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Journal of Experimental Botany
https://doi.org/10.1093/jxb/erab337 Advance Access Publication 24 July, 2021
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REVIEW PAPER
Plant epigenomics for extenuation of abiotic stresses:
challenges and future perspectives
DharmendraSingh1,, PriyaChaudhary1, JyotiTaunk2, ChandanKumarSingh1, ShristiSharma1,
VikramJeetSingh1, DeeptiSingh3, ViswanathanChinnusamy2, RajbirYadav1 and MadanPal2,*
1 Division of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi-110012, India
2 Division of Plant Physiology, ICAR-Indian Agricultural Research Institute, New Delhi- 110012, India
3 Department of Botany, Meerut College, Meerut 250002, India
* Correspondence: madanpal@yahoo.com
Received 24 February 2021; Editorial decision 14 July 2021; Accepted 23 July 2021
Editor: Ramanjulu Sunkar, Oklahoma State University,USA
Abstract
Climate change has escalated abiotic stresses, leading to adverse effects on plant growth and development, even-
tually having deleterious consequences on crop productivity. Environmental stresses induce epigenetic changes,
namely cytosine DNA methylation and histone post-translational modifications, thus altering chromatin structure
and gene expression. Stable epigenetic changes are inheritable across generations and this enables plants to adapt
to environmental changes (epipriming). Hence, epigenomes serve as a good source of additional tier of variability for
development of climate-smart crops. Epigenetic resources such as epialleles, epigenetic recombinant inbred lines
(epiRILs), epigenetic quantitative trait loci (epiQTLs), and epigenetic hybrids (epihybrids) can be utilized in epibreeding
for improving stress tolerance of crops. Epigenome engineering is also gaining momentum for developing sustain-
able epimarks associated with important agronomic traits. Different epigenome editing tools are available for cre-
ating, erasing, and reading such epigenetic codes in plant genomes. However, epigenome editing is still understudied
in plants due to its complex nature. Epigenetic interventions such as epi-fingerprinting can be exploited in the near
future for health and quality assessment of crops under stress conditions. Keeping in view the challenges and oppor-
tunities associated with this important technology, the present review intends to enhance understanding of stress-
induced epigenetic changes in plants and its prospects for development of climate-ready crops.
Keywords: Abiotic stress, DNA methylation, epibreeding, epi-fingerprinting, genome editing, histone modification.
Introduction
The rapidly changing climate poses many challenges ahead,
from loss of diversity to reduced crop production. There
is dwindling agricultural land, and the food grains pro-
duced are too few to keep pace with the upsurge in the
world population. To sustainably reduce food grain demand
over supply, there is a dire need to produce climate-smart
crops. However, conventional breeding approaches have
limited applicability in production of such crops. Therefore,
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Page 2 of 20 | Singh etal.
molecular breeding and reverse genetics approaches are now
being exploited to improve crop productivity. However,
limited genetic resources and narrow applicability of these
approaches limit their application. Exploiting epigenomes
as an additional source of variability for crop trait improve-
ment is seen as the next-generation tool for food produc-
tion, which in the recent past was mostly limited to animal
systems (Gallusci etal., 2017).
The term epigenetics was coined by Waddington in 1942
to describe mitotically or meiotically heritable yet reversible
changes in gene expression (Waddington, 2012). These epigen-
etic changes can be inherited either by mitosis, for example
in case of the vernalization process, or by imprinting which
involves transmission of epigenetic variations to the progeny
directly by meiosis (Gallusci etal., 2017). Epigenetic variations
control a wide range of plant developmental processes, such
as adaptation to abiotic stresses, and thus help in shaping plant
phenotypic plasticity (Gallusci etal., 2017; Lee etal., 2019).
In higher plants, genomic DNA is methylated at cytosine in
symmetrical (CG or CHG) and/or non-symmetrical (CHH)
contexts, where H represents any nucleotide, except guano-
sine (Jin et al., 2011). Apart from DNA methylation, histone
modications at terminal amino acids, RNAi by small RNAs
(sRNAs), etc., also result in epigenetic variations (Fragou
etal., 2011). In plants, DNA methylation is catalyzed by DNA
methyltransferases, namely DNA methyltransferase 1 (MET1),
which is responsible for methylation at CG; chromomethylase 2
(CMT2), chromomethylase 3 (CMT3), and domains rearranged
methyltransferase 2 (DRM2), which cause CHG and CHH
methylations, respectively (Bartels etal., 2018). Mechanisms and
enzymes responsible for epigenetic modications as well as for
maintenance have been extensively reviewed in the literature
(Law and Jacobsen, 2010; Bartels etal., 2018). Segregation of gen-
omes with dierent methylation patterns results in formation
of stably inherited epigenetic variants of genes called epialleles,
which can regulate gene expression (Gahlaut et al., 2020).
Dierences in methylation patterns have contributed many im-
portant agronomic traits (Wang et al., 2015). Epialleles are also
formed as a result of altered environmental conditions, as exposed
plants can develop epigenetic memory (epimarks occurred due
to exposure to stress) (Bartels etal., 2018). These epialleles can be
utilized to develop climate-smart crops via epibreeding.
Advancement of resources for epibreeding employs epialleles
for developing epigenetic recombinant inbred lines (epiRILs)
and epigenetic hybrids (epihybrids). Chemical modiers and
epigenetic quantitative trait loci (epiQTLs) can also be util-
ized in epibreeding. Epigenetic modications and epigenome
editing can be used to engineer plants for climate resilience.
These epigenome modications are not just limited to creating
epialleles, but they can also be utilized in epi-interventions.
Epi-interventions, such as epipriming for stress memory reten-
tion (Ueda and Seki, 2020), epiQTL mapping for stress-related
complex agronomic traits such as yield, seed development,
and plant growth, and epi-ngerprinting for product quality
assessment (Rodríguez López and Wilkinson, 2015), can help in
production of future-ready crops. Epigenome editing employs
advanced tools such as clustered regularly interspaced short
palindromic repeats (CRISPR)/CRISPR-associated protein 9
(Cas9) which is involved in targeted editing of the epigenome
(Miglani et al., 2019). Most of such studies were only tar-
geted in the model plant Arabidopsis, which has been edited
to produce the developmental phenotype and late owering
phenotype, and for improved drought stress tolerance, etc.
(Gallego-Bartolomé etal., 2018; Lee etal., 2019; Papikian etal.,
2019; Roca Paixão etal., 2019). However, epigenome editing
for abiotic stress mitigation is still understudied in cropplants.
Lack of understanding of epimarkers has resulted in limited
use of this potent tool. Development of C-methyl markers is
compelling for epipriming as well as epi-ngerprinting pur-
poses. Overcoming the limitations associated with epigenome
engineering will denitely help in producing climate-smart
crops. Keeping in view the above facts, the present review
highlights epigenetic mechanisms of plants in response to
abiotic stresses and emphasizes the employment of next-
generation tools for precise epibreeding. Also, epigenome
editing of targeted regions can be employed to ameliorate the
climate resilience response in crops. Since the understanding
of epi-intervention is unclear in plants, this review will help
in fostering the idea of employing these epi-interventions in
agriculture for generating prediction models and to develop
climate-smart crops.
Sources of epigenetic variation inplants
The three main sources of epigenetic diversity are (i) environ-
mentally induced variations resulting from interaction between
the environment and epigenetic processes; (ii) genetically induced
variations resulting from interaction between genotype and epi-
genetic processes; and (iii) chemical mutation-induced variations
(Angers etal., 2020). In broad terms, epigenetic variations in plants
can arise from genetic as well as non-genetic sources (Springer
and Schmitz, 2017). Non-genetic sources which include devel-
opmental and environmental factors can interfere with mainten-
ance of methylation states, stress-triggered chromatin changes, or
o-target eects of sRNAs. Genetic sources of epialleles consti-
tute chromatin changes in methylation patterns and/or produc-
tion of siRNAs which triggers RNA-directed DNA methylation
(RdDM) (Springer and Schmitz, 2017). Natural epialleles are
sustainable sources of epigenetic variations in plants, and only a
few have been described in the literature (Quadrana etal., 2014;
Xianwei etal., 2015; Song etal., 2017). Areservoir of these vari-
ations associated with specic agronomic traits together with abi-
otic stress-induced variations provides an interesting source for
climate-resilient epibreeding. However, the stability and mainten-
ance of abiotic stress-induced variations requires much attention.
Several strategies can be used to induce epigenetic variations
since naturally occurring epigenetic variations are limited.
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Epigenomics to improve abiotic stress tolerance in plants | Page 3 of 20
Targeted epigenetic variations require a prior knowledge of
target sequences and epigenome editing tools which are not
very developed in plant systems. Moreover, it also needs trans-
formation protocols which are limited by lack of inheritance
of induced modications (Kungulovski et al., 2015). Non-
targeted epigenetic diversity can be achieved by generating
epiRILs. Two independent Arabidopsis epiRILs were created
using mutants with variable epigenomes which laid the foun-
dation of non-targeted epigenetic diversity (Schmitz and Ecker,
2012). However, in the case of crop plants where no such mu-
tants exist, chemically induced epigenetic variations (e.g. DNA
methylase inhibitors such as azacytidine or zebularine) can be
produced in the parental epigenome (Osorio-Montalvo etal.,
2018).
Abiotic stress-induced epigenetic changes
in cropplants
Change brought about in the epigenome of plants exposed to a
multitude of stresses is a very well known phenomenon (Bressan
et al., 2014; Springer and Schmitz, 2017). Such epigenetic
variations provide a theoretical basis for stress memory, which
helps plants to respond eciently towards recurring stresses.
Analyzing stress-associated changes in the epigenome and
related epigenetic responses can help in utilizing epigenetic
changes as a tool for retaining stress memory in crop plants.
Abiotic stress signals induce changes in the expression and/or
activity of epigenetic regulators, namely sRNAs, RdDM com-
ponents, histone variants, histone modication enzymes, and
chromatin-remodeling factors (Chinnusamy and Zhu, 2009) .
These epigenetic regulators modify histones and cause DNA
methylation. Some of these are heritable epigenetic modica-
tions, while others are transient changes. Transient chromatin
modications mediate the acclimation response. Heritable
epigenetic modications provide within-generation and
transgenerational stress memory, which is stable and thus can
be utilized for crop breeding (Fig. 1). In plants, stress memory
retention for a short duration against abiotic stress(es) is caused
by transient acclimation due to stress-induced proteins, RNAs,
and other plant metabolites. This short-term stress memory
depends on the half-life of these stress-induced biomolecules.
On the other hand, processes which involve reprogramming
of phenology and morphology result in stable stress memory.
Fig. 1. Abiotic stress(s) memory development in plants due to epigenetic variations. Epigenetic changes can be inherited mitotically and/or meiotically.
Within-generation and transgenerational stress memory developed in this way is stable and can be utilized for crop amelioration. Me, methylation; RdDM,
RNA-directed DNA methylation; TF, transcription factor. This figure was created with Biorender.com.
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Page 4 of 20 | Singh etal.
Epigenetic changes generate stable memory and stress-induced
gene expression, and thus can be used as a tool for retaining
stress memory for a longer period oftime.
Regulatory DNA sequences such as enhancers are involved
in enhancing the transcriptional activity of a gene when bound
by specic transcription factors (TFs) or proteins. Also, changes
in chromatin structure can bring the enhancer and coding
sequences in close proximity to each other and thereby af-
fect the gene function (Grant-Downton and Dickinson, 2006) .
Chromatin structure is also inuenced by epigenetic changes,
and thus utilization of epigenetic modications for enhancer
activation can help in directing the gene function for improved
croptraits.
Abiotic stresses result in hyper- or hypomethylation of DNA
and other modications which regulate expression of genes
involved in stress tolerance mechanisms. Gene methylation
and modications at the promoter region directly impact gene
expression. These epigenetic modications shape the stress
plasticity of plants, and their cumulative eect results in stress
acclimation in plants (Ashapkin etal., 2020).
Salinity is one of the major abiotic stresses aecting prod-
uctivity of important food grains worldwide (Karan et al.,
2012). In the case of rice, varied DNA methylation patterns
were reported in salt stress-responsive genes (Karan etal., 2012;
Rajkumar et al., 2020), whereas increased hypermethylation
was observed in shoots of barley (Demirkiran et al., 2013).
In a study by Forestan etal. (2016), long non-coding RNAs
(lncRNAs) were found to play an important role in salinity
tolerance of maize plants, while Mousavi etal. (2019) reported
increased DNA methylation of retrotransposons in olives
under thisstress.
Epigenetic regulation of stress-responsive genes also help
plants to acclimate under temperature extremes. In a study
involving heat stress, it was found that DNA methylation and
histone methylation (H3K9me2) were two important factors
controlling expression of the OsFIE gene which resulted in
reduced seed size in rice (Folsom etal., 2014). Heat-induced
changes lead to hypo- or hypermethylation of dierentially
methylated regions (DMRs) of plant genomes. Dierential pat-
terns of DNA methylation were observed in heat-sensitive and
heat-tolerant genotypes of Brassica (Gao etal., 2014). Similar
changes were also observed in cold stress, which resulted in
disturbed productivity, reduced nutrient uptake, and chilling
injuries in plants (Banerjee et al., 2017). In another study,
up-regulation of histone deacetylases (HDACs) was observed
in maize under cold stress, which led to DNA demethylation
and histone methylation, causing activation of stress-responsive
genes (Hu et al., 2011). Histone acetylation was observed in
cold-responsive genes such as drought-responsive element
binding 1 (DREB1) and cold-regulated 413 protein (COR413)
in maize, which resulted in up-regulation of cold-responsive
genes (Hu etal., 2011; Banerjee and Roychoudhury, 2017).
Drought stress generally increases demethylation in the
genome (Singroha and Sharma, 2019). Dierential DNA
methylation patterns under drought stress have been reported
in many studies (Wang etal., 2011; Gayacharan and Joel, 2013;
Sallam et al., 2019). Sallam et al. (2019) reported increased
DNA demethylation in maize under drought stress condi-
tions. Reduced DNA methylation or hypomethylation is also
reported in plants. In a study by Gayacharan and Joel (2013),
hypermethylation of DNA was found in drought-sensitive
rice genotypes, whereas tolerant genotypes showed reduced
methylation. Increased DNA methylation results in gene si-
lencing, and thus severely aects plant growth parameters.
Drought stress-related epigenetic variations were also studied
in barley, which showed methylation of the DEMETER
(DME) homolog, resulting in improved seed development
under drought stress (Kapazoglou et al., 2013). In soybean,
high resolution DNA methylome analysis revealed that DNA
demethylation enhances adaptability towards continuous
cropping stress. Continuous cropping stress is a comprehen-
sive stress resulting from continuous cropping of a single crop
over and over again, causing soil deterioration and overall re-
duced yields in crops (Li etal., 2016). The study suggests DNA
demethylases as an alternative for developing continuous crop-
ping stress-tolerant varieties (Liang etal., 2019).
Like any other stress, heavy metal toxicity also aects a plant’s
epigenome and causes many epigenetic variations (Cong etal.,
2019). In rice, a few studies have reported transgenerational
memory development against heavy metal-induced stress (Ou
etal., 2012; Cong etal., 2019). In most plants, heavy metals in-
duce hypermethylation of stress-responsive genes. Zinc- and
lead-induced epigenetic variations caused hypermethylation of
DNA in maize (Erturk etal., 2014, 2015). Similarly, He etal.
(2019) reported increased methylation in response to stron-
tium stress in soybean.
Thus, epigenome modications can be employed to develop
a better understanding of transgenerational inheritance in im-
portant crops susceptible to damage caused by abiotic stresses.
These variable epigenetic modications in plants can be used
to develop epigenetic markers for assessment of abiotic stresses
in advance. Various types of abiotic stress-induced epigenetic
variations are given in Table 1.
Inheritance of epigenetic variations
Plants, in contrast to animal species, can be used to study inher-
itance of epigenetic changes as they are a rich source of epigen-
etic variations such as paramutations resulting from interactions
between two alleles in which one of the paramutagenic alleles
permanently alters the other allele. Also, transposable elements
(TEs), siRNAs, and RdDMs cause altered gene expression or
silencing of genes (Henderson and Jacobsen, 2007). An im-
portant concern for using epigenetic information in crop im-
provement programs is related to the stability of epigenomic
patterns. The methylome from any tissue can be used accur-
ately to describe the epigenetic prole of an individual if
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Epigenomics to improve abiotic stress tolerance in plants | Page 5 of 20
Table 1. Abiotic stress-induced epigenetic changes in crops
Crop Abiotic stress Epigenetic modification in
response to abiotic stress
Technique used to decipher epigen-
etic changes
Response to abiotic stress Reference
I. Cereals
Barley Salinity Hypermethylation of DNA RAPD Increased hypermethylation in shoots. Demirkiran etal.
(2013)
DNA methylation NGS Changes in non-CG methylation. Konate etal.
(2018)
Drought DNA methylation Methylation enzyme-dependent digestion of gen-
omic DNA
Methylation of DME homolog during drought stress respon-
sible for seed development.
Kapazoglou
etal. (2013)
Maize Salinity Histone acetylation ChIP-PCR Up-regulation of cell wall-related genes involved in salinity-
induced root swelling.
Li etal. (2014)
lncRNAs Transcriptome annotation and differential expres-
sion analysis
LncRNAs that may play critical roles in regulating gene
expression.
Forestan etal.
(2016)
DNA methylation MeDIP-seq Changes in methylation pattern. Sun etal. (2018)
Heat, cold, UV DNA methylation MeDIP and microarray hybridization Changes in methylation pattern under different stress
conditions.
Eichten and
Springer (2015)
Cold DNA methylation MSAP Demethylation of cold-responsive genes. Shan etal.
(2013)
Heavy metals (Zn) DNA methylation CRED-RA Increased DNA methylation due to Zn stress. Erturk etal.
(2015)
Heavy metals (Pb) DNA methylation RAPD and CRED-RA analysis DNA hypermethylation and increased ABA levels. Erturk etal.
(2014)
Rice Salinity DNA methylation MSAP Cytosine methylation changed under salinity stress. Karan etal.
(2012)
Hyper- and hypomethylation of DNA
in different genotypes
Wh4ole-genome bisulfite sequencing Increased expression of genes related to stress tolerance. Rajkumar etal.
(2020)
Drought DNA methylation MSAP Regulation of drought stress responsiveness by DNA
methylation in different genotypes.
Wang etal.
(2011)
DNA methylation MSAP Hypermethylation in drought-sensitive genotypes while
drought-tolerant genotypes showed hypomethylation.
Gayacharan and
Joel (2013)
Heavy metals DNA methylation Bisulfite sequencing Transgenerational memory development of DNA methyla-
tion changes.
Cong etal.
(2019)
DNA methylation Methylation-sensitive DNA gel-blotting Transgenerational memory development of DNA methyla-
tion changes and development of tolerance to heavy metal
stress.
Ou etal. (2012)
Heavy metals (Cd) DNA methylation Bisulfite sequencing Differential expression of genes under Cd exposure. Feng etal.
(2016)
Wheat Salinity DNA methylation MSAP Genome-wide hypomethylation. Zhong etal.
(2009)
Cytosine methylation
polymorphisms
HPLC and MSAP Increased methylation of stress-responsive genes. Wang etal.
(2014)
DNA methylation MethylFlash methylated DNA quantification (col-
orimetric) kit
Tissue-specific cytosine methylation down-regulated ex-
pression of HKT genes
Kumar etal.
(2017)
II. Pulses
Chickpea Cold DNA methylation MSAP Prolonged cold stress induced DNA demethylation in tol-
erant genotypes.
Rakei etal.
(2015)
Faba bean Drought DNA methylation MSAP Drought induced increased DNA methylation in root tissues. Abid etal. (2017)
Pea Drought DNA methylation MSAP Drought stress induced increase in DNA methylation. Labra etal.
(2008)
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Page 6 of 20 | Singh etal.
Crop Abiotic stress Epigenetic modification in
response to abiotic stress
Technique used to decipher epigen-
etic changes
Response to abiotic stress Reference
Pigeon pea Salinity DNA methylation MethylFlash methylated DNA quantification (colori-
metric) kit and bisulfite sequencing
Increased methylation in CHH context resulted in
up-regulation of genes under salinity stress.
Awana etal.
(2019)
Soybean Salinity DNA methylation and histone modi-
fications
ChIP and bisulfite sequencing and DNA
methylation-sensitive DNA gel- blot analysis
DNA methylation and histone modifications of salt-
responsive transcription factor gene.
Song etal.
(2012)
DNA methylation Bisulfite sequencing DNA methylation of salt stress-associated lncRNAs. Chen etal.
(2019)
Histone methylation ChIP Changes in H3K27me3 deposition during salt stress
leading to inactivation of genes.
Sun etal. (2019)
Heavy metals (Sr) DNA methylation MSAP Increased DNA methylation. He etal. (2019)
Heavy metals (Cd) DNA methylation ELISA Changes in DNA methylation. Holubek etal.
(2020)
III. Oilseeds
Brassica napus Salinity DNA methylation MSAP Changes in methylation pattern. Guangyuan etal.
(2007)
Histone methylation Immunofluorescent staining and HPLC-assisted
quantification
H3K4me3, H3K9me2, and 5-methylcytosine (5-mC) methy-
lation.
Fang etal.
(2017)
Heat DNA methylation MSAP Increased DNA methylation in heat-sensitive genotypes and
DNA demethylation in tolerant genotype.
Gao etal. (2014)
Brassica napus
var. oleifera
Salinity DNA methylation MSAP Cytosine methylation changes under salinity as well as gene
expression.
Marconi etal.
(2013)
Castor bean Salinity Histone methylation ChIP-Seq Salt stress-responsive regulators were regulated by bivalent
H3K4me3–H3K27me3 modifications.
Han etal. (2020)
Olive Salinity DNA methylation MSAP DNA methylation of several retrotransposons in response
to salinity.
Mousavi etal.
(2019)
IV. Fiber crop
Cotton Salinity DNA methylation MSAP Increase in salt stress induced DNA demethylation. Li etal. (2009)
DNA methylation MSAP Salt induced demethylation positively contributed towards
salt tolerance, whereas hypermethylation showed negative
effects.
Zhao etal.
(2010)
Heat DNA methylation Whole-genome bisulfate sequencing High temperature induced de /methylation of genes and
their promoters affected anther development.
Zhang etal.
(2019)
Cold DNA methylation MSAP Prolonged cold stress induced demethylation of
hemimethylated or fully methylated cytosine.
Fan etal. (2013)
Drought DNA methylation Whole genome bisulfate sequencing Drought stress induced hypermethylation and full restor-
ation during re-watering.
Lu etal. (2017)
Salinity and alkalinity DNA methylation MSAP Alkali stress induced more hypomethylation in leaves and
roots than salt stress.
Cao etal. (2011)
RAPD, random amplified polymorphic DNA; NGS, next-generation sequencing; CRED-RA, coupled restriction enzyme digestion-random amplification; CHH, cytosine (H can be
any base except G); lncRNA, long non-coding RNA; ABA, abscisic acid; DME, DEMETER; MSAP, methylation-sensitive amplification polymorphism; MeDIP-seq, methylated DNA
immunoprecipitation sequencing; Sr, strontium; Cd, cadmium; Pb, lead; Zn, zinc.
Table 1. Continued
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Epigenomics to improve abiotic stress tolerance in plants | Page 7 of 20
DNA methylation patterns are stable throughout its develop-
ment. However, if DNA methylation is inuenced by develop-
mental or environmental factors, then the methylome of that
organism will be dierent from what is reected by its geno-
type (Springer and Schmitz, 2017). Epigenetic changes can be
inherited both mitotically and meiotically (Fig. 1), yet they are
often inecient as ospring sometimes show greater epigenetic
variations than their parents (Becker etal., 2011). Sometimes,
due to negative impacts on the phenotype, there are chances
of resetting epigenetic changes to their original states (Iwasaki,
2015). Moreover, re-programming of epigenetic variations is
not very ecient in plants (Hofmeister etal., 2017), although
some epimutations are stably inherited through generations.
Traditional plant breeding has relied mainly on meiotic
recombination for the generation of new variability for the
various characters under selection. However, meiotic recom-
bination is known to dier between and within chromosomes.
Epimarks are known to inuence meiotic recombination
patterns in plants and aect plant genomic variability. Many
studies have shown that the frequency of meiotic recombin-
ation in plants is highly inuenced by epigenetic variations
(Mirouze et al., 2012; Yelina et al., 2012; Choi et al., 2013;
Termolino et al., 2016; Underwood et al., 2018). Epigenetic
variations can both activate (Choi et al., 2013; Underwood
etal., 2018) and repress (Termolino et al., 2016) the meiotic
recombination process in plants. Study of epigenetic control of
meiotic recombination will help in further understanding the
key regulators of genetic diversity which can be exploited for
crop breeding applications.
Maintenance of DNA methylation in plants is believed to
be based on strand symmetry (Almouzni and Cedar, 2016).
Methylation maintenance occurs at CG and CHG sites, which
are symmetrical sites (read in the 5′ to 3′ direction) and the sym-
metry is important for semi-conservative copying of methy-
lation patterns (Pikaard and Mittelsten Scheid, 2014). Plant
DNA can maintain methylation at symmetrical dinucleotide
CpG residues as well as trinucleotide sequences with symmet-
rical C residues (Almouzni and Cedar, 2016). The existence of
methylation maintenance enzymes for such sites has been con-
rmed in the study by Pikaard and Mittelsten Scheid (2014).
Transgenerational inheritance of epimarks is still in its infancy
and is considered controversial, as epimarks are removed after
DNA replication. However, some recent studies suggest the
transgenerational inheritance of epimarks, which forms the
basis of inheritance of acquired epimarks (Jablonka and Lamb,
2015). The model by Jablonka and Lamb (2015) suggests the
inheritance of environmentally induced heritable chromatin
modications which might result in adaptive response to that
environmental stimulus. Similarly, Sandholtz etal. (2020) pro-
vided a physical model for heritability and maintenance of
epimarks in successive generations. The methylation model
re-establishes the methylation marks following DNA replica-
tion over the generations. Development of such models for
plant species is required for wider applicability of epipriming as
a sustainable tool for developing climate-smart crops.
Epipriming for generating abiotic stress
tolerance
Plants are exposed to various types of environmental stresses and
they develop ‘memory’ at the very rst exposure. Recurring
abiotic stresses throughout their life span enhances a plant’s
resistance to the changing environments. Priming in plants has
been described as a physiological mechanism by which a plant
quickly responds to a future stress condition. Stress priming
and memory development help plants to survive in rapidly
changing environments. Priming in plants can be induced by
benecial microorganisms or treatment with natural or syn-
thetic compounds. Epigenetic regulation is considered as one
of the molecular mechanism responsible for priming (Laura
et al., 2018). Environment-induced chromatin modications
in stress-responsive genes contribute to the development of
environmental memory in plants (He and Li, 2018). When epi-
genetic memory improves the plant’s response to subsequent
exposure to stress, the plant is said to be epiprimed or epigen-
etically primed (Fig. 2). Compared with other stress-responsive
biomolecules such as amino acids and ions, which provide
transient stress priming due to their short half-life, chromatin
modications such as methylation have a much longer half-life
which provides a long-lasting and stable stress memory (Ueda
and Seki, 2020). In the recent past, the phenomenon of priming
was utilized to prime plants against various biotic and abiotic
stresses (Hilker etal., 2016; Laura etal., 2018; Ramirez-Prado
et al., 2018). Priming in plants can be induced by elicitors,
for example volatile organic compounds or hormones (Lee
and Seo, 2014; Laura etal., 2018). Application of epipriming
to generate locally adapted crop varieties with enhanced re-
silience can be used as an alternative to genome editing. Key
crop characteristics pertaining to yield under changing envir-
onments can be improved through recurrent epigenetic se-
lection of isogeneic lines with novel epialleles (Varotto et al.,
2020). Hauben etal. (2009) conducted a study on high energy
use eciency and increased seed production, and found that
articial selection of canola parental populations with specic
epigenomic states improved its yield potential. However, in
crops with long juvenile phases, recurrent selection for specic
epigenetic features is limited. For such cases, epipriming can be
utilized for obtaining epigenetic variability.
Epipriming benets from changes in environmental factors
which alter the epigenetic shape of plants and drive them towards
adaptation against phenotypic changes. Epigenetic response to
abiotic stresses can lead to transgenerational stress memory
which may increase the chances of adaptation to the stress
(Fig. 2). In a study by Molinier etal. (2006), Arabidopsis was
exposed to UV-C or agellin, which resulted in a hyper-
recombination state even in subsequent untreated gener-
ations. Similarly, drought stress-related epimarks were stably
inherited in subsequent generations in the case of Arabidopsis
(Ganguly et al., 2017). These studies suggest that epipriming
can be induced by deliberate stimulation of epigenetic vari-
ations by changes in the growth environment during crop
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Page 8 of 20 | Singh etal.
breeding. Epipriming can also be used as an alternative to
natural germplasm for breeding purposes and additionally for
the production of locally adapted varieties. Determination of
meiotic inheritance of epimarks and their maintenance in the
next generation is an important key factor for determining
epiprimes against abiotic stresses. Epigenetic markers can be
retained or erased in plants; therefore, epigenetic editing can be
used to produce and maintain epimarks of interest.
Epigenetic mechanisms of plants against
abiotic stresses
Plants utilize epigenetic regulatory mechanisms, namely DNA
methylation including RdDM, histone modications, and epi-
genetic modication of mRNA bases, to survive adverse con-
ditions posed by climatic changes. DNA methylations occur at
cytosine residues by methyltransferases, resulting in modied
chromatin (Saxena and Carninci, 2011). Histones also oer a
range of post-translational modications (PTMs) that regu-
late the transcription machinery. Histone modications im-
pinge on the activities of specic factors or proteins involved
in transcription, DNA repair, etc. Apart from methylation,
acetylation, or any other modication, sRNAs and lncRNAs
can also modify the chromatin structure and they additionally
regulate transcription of genes through recruitment of DNA
methyltransferases or by modifying histone variants (Saxena
and Carninci, 2011). Both sRNAs and lncRNAs are involved
in RdDM-mediated epigenetic regulation in owering plants
(Matzke etal., 2015). Chromatin architecture and gene expres-
sion are greatly impacted by activation of one of these mech-
anisms. DNA methylation results in deamination of cytosine to
uracil which increases the mutation rate causing disturbance in
gene expression and regulation. In addition, it also suppresses
gene transcription, whereas DNA hypomethylation results in
active gene transcription. DNA methylation mostly occurs in
CpG dinucleotides and most of the recognition sites of TFs
are rich in these dinucleotides. Due to addition of methyl
groups, TFs cannot bind at their recognition sites, resulting in
suppressed gene transcription (Fragou etal., 2011).
Epigenetic modications of histones include histone
acetylation, methylation, phosphorylation, ubiquitylation,
and sumoylation. Enzyme histone acetyltransferases (HATs)
transfer an acetyl group from acetyl-CoA to the ε-amino
group of the histone lysine residues, which reduces the overall
positive charge of histones and thus diminishes their anity
towards DNA. By this mechanism, it brings chromatin to its
open state, allowing TFs to access recognition sites, and thus
activate transcription (Choudhuri et al., 2010). On the other
hand, histone deacetylation catalyzed by HDACs results in
closed chromatin states, hindering the access of TFs to recog-
nition elements. Histone acetylation, methylation, phosphoryl-
ation, and ubiquitination activate transcription, whereas DNA
methylation, histone deacetylation, and demethylation result
in gene silencing (Fragou et al., 2011). sRNAs and lncRNAs
also function as transcription switches which aect gene func-
tioning. lncRNAs binds to histone modication complexes,
DNA-binding proteins (e.g. TFs), and RNA polymerases to
Fig. 2. Epipriming for crop resilience towards abiotic stresses. Retained epigenetic memory helps in plant sustenance under subsequent stress, making
the plant epigenetically primed. This figure was created with Biorender.com.
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Epigenomics to improve abiotic stress tolerance in plants | Page 9 of 20
aect transcription (Long etal., 2017). Such epigenetic modi-
cations result in either gene activation or silencing (Fragou
etal., 2011). Another type of epigenetic regulatory mechanism
involves RNAi, resulting in altered gene expression (transcrip-
tional gene silencing) through DNA methylation. It requires
siRNAs which target genes and inactivates gene expression at
the transcriptional or post-transcriptional levels (Holoch and
Moazed, 2015).
Epibreeding
The epigenome of any plant can be permanent or transient
in nature, which is shaped by its interaction with the growth
environment. Epigenetic variations serve as novel genetic re-
sources for crop improvement programs. Epibreeding, which
is a next-generation plant breeding tool, capitalizes on these
environment-induced variations in chromatin. Epibreeding
tools utilize selective application of novel epialleles favoring
a trait of interest, and can be applied in climate-smart agri-
production without introducing any foreign genes into plants.
Many studies have utilized epibreeding for improving trait
characters, stress tolerance, and enhanced yield (Hauben etal.,
2009; González etal., 2013; Schmidt etal., 2018).
Epibreeding depends on the variability of the epigenome
and the stability of epimarks in subsequent generations.
Engineering the epigenome for elicitation of stress or epigen-
etic memory, epigenome variability, and maintenance of stable
epimarks can open up new avenues for using epigenomics as a
tool for next-generation crop breeding. Evaluation and utiliza-
tion of functional diversity in the epigenetic make up of a plant
can serve as new resource to enhance abiotic stress adaptation
and to improve productivity of crops. However, their exploit-
ation for production of future crops is still in its infancy due
to lack of understanding of epigenetic variations, epimark sta-
bility, and their reprogramming for stress adaptation. Abetter
elucidation of these epigenetic features can help in utilizing
epibreeding for developing climate-ready crops.
Epigenetic resources
Epigenetic resources such as epialleles, epiQTLs, epihybrids, and
epiRILs associated with traits of interest are used for developing
crops with desired characters. Quantitative epigenetics is the
new approach through which the relationship between epialleles
and phenotypic traits can be elucidated by mapping epiQTLs.
This section of the review deals with the epigenetic resources
and application of quantitative epigenetics in crop improvement
programs for abiotic stress tolerance and associatedtraits.
Epialleles andepiQTLs
Epialleles are dened as transgenerationally inherited stable
changes in DNA methylation patterns which lead to trait
variability in plants. Epialleles can be developed in plants
naturally or can be induced chemically. The rst epiallele
known as clark-kent (clk) with cytosine hypermethylation at
the ower development locus ‘SUPERMAN’ was identi-
ed in an Arabidopsis epimutant. It was a natural epimutant
with an increased number of stamens and carpels. However,
hypomethylation of cytosine in this epimutant resulted in
the wild-type phenotype (Jacobsen and Meyerowitz, 1997) .
Many stably inherited epialleles were found in peloric mu-
tants of Antirrhinum that have radial symmetry of owers
compared with the wild-type phenotype (bilateral sym-
metry) due to hypermethylation in the promoter region of
the gene Lcyc (Cubas et al., 1999). Epialleles can control
a large number of agronomic traits such as plant develop-
ment, plant architecture, and nutrient accumulation in dif-
ferent species (Table 2). Epialleles were also found to be
involved in abiotic stress response in many plants, such as
Asr2 epialleles for drought response in tomato (González
etal., 2013), epialleles for improved cellular respiration rates
and enhanced energy use eciency in rice epilines (Schmidt
et al., 2018), and salinity-responsive epialleles related to
hypermethylation of the promoter region of the MsMYB4
gene in alfalfa (Dong etal., 2020). Additionally, epialleles can
also regulate the homeostasis between euchromatin and het-
erochromatin regions to maintain genomic stability, as loss
of heterochromatin regions would expose the genes to the
DNA methylation machinery (Zhang etal., 2020).
Epimarks can be used for identication of epiQTLs which
are epigenomic loci with no DNA polymorphism, but they
dier for cytosine methylation patterns which regulate
phenotypic variations of quantitative traits. Many studies on
Arabidopsis found epiQTLs associated with complex traits or
with adaptation to changing environments with stable her-
itability (Cortijo et al., 2014; Kooke and Keurentjes, 2015) .
Identication of epiQTLs in plants is of major importance
before implementing epibreeding. The accuracy of identi-
ed epiQTLs is usually hampered by factors such as unstable
epialleles or recombination, which result in false negatives
or false positives. To avoid false results, crossing between iso-
genic lines diering in epimarks should be done (Johannes and
Colomé-Tatché, 2011). Epigenome-wide association studies
(EWAS) can help in shing out important epibreeding re-
sources in various crops.
Epigenetic RILs and epihybrids
EpiRILs can be dened as RILs which dier in DNA methy-
lation patterns and have no genetic variations. They serve as
an excellent source of epigenetic variations for successful and
stable, trans-generational epigenetic modications during
epibreeding. EpiRILs developed in the model plant Arabidopsis
have been used to study the eects of environmental factors
such as local climate adaptation, and heritability variations of
complex traits such as plant height, based on the development
of phenotypic plasticity. In these studies, epiRIL populations of
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Page 10 of 20 | Singh etal.
Arabidopsis were derived from hypomethylated mutants met1
and ddm1 (Vongs etal., 1993; Kakutani, 1997). Similarly, in an-
other study, epiRILs were created using the ddm1 mutant line
to study trans-generational inheritance of complex traits such
as plant height or owering time (Johannes etal., 2009).
Epihybrids can be dened as the plants derived from near
isogenic but epigenetically dierent parental lines (Lauss et al.,
2018). Epihybrids can be utilized to reveal novel epialleles as-
sociated with abiotic stress tolerance. Just like genetic hybrids
which show heterosis, epigenetic hybrids derived from epigen-
etically divergent parents show hybrid vigor. There is increasing
evidence supporting the role of epigenetic variations in hybrid
vigor (Rigal etal., 2016; Lauss etal., 2018). Using epiQTL map-
ping, Lauss etal. (2018) were successful in identifying DMRs in
parental lines which are associated with epihybrid traits. Further,
sequencing of parent–epihybrid combinations of the genome,
methylome, and transcriptome showed the role of DMR in
epigenetic remodeling of transcription and methylation states
at specic loci controlling various traits in epihybrids. These re-
sults prove that epigenetic factors are also responsible for hy-
brid performance. Such studies can help in determining novel
epialleles in other crops and subsequent creation of climate-ready
epihydrids. Development of epiRILs and epiQTL association
studies will further improve our understanding of inheritability
and retention of complex traits in epibreeding programs.
In some studies, it was revealed that polyploid hybrids oer
yield advantage through hybrid vigor (He etal., 2010; Guo
etal., 2017; Ding etal., 2021). High-yielding varieties are re-
quired to cater for the needs of the still-growing population
under a constantly changing environment. In some studies, on
rice polyploids, epigenetics was found to play a pivotal role in
whole-genome duplication and hybrid vigor expression in rice
(He etal., 2010; Guo etal., 2017). Epigenetic modications of
important regulatory genes in hybrids and polyploids leads to
heterosis, and more importantly under stress conditions (Chen,
2013). In a study on rice hybrids, He etal. (2010) found epi-
genetic marks correlated with change in transcription levels
among parental and hybrid lines. Dierential gene expres-
sion in heterotic rice also revealed the usefulness of epigenetic
modication in realization of heterosis and, therefore, epigen-
etic modications can be used as an ecient tool in hybrid
breeding.
Epi-interventions for improved epibreeding
Epigenetic interventions include tools and techniques for
overall improvement of epibreeding resources for wider ap-
plicability. Epi-interventions are not only restricted to protocol
improvement but also can be used for revealing links between
environmental factors and epigenomes, transgenerational inher-
itance, epipriming, and epigenome stability. Epi-interventions
such as association mapping studies for epimarks or epiQTL
mapping can be used to identify quantiable traits and novel
epialleles associated with abiotic stress tolerance. Use of epi-
interventions is not only limited to epibreeding but can also be
used in product quality assessment, stressor assessment, as well
as in plant health status assessment via epi-ngerprinting based
on methylation markers. Epi-interventions also employ editing
tools or chemical inducers for diverse crop improvement ap-
plications. However, the concept of epigenetic intervention is
quite new and a lot of development is still required for its
application in epibreeding. The present section of the review
delineates the use of epigenetic interventions such as epiQTL
mapping and epi-ngerprinting as a tool for epibreeding.
EpiQTL mapping for uncovering quantifiabletraits
Recent advancements in epiQTL mapping have opened up
new avenues for future epibreeding via associating epialleles
with important phenotypic traits (Lauss and Keurentjes, 2017) .
Table 2. Epibreeding sources for climate-ready agriculture
Plant Epibreeding source Characteristics Reference
Arabidopsis thaliana Naturally occurring epiallele Responsible for local climate adaptation and leaf senescence. He and Li (2018)
epiQTLs Adaptation to changing environments. Kooke and Keurentjes
(2015)
EpiQTLs governing flowering time and primary root length
with 60–90% heritability.
Cortijo etal. (2014)
Col-0 wild-type epiRILs Heritable variations of complex traits such as plant height and
flowering time.
Johannes etal. (2009)
Rice Naturally occurring epiallele RAV6 Large lamina inclination and small seed size. Xianwei etal. (2015)
Naturally occurring epiallele Nitrogen deficiency stress tolerance. Kou etal. (2011)
Tomato Methylated epialleles of Asr2 Water stress tolerance. González etal. (2013)
Naturally occurring epiallele Vitamin E accumulation in tomato fruits. Quadrana etal. (2014)
Cotton Naturally occurring epiallele COL2 Demethylation of COL2 increased its expression, inducing
photoperiodic flowering.
Song etal. (2017)
Brassica napus epiQTL Several agronomic traits in different environmental conditions. Long etal. (2011)
Brassica rapa Chemically induced epiallele Hypomethylated epialleles for epibreeding Amoah etal. (2012)
epiQTLs, epigenetic quantitative trait loci; epiRILs, epigenetic recombinant inbred lines.
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Epigenomics to improve abiotic stress tolerance in plants | Page 11 of 20
EpiRILs are the most common resources used for studying
and quantifying epigenetic variations and their association
with phenotypes. Alinkage map is required to associate epi-
genetic loci with phenotypic variation in an epiRIL. In RILs,
a single nucleotide polymorphism is used as a genetic marker
to determine which fragment is derived from which parent.
Similarly, in epiRILs, DMRs are used as markers. Identication
of DMRs and their stable inheritance between dierent gen-
erations is required for mapping of traits. To identify DMRs
which are stably inherited, genome-wide DNA methylation
analysis is performed in parental lines and data are compared
with those of epiRILs. These data can be utilized to determine
those DMRs which are stably inherited from either parent.
To perform epiQTL mapping, marker density should be su-
ciently high and spacing between markers should be consistent.
Stably inherited DMRs (markers) can be used to create maps
using software packages such as BioMercator 2.0 (http://www.
generationcp.org/), MAPQTL 6.0 (Kyazma.nl), or JoinMap 4
(Kyazma.nl). After creation of an epigenetic linkage map for
each selected epiRIL, epiQTL analysis can be performed like
normal QTL analysis (Lauss and Keurentjes, 2017). Many
studies used epiQTL analysis for identifying important agro-
nomic traits such as owering time and primary root length
in Arabidopsis (Cortijo etal., 2014); traits related to seed, yield,
quality, and plant development under dierent conditions in
Brassica (Long etal., 2011); and traits associated with growth and
wood properties in poplar (Lu etal., 2020). Thus, epiQTL map-
ping has a great potential to be used in epibreeding for climate-
smart agriculture and for identication of novel epialleles
associated with improved traits under stressed conditions.
Epi-fingerprinting
The concept of epi-ngerprinting was rst explained by
Rodríguez López and Wilkinson (2015) as a relationship be-
tween the epigenetic state and the response of a plant to its
growing environment. Epi-ngerprinting is not only limited
to exploring relationships between environmental factors
and epigenetic variations, but it can also be used as a plant
health indicator for plant product quality assessment. Epi-
ngerprinting requires development of methylation markers
which could be used in the near future for quality agriculture
produce. Previous studies showed that changes in the methyla-
tion pattern are linked with exposure to various abiotic stresses
(Table 1). These markers can be used as pre-stress interventions
to control overall productivity of crops during increased stress
periods. Epi-ngerprinting can also be employed as a quality
assessment tool based on the methylation prole of plants as it
denes the crop’s growth environment (Rodríguez López and
Wilkinson, 2015). Organ-specic epigenetic markers can be
used to assess food quality parameters as dierent tissues and
organs have dierent epimarks (Rodríguez López etal., 2010).
Thus, epi-ngerprinting holds great potential in achieving
sustainability in food production and quality assessment under
stress conditions.
Chemically induced epigenetically modifiedplants
The basis of epiRILs was discovered by utilizing hypomethylated
mutants met1 and ddm1 in Arabidopsis, as described previously
in this review. However, such mutants are not available in other
crop plants, therefore chemical agents can be utilized to produce
epigenetic variations. Various chemical compounds are known
to induce epigenetic modications such as DNA methylation
or histone modications. Chemical methyltransferase inhibitors
such as 5-azacytidine, 5-aza 2′-deoxycytidine, and zebularine
inhibit transfer of methyl group from S-adenosylmethionine
to cytosine (Haaf, 1995; Amoah etal., 2012; Akone etal., 2020).
Using 5-azacytidine, hypomethylated epialleles were developed
in Brassica rapa (Amoah etal., 2012). These chemical mutagens
lead to hypomethylation and these hypomethylated popula-
tions can be used for epiRIL development. HDAC inhibitors
such as diallyl disulde sodium butyrate, Helminthosporium
carbonum (HC) toxin, trichostatin-A (TSA), and nicotinamide
were also used to induce epigenetic variations in non-model
plants (Akone et al., 2020). Thus, chemical mutagenesis can
serve as a new source for developing epibreeding populations
for improving abiotic stress tolerance.
Epigenome engineering for crop
improvement
Epigenome editing or engineering is a branch of epigen-
etics which involves chromatin engineering in which the
epigenome is modied using epigenome editing tools such as
various enzymes, activators, or eector molecules. The process
is called epigenome writing, which involves a catalytic domain
called an eector domain (ED) writer, or eraser. The ED is
targeted towards a DNA-binding domain (DBD) and con-
nected to it by a linker peptide. EDs involved in epigenome
editing lack nuclease activity, unlike genome editing in which
an ED has its own nuclease activity (Miglani etal., 2019). Also,
in contrast to genome editing, epigenome editing does not
employ endogenous DNA repair mechanisms. Epigenome
editing has been successfully used in Arabidopsis but it still
needs some important renements such as uncovering the role
of the eraser, writer, and reader, and in understanding DNA
methylation patterns and histone modications during stress
conditions together with stability in clonally and sexually re-
producing plants, for its wider applicability. Afew applications
of epigenome engineering for crop improvement in the recent
past have been reviewed by Agarwal etal. (2020).
Writing, erasing, and rewriting epimarks for improved
stress tolerance
Epigenetics is gaining momentum for improving crop traits
and productivity. However, substantial knowledge about
writing, erasing, and rewriting epimarks for improved stress
tolerance is still lacking. Epimarks can be modied using
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Page 12 of 20 | Singh etal.
targeted epigenetic editors. Rewriting epimarks can be helpful
in modifying gene expression under dierent stress conditions
such as up- and down-regulation of gene expression. Epimarks
are maintained by DNA methylases (writers) and demethylases
(erasers) together with reader proteins (Fig. 3). Writers are
the enzymes or catalytic domains which write the epigen-
etic signature of an organism by modifying its DNA or his-
tones with methylation, acetylation, and/or phosphorylation.
Enzymes involved in writing epigenetic signatures are histone
acetyltransferases (HATs), DNA methyltransferases, and kin-
ases which are involved in phosphorylation, together with
ubiquitinases responsible for ubiquitination. Conversely, erasers
remove the epigenetic modications with the help of HDACs,
DNA demethylases, and phosphatases (Xu et al., 2017). The
presence or absence of histone methylations at Lys and Arg can
alter association between histones and reader proteins, resulting
in activation or deactivation of genes. Various domains which
identify modications at Lys and Arg have been identied in
plants, such as chromodomain (CD), Tudor, and ADD (ATRX-
DNMT3-DNMT3L) (Ueda and Seki, 2020). Xu etal. (2017)
described a bromodomain and a tandem plant homeodomain
(PHD) in Arabidopsis. Recent studies have made progress in
understanding development of epigenetic signatures due to
abiotic stresses (Table 1). Components of targeted epigenetic
modeling such as erasers, writers, and readers are now being
identied, which will help in development of articial modelers
for enhanced abiotic stress tolerance in crops. Development of
synthetic erasers, writers and eectors will help in producing
variable epiallelic sources for epibreeding.
Targeted rewriting of epigenomes will help in modica-
tion of epigenetic signatures in response to the environment.
Targeted epigenetic silencing has been widely exploited in
medicine, but limited applications are observed in plants
(Ahuja etal., 2016). Targeted regulation of genes by modi-
fying chromatin using writer enzymes can open up new
avenues in the eld of epibreeding. Adetailed review of this
type of targeted modication has already been published
(de Groote etal., 2012). Exploitation of enzymes or cata-
lytic domains for modication of epimarks can have a var-
iety of applications in plant breeding such as stress priming,
gene silencing, or crop amelioration. Most of the studies
on epigenetic modications include utilization of enzym-
atic tools such as CRISPR/Cas, dCas9 (dead or inactivated
Cas9), zinc ngers (ZFs), siRNA, or TEs. Targeted modi-
cation of the epigenome using such tools has been ex-
ploited in crops such as tomato for repressed ripening (R. Li
etal., 2018), articial sport cultivar development in potato
(Kasai etal., 2016) and in other crops as depicted in Table
3. Advancement of tools suitable for dierent crop plants is
still required.
Epigenome editingtools
Various epigenetic tools have been developed for inducing
epigenetic changes at specic targets in plants. Most editing
tools including readers and erasers of epimarks, chromatin, and
modelers target transcripts involved in epigenetic processes.
Previously, ZF proteins, transcription activation domains, and
Fig. 3. Epimark modification by targeted epigenetic editors. Editors help in dynamic epigenetic regulation. Residues on histone tails are marked by
writers. Readers bind with these epigenetic marks, whereas erasers help in removing them. This figure was created with Biorender.com.
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Epigenomics to improve abiotic stress tolerance in plants | Page 13 of 20
Table 3. Modified tools for targeted epigenome editing in plants
Plant Gene/loci
edited
Epigenetic alteration Tool used Phenotype Reference
Solanum
lycopersicum
lncRNA145 Down-regulation of SlDML2, that is responsible for
nearly all DNA demethylation during the process of
ripening and was critical for tomato fruit ripening.
CRISPR/Cas9-mediated editing of
lncRNA145
Ripening of tomato was repressed
in LncRNA1459 mutants due to
repressed ethylene and lycopene
accumulation.
R. Li etal. (2018)
Solanum
tuberosum
Granule-
bound starch
synthase
I(GBSSI) and
transgene
from tobacco
The transcriptional gene silencing was maintained in the
progeny tubers lacking transgenic siRNA.
Transcriptional gene silencing by
siRNA from transgenic tobacco.
Low amylose and high amylo-
pectin potato starch was pro-
duced in the transgenic potato
which could be used in artificial
sport cultivar development.
Kasai etal. (2016b)
Chrysan-
themum
morifolium
CmMET1
gene
RNAi-induced CmMET1 gene silencing resulting in
demethylation
RNAi Early flower phenotype. Li etal. (2019)
Zea mays Mu elements The Mu elements were targeted by different small RNAs
which resulted in silencing of different regions of these
elements, and thus produced different methylation
patterns.
Small RNA-mediated silencing Pale kernel Burgess etal. (2020)
Triticum
aestivum
TaEXPA1
homoeologs
(expansin
genes)
Silencing of TaEXPA1 homoeologs in roots resulted in
increased level of H3K9 dimethylation; decreased levels
of H3K4 trimethylation and H3K9 acetylation.
Cloning of wheat TaEXPA1
homoeologs
Formation of hexaploid wheat. Hu etal. (2013)
rRNA genes Increased CHG and CHH DNA methylation in pro-
moters (rRNA genes) was accompanied by asymmetric
silencing of nucleolus-organizing regions.
Polyploidization (synthetic allopoly-
ploids studied for epigenetic silencing
of nucleolus-organizing regions).
Stable allopolyploid wheat with
increased differentiation and di-
versity.
Guo and Han (2014)
Oryza sativa OsMet1‐1,
OsMet1‐2,
and OsRac5
Exogenous siRNAs induced de novo DNA methylation
of CpG dinucleotides in transcribed region of rice en-
dogenous genes.
RNAi – Miki and Shimamoto
(2008)
Su(var)3–9
homolog
(SUVH)
Histone demethylation resulted in transposon repres-
sion and chromatin modifications.
RdDM of rice Su(var)3–9 homolog
(SUVH)
Altered seed morphology. Qin etal. (2010)
LDMAR (a
lncRNA)
Increased methylation of the LDMAR promoter which
reduced its expression.
Cloning of pms3, a locus controlling
photoperiod-sensitive male sterility
Change in critical day length for
fertility recovery and delayed
fertility recovery under short-day
conditions.
Ding etal. (2012)
OsDRM2 De novo methylation. RdDM of target disruptants of
OsDRM2
Plant growth impairment. Moritoh etal. (2012)
OsMet1-2
mutants
Loss of mCG. Tos17 insertion in OsMet1-2 mutants Impaired seed development and
swift necrotic death in seedlings.
Hu etal. (2014)
ESP (Epigen-
etic Short
Panicle)
Hypomethylation of ESP gene resulted in ectopic
expression of ESP in Epi-sp plants which resulted in
specific rice panicle architecture.
Epi-sp (a gain-of-function epiallele)-
induced epigenetic modification
Short and dense panicle pheno-
type inherited in semi dominant
manner.
Luan etal. (2019)
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Page 14 of 20 | Singh etal.
transcription activator-like eectors (TALEs) were utilized in
genome targeting; however, due to their o-target hits, they
are not frequently used as compared with the CRISPR/Cas
system. CRISPR/Cas and dCas9 have proved to be revolu-
tionary in the eld of epigenetic modications. Now, gene ex-
pression levels can be easily modied using fusion of dCas9
with transcriptional regulators. Also, fusing dCas9 with epi-
genetic modulators can be used for precise epigenome editing,
guided by single-guide RNA (sgRNA) which recognize
the target sequence that needs to be edited (Moradpour and
Abdulah, 2019). The main advantages of CRISPR/dCas9
over previous genome editing techniques are target specicity,
adaptability, and reversibility. It consists of three major com-
ponents: a nuclease-dead Cas9, sgRNA, and transcriptional
activators. Transcription regulators including transcriptional
activators are fused to dCas9 or sgRNAs to modulate expres-
sion of the gene of interest. Synthetic transcriptional regulators
may include the transactivation domain of ZF protein, TALEs,
or Herpes simplex viral protein 16 (VP16), VP64, or VP160
(Cheng etal., 2013). Plant-specic transcriptional eectors in-
clude response factors belonging to the ERF/EREBP family
as they are actively involved in stress regulation, and the SRDX
domain derived from the ERF-associated amphiphilic repres-
sion domain (EAR) (Moradpour and Abdulah, 2019). The
dCas9 transcription activation domains potentially oer sim-
plicity and multiplex ability compared with transcription ac-
tivation domains of ZF proteins and TALEs as sgRNAs can
be easily modied to achieve new targeting specicities, and
dCas9 guided by multiple sgRNAs can simultaneously bind
to dierent target loci (Gao etal., 2014; Didovyk etal., 2016).
Therefore, dCas9 activators are now being exploited for max-
imal promoter activity.
Engineered dCas9 for epigenome editing inplants
Several tools are reported in Arabidopsis that allow site-specic
manipulation of DNA modications using the dCas9 system
(Table 3). In a study, an articial ZF fused with RdDM com-
ponent SU(VAR)3–9 HOMOLOG 9 (SUVH9) was used to
methylate the promoter of Flower Wageningen (F WA ) gene
in Arabidopsis, which rescued the late owering phenotype
of the fwa-4 epiallele. It is one of the tools developed in plants
for targeted DNA demethylation using recombinant eector
molecules (Johnson et al., 2014). A CRISPR/dCas9-based
SunTag system has been developed in Arabidopsis using the
catalytic domain of tobacco DRM to silence the F WA locus
(Papikian etal., 2019). Moreover, targeted DNA demethylation
was achieved in Arabidopsis by fusing human TEN-ELEVEN
TRANSLOCATION 1 catalytic domain (TET1cd) with
DBDs such as ZF protein and the CRISPR/dCas9-SunTag
system. Demethylation was stable, specic, and heritable in na-
ture (Gallego-Bartolomé et al., 2018). A modied CRISPR
activation (CRISPRa) system that targets ABA-responsive
element-binding protein 1 (AREB1) was fused with modied
Plant Gene/loci
edited
Epigenetic alteration Tool used Phenotype Reference
Arabidopsis
thaliana
PPOX locus Loss of CG methylation at targeted locus. Gene targeting No change in expression of the
PPOX gene in seedlings.
Lieberman-Lazarovich
etal. (2013)
FWA gene Methylation of FWA promoter resulted in silencing of
FWA.
Artificial ZF fused with RdDM com-
ponent SU(VAR)3–9 HOMOLOG 9
(SUVH9)
Late-flowering phenotype. Johnson etal. (2014)
FWA gene Removal of cytosine methylation in targeted locus of
genome (here, FWA promoter).
CRISPR/dCas9-based targeted
demethylation system (artificial ZF
fused with human TET1 catalytic do-
main and a modified SunTag system)
Late-flowering phenotype. Gallego-Bartolomé
etal. (2018)
AREB1 Histone acetylation by dCas9HAT at AREB1 loci was
found to be a determinant parameter in AREB1 expres-
sion.
CRISPRa dCas9HAT Dwarf phenotype with improved
drought stress tolerance through
positive regulation of AREB1.
Roca Paixão etal.
(2019)
FWA gene Induction of site-specific expression or methylation.
FWA gene silencing and methylation.
dCas9–SunTag system Early flowering phenotype. Papikian etal. (2019)
FLOWERING
LOCUS T (FT)
Modulation of transcriptional activity and epigenetic
status of specific target genes in plants
MS2–CRISPR/dCas9 system Altered flowering time phenotypes. Lee etal. (2019)
CRISPR/Cas9, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9); lncRNA, long non-coding RNA; AREB1, abscisic acid (ABA)-
responsive element binding protein 1; RdDM, RNA-directed DNA methylation; ZF, zinc finger; mCG, methylated CG; CRISPRa, CRISPR activation; FWA, Flowering Wageningen; dCas9,
dead Cas 9; mCHH, methylated CHH (H can be any base except G).
Table 3. Continued
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Epigenomics to improve abiotic stress tolerance in plants | Page 15 of 20
dCas9HAT to alter the target, and the resultant plant showed
better adaptability towards drought stress (Roca Paixão etal.,
2019). Therefore, epigenome editing with dCas9 holds good
potential to be used as a stress mitigation strategy in future
breeding programs.
Details of modied epigenome editing systems used in
Arabidopsis are given in Table 3. The modied CRISPR/dCas9
system for application in plant systems has been reviewed ex-
tensively by Moradpour and Abdulah (2019) and Miglani etal.,
(2019). Further development of these tools together with arti-
cial eector domains and transcription activators for targeted
epigenome editing in crop plants will provide new epialleles
for epibreeding purposes.
Challenges and future perspectives
Major challenges pertaining to epibreeding are the limited
stable epigenetic variations in response to environmental
stresses as plants maintain induced epimarks for only a few
generations. Another major challenge of epibreeding is to pre-
dict the impact of epigenetic variations on plant phenotype
(Gallusci etal., 2017). Afew attempts have been made to assess
the impact of epigenetic variations on plant phenotype through
statistical modeling to evaluate the overall improvement of
plant due to epigenetic variations (Coustham et al., 2012;
Colicchio etal., 2015; Hu etal., 2015). The statistical models
rely on a large number of datasets from plant methylomes but
lack programming based on data from the mechanism of epi-
genetic linkages, and the predictions are based on the datasets
used for making models which gives only a generalized pre-
diction and not specialized ones (Richards et al., 2012). The
challenges associated with the prediction models for epigenetic
performance of crops include identication of epimarks, char-
acterization of transmission features (meiotic or mitotic), and
quantication of linkage between epigenetic and phenotypic
variations. Tracking epigenetic variations and characterization
of epimarks for phenotypic performance of crops is very com-
plex and, thus, prediction modeling is very limited and scarcely
reported in the literature.
There is a dire need for development of methylation markers
which can identify important epigenetic variations in crops
for epibreeding programs. These markers will be benecial for
identication of novel epialleles, stress priming, and evaluation
of crop health status. Yet, due to limited understanding and sta-
bility of epimarks in plants, the development of methylation
markers for crops in response to abiotic stresses is still in its
preliminarystages.
Chemically induced epimutations have their own limi-
tations in the case of plants, such as toxicity associated with
chemicals, viability of plant cells, inability to control epigen-
etic variations which negatively impacts growth and develop-
ment of the plant, and the hazardous nature of chemicals for
the environment. The potency of chemical mutagens varies for
dierent genome sizes (Amoah etal., 2012); thus, it is impera-
tive to develop dose-dependent protocols for dierent crops.
Lack of protocols for induction of stable epialleles in dierent
crops using chemicals is another major challenge. The safety
of agri-products generated from such crops for human use is
understudied and requires legislation for commercialization.
Further, epigenome editing in plants is restricted by the
editing eciency of the applied tools, cytotoxicity, epigenome
stability, and limited cellular delivery protocols. Use of engin-
eered CRISPR/Cas and other transgenic methylation main-
tainers can improve the applicability of epigenome editing in
crops with complex inheritance of agronomic traits. However,
most of the studies with engineered Cas9 have been done in
the model plant Arabidopsis, and engineered Cas9 for other
crop plants is still under development. Also, the applicability of
engineered Cas9 and other editing tools in crop plants under
eld conditions is yet to betested.
As discussed, the stable inheritance of epimarks is crucial for
epibreeding; however, the transmission of epimarks to meiotic
descendants through sexual reproduction is unstable due to re-
setting of some epigenetic modications during meiosis. On
the other hand, the transmission of epimarks through mitosis
is stable, and thus clonally propagated crops can be utilized
for epibreeding programs (Latutrie etal., 2019). There is sup-
porting evidence in the literature on the stability of epimarks
(genome-wide DNA methylation) resulting from drought,
shading, and soil contamination lasting up to ve rounds of
vegetative propagation of Trifolium repens L.(Rendina González
et al., 2018). Clonal propagations of epigenetic variants thus
provide an opportunity to multiply epigenomes harboring
traits of interest for many generations, and the resource can
therefore be utilized for epibreeding. According to a study
done by Meyer et al. (2012), ~51.8% of a total of 203 crops
under study can be propagated both sexually and vegetatively.
Thus, these crops can be employed in epibreeding programs
because of the stability of epimarks and the ability to the x
the trait of interest for several generations oered by clonal
propagation. Vegetative propagation of plants which are propa-
gated sexually can provide opportunities for small-scale farmers
and crop breeders. Capitalizing on clonal propagation for stable
inheritance of epimarks requires extensive research for crops
which are propagated viaseeds.
Further, to date, epigenetic resources such as epiRILS,
epiQTLs, and epihybrids were mainly developed to study
complex agronomic traits, hybrid vigor, etc. These resources
can also be developed to produce climate-resilient crops via
epibreeding. EWASs—which are commonly employed for
epidemiological studies in humans (W. Li etal., 2018)—can be
employed to understand the association between genetic and
epigenetic changes linked to climatic perturbations, pheno-
typic plasticity, and epibreeding. Also, epigenetic editing tools
which are mainly utilized in medical science or animal systems
have vast potential in ameliorating abiotic stress tolerance. In a
nutshell, epibreeding can be seen as the future of climate-ready
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Copyedited by: OUP
Page 16 of 20 | Singh etal.
agriculture, but development of markers, editing tools, and
epibreeding protocols in non-model plants, epigenetic regu-
latory networks, and legislation for its global application still
need to be elaborated further.
Conclusion
Epigenetic changes in response to the changing environ-
ment create epimarks which are transgenerationally inherited
in plants. Stable epimarks can provide new genetic sources
for development of climate-ready future crops. Epigenetic
interventions such as epipriming and epi-ngerprinting for
determining the association between the epigenome and the
growing environment, epiQTL mapping for shing out quan-
tiable traits in food crops, and epigenome editing are novel
areas of epigenetic research which can open up new avenues
for abiotic stress tolerance epibreeding. These interventions
can provide food security to an ever-increasing population,
although novel methylation markers, editing tools with im-
proved eciency, and protocols in non-model crops still need
to be developed rapidly.
Acknowledgements
The authors thank the Director of the Indian Council of Agricultural
Research (ICAR)-Indian Agricultural Research Institute (IARI), New
Delhi, and the Head of the Division of Genetics, ICAR-IARI, New
Delhi for helpful discussions during preparation of this review.
Author contributions
DS, MP, and VC: conceptualization and design; PC, JT, SS, DPS, and DS:
interpretation of the relevant literature; PC, JT, CKS, and DS: writing—
original draft; DS, VC, JT, VJS, MP, and RY: critical revision. All authors
read and approved the nal manuscript.
Data availability
All data presented here are publicly available in published research and
review papers which are cited herein.
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