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Epigenome refers to the sum of all the epigenetic
changes in DNA base (without altering the underlying nucleotide sequence),
histone proteins and small-RNA biogenesis in a cell. Genome-wide epigenetic
changes are being reported during cellular growth and development as well as
during environmental stress, which are often associated with variation in gene
expression. The level of gene expression and epigenetic changes may go back to
the pre-stress state immediately after withdrawal of the stress. A well-known
mechanism of epigenetic change has been the methylation of cytosine at 5th
carbon. Additionally, certain amino acids of histone proteins are
post-translationally modified that may affect transcription, chromosome
segregation/condensation, and/or DNA repair processes. Small-RNAs play a
crucial role in DNA methylation through the RNA-directed DNA methylation (RdDM)
pathway. The epigenetic changes may be inherited over the generation that often
results in phenotypic variations. It is becoming evident that epigenetic changes
play important roles in acclimatization, stress tolerance, adaptation, and
evolution processes. As the growing evidence on epigenetic variations suggest
their effect on gene expression, it would be crucial to investigate the
epigenetic machinery of gene regulation in plants, and its possible use in
epigenome engineering/editing for crop improvement. This mini-review focuses on
the basics of epigenomics, followed by the present status and prospects towards
its usage for crop improvement to meet the challenges of sustainable food
security for the global population.
Keywords: Crop improvement, DNA methylation, Epigenomics, Gene regulation,
Histone modification, Stress memory
INTRODUCTION
Explaining genotypic variations with the
rapid evolutionary changes under environmental pressure has become difficult
using classical genetics alone. The rate of phenotypic variations and genetic
mutations are considerably different, which cannot be explained merely based on
genetics as the primary molecular mechanism. Additional mechanisms such as
epigenetics can help to explain this enigma [1]. If epigenetics is considered
as a complementary molecular mechanism, many of the phenotypic variations (e.g.
the dissimilarity between the clones) can be explained easily [2].
Plants are sessile in nature and face
multiple environmental stresses [3]. Until the last century, it was thought
that isolation of the gene(s) associated with a trait of interest was
sufficient to transfer the trait to a crop plant and to achieve the expected
phenotype. Recently, definitive evidence has been gathered for the DNA to
provide only part of the genetic information for a trait, and that chromatin
changes also contribute to the expression of the trait. DNA (cytosine)
methylation, post-translational modifications (acetylation, methylation,
phosphorylation, etc.) of histones and regulatory RNAs (small non-coding RNAs
or sncRNAs) define distinct chromatin/epigenetic states of the genome
(epigenome), which vary with the changing environmental conditions [4]. Thus,
chromatin is a highly dynamic structure which carries various information: (i)
the one encoded by the DNA sequence, and (ii) those provided by the epigenetic
states. Since the epigenetic states of chromatin are variable, transfer of a
trait from one species to another not only requires the transfer of the gene(s)
associated with the trait but also the appropriate chromatin/epigenetic states
to enable the trait to express. It is, therefore, essential to study the
epigenetic states in the donor plant/species and to ensure proper
re-establishment of the epigenetic state of the genes in the recipient
plant/species for their expression under the appropriate (de)methylation level
[5]. However, epigenetic mechanisms of gene regulation are yet to be fully
understood and utilized as epialleles (the alleles that are genetically
identical but epigenetically different due to the
Considering the current molecular understanding,
epigenetics can be defined as the studies of the molecular processes in and
around DNA that control genome activity independent of the DNA nucleotide
sequence which may be inherited through mitosis or
meiosis [7]. These epigenetic mechanisms
include DNA methylation, histone protein modifications,
and biogenesis of sncRNAs [1]. Environmental factors have been reported to
promote epigenetic variations. Several researchers have proposed the role of
epigenetics in the evolution process, primarily as a sensible and responsive
molecular mechanism in the natural selection [8].
Many of the
traits of economic importance are complex in nature controlled by the joint
action/interactions of multiple genes. Recent findings indicate that heritable
variations may also be caused by epigenetic changes in the genetic material. Propagation of epigenetic marks in plant takes
much more direct route than that in the animal.
It has also been reported that the rate of spontaneous epimutations is higher
in the CG context because these sites are
not retargeted by RdDM. DNA methylation generally refers to the addition of a methyl group at the 5th carbon of cytosine as a post-replicative event. In plants, cytosine
methylation occurs in CG, CHG and CHH contexts (where
H=A, C or T), while in somatic cells of animals/vertebrates, cytosine
methylation is limited to CG context [9].
On the other hand, CHH methylation is maintained by Domains
Rearranged Methyltransferase 2 (DRM2). Interestingly in Arabidopsis, DRM2 is
responsible for de novo methylation
in all the contexts of cytosine. DRM2 is recruited to the target loci by a
specialized 24 nucleotide small interfering RNA (RNA-directed DNA methylation
pathway) [10]. Cytosine
methylation homeostasis is determined by the DNA methylation and demethylation
processes. Promiscuous methylation is pruned by demethylases to create the
desired methylation pattern. Demethylation of the promoter and/or coding region may also be
required to activate the expression of specific genes under the changing
environmental conditions or during the developmental stages of plant [11].
Histone
proteins have numerous evolutionary conserved lysine (K) residues that are
subjected to acetylation (ac), methylation (me), ubiquitylation (ub), etc. A variety of histone modifications
and their possible combinations (e.g. H3K4me3 and H3K27Ac: activation marks and H3K9me3 and H3K27me3:
repressive marks) regulate transcriptional potential of a gene. The level of histone acetylation is
controlled by histone acetyltransferases (HAT) and histone deacetylases (HDAC).
Histone lysine methylations have differential effects on transcriptional
activity, depending on the site (K4, K9, K27) and mode (me1, me2, me3) of
modifications. Histone lysine methylation can also be reversed by the action of
two different types of histone demethylases [12].
Studies indicate that the genome-wide
hypomethylation induces biogenesis of 24 nt
siRNAs, and activates de novo methylation pathways [13,14]. Studies
suggest that epigenetics is more likely to be involved in the heritability of
phenotypes in plants than in mammals. This might be attributed to two different
activities. First, the RdDM pathway influences de novo establishment and
maintenance of DNA methylation in the plant genome with the help of siRNAs.
Second, unlike resetting of DNA methylation pattern during gametogenesis in
mammals, DNA methyltransferases in plants are active during gametogenesis and
embryogenesis; hence, the patterns of DNA methylation can inherited from parent
to progeny in plant. In-depth studies
would be necessary to understand the role of RdDM
pathway in the epigenetic regulation of
genes and its deployment in epigenetic manipulation.
EPIGENETIC REGULATION OF DEVELOPMENTAL PROCESSES
Epigenetic changes in DNA
methylation, histone modifications and
ncRNA expression cause important biochemical, physiological and molecular
consequences in plants. The epigenetic-phenotypes are now being explained based on the fundamental
discoveries such as activation, excision and translocation of transposable
elements, allelic interactions, transgene silencing and epialleles of the endogenous genes. In Arabidopsis thaliana, four bifunctional
DNA glycosylases and AP lyases, namely DME (Demeter), DML2 (Demeter-Like 2),
DML3 (Demeter-Like 3) and ROS1 (Repressor of Silencing 1) are known to
recognize and remove methylated cytosines. ROS1, DML2, and DML3 generally
function in vegetative tissues and demethylate specific loci in the genome
[10]. These enzymes appear to counterbalance the RdDM pathway to fine-tune the
methylation levels at particular genomic locations.
Data indicate that apomictic seed development in plants is associated with
dynamic transcriptional activity in ovule probably regulated through
epigenetic mechanisms. Epigenetic model of regulation of apomixis shows that
reversible changes in chromatin configuration might alter the expression of
essential genes of the apomictic pathway
at the different developmental stage or
in different cell types [1].
Since
the discovery of imprinted R gene in
maize, dozens of imprinted genes have been identified
in plants and epigenetics has been found to play a crucial role in this process
[15,16]. Silencing of
transposable elements in the male gametes is essential for genome stability and
integrity. The decrease in methylation in pericarp on ripening of tomato
suggests the involvement of DNA demethylation in fruit ripening [17]. Gliadins, the storage proteins in
wheat and barley endosperm, require TaDME for their expression. RNAi-mediated suppression of DME resulted in a significant reduction
in gliadins and LMWgs, but HMWgs remained unchanged [18]. In a recent study, it
was revealed that MtDME gets strongly
induced in Medicago truncatula during nodule differentiation,
and knockdown of MtDME resulted in
morphological and functional alterations in the nodule [19]. Variation
in DNA methylation and its effect on the expression of high-affinity potassium
transporter under salt stress was reported to provide salt tolerance in wheat
[20]. There
is increasing evidence for the
involvement of epigenetic regulations in various developmental processes in
plants. Thus, understanding epigenetic regulation and functions of the machinery involved would be very much essential
for epigenetic
manipulation of plants for the trait of interest [21].
APPLICATIONS IN CROP IMPROVEMENT
Epigenetic
changes can affect important traits in crop plants; therefore,
creation/manipulation of stably inherited epigenetic variation could be a
powerful tool in plant breeding. It can enable modification of traits in plant without altering the DNA sequence of the
gene. Similarly, understanding the bases of phenotypic plasticity is crucial
for crop breeding. Zheng et al. [22] reported that the genes of
stress-responsive pathways showed accumulation of transgenerational epimutations
and the DNA methylation patterns in the drought-responsive genes were affected
by multi-generational drought. They reported that about 30% of the changes in
methylation were stable and inherited, which corroborated with the earlier
findings of Wang et al. [23] who reported 29% of the drought-induced DNA
methylation to be maintained even after recovery to the normal condition. Kumar and Singh [7] also observed that
25% of the increase in methylation was
retained in a rice genotype IR-64-DTY1.1 even after recovery from
the drought stress. Thus, epigenetics can be considered as an important
regulatory mechanism in plant’s long-term adaptation and evolution under
adverse environments. In Arabidopsis, DNA demethylases target promoter TEs to
regulate stress-responsive genes. Therefore, manipulating DNA methylation of
TEs in the promoter region (by recruiting DRM2 to the target loci) could be
considered for epigenetic manipulation of stress tolerance in plants [24].
Certain
epigenetic changes in plants persist even after
withdrawal of the stress and may inherit over the generation in the form of
epigenetic alleles. These heritable epigenetic alleles (epialleles) are now
considered as another source of polymorphism which may be utilized in the breeding program. It is now apparent that
somatically-acquired epigenetic changes in plants may be mitotically stable and
meiotically heritable; hence the emphasis is given to the variations in DNA
methylation as a source of variation [5]. A better understanding of the role
and significance of this new source of genetic and phenotypic diversity in
plants would be achieved as more data accumulates about the role of DNA
methylation in plant evolution, domestication,
and breeding. Identification and assessment of the importance of epialleles in
plant breeding require determination of (i) the extent of variation in
epigenetic marks among the individuals, (ii) the degree to which the epimarks
affect phenotype, and (iii) the extent to which the epimark-linked superior phenotypes are stably inherited. Although
there are several challenging tasks, the technical potential to assess
epigenetic variations between individuals and the estimation of the levels of epimark-associated phenotypic diversity does exist. With the increasing understating of
epigenetic phenomena, it is expected that our potential to exploit
epigenetics in crop improvement and nutritional management would get better,
and will have significant implications in plant
breeding [25].
Data indicates that F1
hybrids are, in general, less methylated than their parental inbred. In
general, hybrids are less methylated than their parental inbred, (ii) heterotic
hybrids are less methylated than related non-heterotic hybrids, (iii) old and
low-yielding inbred are highly methylated, (iv) new inbred, especially those selected for high and stable yield, have lower methylation
level in comparison to their progenitors. DNA methylation can be considered as
a regulatory mechanism that affects the expression
of several genes important for the manifestation of heterosis. Repeated selfing
carried out during the development of inbred, with
more emphasis on combining ability of the
inbred, leads to the gradual accumulation of methylated loci, which is released
and/or re-patterned when the inbred are crossed to develop hybrids. The
stressful growth conditions during the development of inbred result in more
methylated DNA, and these stress-induced methylations and the linked
suppression of genome activity could be at the core of higher yield of the
hybrid [5].
Manipulation of parental imprinting by epigenetic
alteration may lead to the development of a superior endosperm, which has
become a necessity for the improvement of seed crops. Understanding the
epigenetic regulation of seed development would eventually uncover the
mysteries behind apomixis, the asexual mode of reproduction through seeds
wherein embryo develops without meiosis and double-fertilization leading to the
production of progenies genetically identical to the
mother plant [26]. If this mechanism could be deployed successfully in the
commercial seed crops, hybrid vigor can
be maintained indefinitely which may help to
overcome the current limitations of plant breeders in maintaining hybrid
vigor for more than one generation.
Zhang et al.
[15] reported tissue-specific differentially methylated regions in sorghum and
suggested that DNA methylation play an important role in regulating
tissue-specific expression of the genes. Polycomb group (PcG) proteins are
involved in controlling the expression of
homeotic genes that are essential for the proper developmental processes in
plants. The main component of the PcG
complex in plants is methyltransferase (e.g. MEA in Arabidopsis) that methylate histone to regulate expression of
the homeotic genes for development of plant. In most of the plants,
embryogenesis starts with asymmetric cell division, which gives rise to a polar
embryo having a larger basal cell and a smaller apical cell. Cell division and
differentiation during these processes are highly regulated that are influenced
by epigenetic mechanism [1]. Demethylation of the promoter
of the gliadins and LMWgs encoding genes
in barley was reported to be important for the accumulation of gliadins
and LMWgs. However, regulation of HMWgs expression was found to be independent
of DNA (de)methylation. Due to the differential regulation of gliadin/LMWg and
HMWg expression in wheat and barley, suppression
of TaDME and HvDME has been proposed to be
a potential strategy to eliminate gliadins and LMWgs that cannot be digested/tolerated by many
people suffering from celiac disease
[11].
Silencing of
the transgene has frequently been
observed as a major commercial risk of the transgenic technology, creating
hindrance in the economic exploitation of transgenic plants [27]. Several
strategies have been suggested to minimize silencing of the transgene at
different stages of transgenic development. Silencing of transgenes also correlated with methylation of the
transgenes. Methylation of the promoter correlates with transcriptional gene
silencing, and methylation of the coding region is generally associated with
post-transcriptional gene silencing. A better understanding of the mechanisms
of epigenetically-enforced transgene silencing might help avoiding silencing of
the gene of interest. One of the strategies suggested to avoid transgene silencing has been the
careful designing of the transgene and thorough analyses of transformants at
the molecular level [7].
Under osmotic stress, P5CS and δ-OAT genes were found to show DNA
demethylation in mother plants, but it disappeared in the next generation,
suggesting that DNA demethylation regulated expression of the genes [28]. One
of the ways for plants to adapt to environmental stress is to remember a stress
episode and to react more efficiently (faster and more strongly) upon
subsequent exposures to the stress. At the molecular level, short-term memory
results from a combination of mechanisms, including modification of the levels
of stress-associated receptors, signaling
components, and transcription factors. Multiple lines of evidence indicate that
both short-term and transgenerational memories mainly rely on epigenetic
modifications, and it can be exploited in developing tolerant crop plants [12].
However, fundamental investigations are required to understand whether
stress-induced epialleles can be stabilized over several generations and
consequently be utilized in crop breeding programs. The research challenges
ahead include improving our understanding of the stability, reversibility, and heritability of epialleles.
Epigenetic manipulation may become a valuable strategy in the future for crop
improvement, as the approaches are available for stochastic modulation of DNA
methylation using chemical or by genetic means, followed by the forward or
reverse selection of epialleles. However, we need to devise strategies to
ensure stable retention of desirable epialleles within breeding materials and
to develop techniques for targeted epigenetic manipulation. Eukaryotic genomes
are complex in nature and genome complexity of many crop plants increases
further because of their polyploid origins, which makes gene interaction
networks complicated, and difficult to modulate for improved plasticity with
inbuilt gene redundancy. Understanding how epigenetic changes are superimposed on the multiple gene copies to
confer plasticity may provide a framework for the development of desirable crop
variety enabled to cope up with the harsh multiple-stresses the crops are
facing now due to the global climate changes. Currently, it is
difficult to control epigenetic variations; mobilization of stress-responsive
epigenetically-silenced TEs may contribute to
the stable inheritance of stress-induced
epigenetic changes.
Over the last
century, genetic improvement of crops and modern agronomic practices has
underpinned a massive increase in crop
yield and productivity. However, most of these gains have been achieved by utilizing
the ‘Green Revolution’ technologies in a period of relative climate stability
[29], compared to the current period of increased climate change and
variability. To facilitate climate resilient agriculture in the future, we need
to understand the molecular and mechanistic basis of genotype × environment
interactions (G × E) and the emergent property of crop plant plasticity
facilitated by epigenetic mechanisms. Epigenetic manipulation may provide a way to achieve the desired
variations and adaptive advantages without manipulating DNA sequence.
Importantly, epialleles may alter the expression of the gene(s) controlling cellular/physiological processes during plant
development. Stable inheritance of such adaptive epialleles may provide
increased fitness/adaptability to the plant in the changing environmental
conditions.
FUTURE PERSPECTIVES
In recent years, tremendous progress has been
witnessed towards understanding the epigenetic regulation of gene expression in
plants, particularly in Arabidopsis. The proteins
involved in DNA (de)methylation, histone
modification, and the mechanisms of ncRNA mediated regulation of developmental processes in plants are becoming clear
day-by-day. However, many areas of epigenetics remain to be explored. We
still know only a little about the factors that regulate the targeting of
active DNA demethylation during developmental stages. Does
DNA (de)methylation interplay with other epigenetic features or chromatin
features? Future research should aim at identifying more developmental
processes in different species that
involve epigenetic regulation. Assessing
the contribution of transgenerational epimarks
to heritable phenotypic variation has been a
major challenge as many of the chromatin (DNA
methylation and histone modification) changes
and gene expression variants co-segregate with DNA
sequence polymorphisms.
Nonetheless, there is evidence
that plants possess heritable epiallelic variations that can be associated with
the trait of interest and utilized for crop improvement. Although it had been
difficult to alter DNA methylation and chromatin states in a locus-specific
manner, the situation is changing rapidly with the advances in genome editing
tools like the CRISPR-Cas9 system. Catalytically inactive Cas9 (dCas9) can be fused with methylases and/or
demethylases to manipulate DNA methylation in a site-specific manner [8]. Thus, we can anticipate that soon epigenome editing will
provide a means to assess the role of a QTL in epiallelic variations which may
provide an exciting new route for the improvement of crop plants. With the
modern tools and techniques in molecular biology and biotechnology, it is
expected that soon we may achieve a comprehensive
understanding of this amazing biological
phenomenon, and we might be able to use it for the development t of
climate-resilient crops for the benefits of humankind. However, this will
need a deeper understanding of the
interactions between crop genomes and how their genomic regulatory networks
contribute to the plasticity of phenotype.
Genetic engineering technology offers novel
approaches for biotic and abiotic stress management with several advantages
over the conventional methods. However, a few drawbacks like gene silencing due
to the epigenetic changes are also there which can be managed as mentioned
above. Plant-incorporated protectants like Bt
gene have been one of the modern biotechnology approaches to protect crop
plants which have provided several products in the global market [30,31]. Considering the biosafety uses of genetically
modified organisms developed through the genetic manipulation of crop plants [32-34], the
epigenetic engineering (which is supposed to have limited biosafety issues, if
any) would be a preferred approach [2]. However, necessary safety guidelines framed in the
country by the regulatory authorities must be followed for personnel,
laboratory and environmental safety [35,36]. Thus,
epigenome engineering not only provides unprecedented opportunities for
understanding the epigenetic mechanisms of growth and development, but also to
manipulate the biological system to improve stress tolerance against the
changing climatic conditions.
CONCLUSION
Our understanding of the foods,
their production, and uses in maintaining and optimizing health is continuously
being improved. The global population is speculated to reach 9 billion by 2050.
This 2-3 billion upsurge in the global population would require increasing food
production by 70% [37]. Providing adequate food to the global population is
only the preliminary challenge; the major challenges would be to produce the
food in a safe and sustainable manner [38,39] under the increasingly
unfavorable environmental conditions [36]. Although pants have the innate
capability to survive under adverse climatic conditions, yet crop plants need
improvement in their efficiency to produce more and more nutritious food even
under unfavorable climatic conditions. Properly harnessing the epigenetic
variation is must to provide new opportunities for crop improvement and boost
the production. The coming years are likely to realize increased opportunities
for monitoring and manipulating crop epigenomes. Because gene expression
profile provides the primary account of the epigenotype to phenotype effect, it
becomes essential to dissect the relative contributions of genetic and
epigenetic variations on gene expression. The knowledge of epigenetic variation
might allow exploitation of different epigenetic marks towards the
development/selection of superior genotype at the early stage of plant growth.
The views expressed are those of
the author only. These may not be the views of the institution or organization
the author is associated with.
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S (2017) Epigenetic control of apomixis: A new perspective of an old enigma.
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S, Chinnusamy V, Mohapatra T (2018) Epigenetics of modified DNA bases: 5-methylcytosine
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30. Sharma
A, Kumar S, Bhatnagar RK (2011) Bacillus thuringiensis protein Cry6B (BGSC ID
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Curr Microbiol 62: 597-605.
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111-124.
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S, Arul L, Talwar D, Raina SK (2006) PCR amplification of minimal gene
expression cassette: An alternative, low cost and easy approach to ‘clean DNA’
transformation. Curr Sci 91: 930-934.
33. Kumar
S, Chandra A, Pandey KC (2008) Bacillus thuringiensis (Bt) transgenic crop: An
environment friendly insect-pest management strategy. J Environ Biol 29:
641-653.
34. Kumar
S (2014) Biosafety issues of genetically modified organisms. Biosafety 3: e150.
35. Kumar
S (2012) Biopesticides: A need for food and environmental safety. J Biofertil
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36. Kumar
S (2015) Biopesticide: An environment friendly pest management strategy. J
Bifertil Biopestide 6: 1.
37. Kumar
S (2013) The role of biopesticides in sustainably feeding the nine billion
global populations. J Biofertil Biopesticide 4: e114.
38. Kumar
S (2012) Biosafety issues in laboratory research. Biosafety 1: e116.
39. Kumar
S (2015) Biosafety and biosecurity issues in biotechnology research. Biosafety
4: e153.
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