Control Control Primed

Figure 9. Acquired thermotolerance of cmt3 and wild-type. Figure showing the

3.7 Alteration in priming and triggering stimulus by decrease in 1°C ,

WT hda6 kyp6 WT hda6 kyp6

WT hda6 kyp6 WT hda6 kyp6

3.8 Does the HDA6-CMT3-KYP regulate priming-mediated acquired

Double mutants Formation of siliques

cmt3♀ × kyp6♂ Yes

kyp6♀ × hda6♂ Yes

hda6♀ × cmt3♂ Yes

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