Epigenetics, types of stress memory, DNA methylation techniques

1. DEPARTMENT OF MOLECULAR BIOLOGY AND
BIOTECHNOLOGY
COURSE NO : MBB-691
COURSE TITLE : Doctoral Seminar I
TOPIC : Exploration of Epigenetic memory in Crop
improvement
PRESENTED TO
Dr. V.L.N Reddy,
Associate Professor and Head
Department of Molecular Biology and
Biotechnology
PRESENTED BY
A. Govardhani
TAD/2020-028
1

2. Contents
 History and Introduction
 Types of stress memory
 Epigenetic Mechanism
 Role of epigenetics in crop improvement
 Case studies
 Conclusion 2

3. History and Introduction
• Epigenesis appeared in the literature in the mid 19th century,
although the conceptual origins date back to Aristotle (384-
322 BC).
Epigenesis: the development of individual organic form
from the unformed.
• Lamark (1744-1829) : ‘soft inheritance’ or ‘inheritance of
acquired characters’ describing that an organism can pass on
the characters to its offspring.
Epigenetics rehabilitating Lamarckian inheritance
3

4. What is Epigenetics ?
• Epigenetics- changes in gene expression that are not
associated with changes in DNA sequence
• Epigenetics = epigenesis + genetics
• Epigenetics means ‘above’ or ‘on top of genetics’
• Epigenetics’ was coined by Conrad H. Waddington in
1942.
• Waddingtion-epigenetics as “the branch of biology
that studies the causal interaction between genes and
their products, which brings the phenotype into
being”
Iwasaki and Paszkowski et al., 2020
4

5. What Does “Epigenetics” Mean?
Epigenetics describes phenomenon in which
genetically identical cells or organisms express their
genomes differently, causing phenotypic differences
5

6. Galviz et al., 2020
Previous responses can affect the subsequent ones, which is referred
as priming
Plant do you
have Memory?
Memory is a basic capacity to store and eventually recall information
6

7. Conrath et al. (2006)
Epigenetic mechanisms are known as events that may provide
mechanistic basis for the memory formation (Bruce et al.,
2007)
Rustification improves the performance of both crops and forest
trees under field conditions (Ferna´ndez et al., 2013; Bompadre
et al., 2018).
Galviz et al., 2020
7

8. STRESS MEMORY
• Stress memory can be described as the phenomenon through
which information on a past stress cue is retained and results
in a modified response upon a recurring stress (Lakme et al.,
2017).
Diagrammatic representation of
stress memory- Plant that
experiences a period of drought
wilts under the dehydration
stress and then recovers after
rehydration during a second
drought stress, the plant
‘remembers’ the past drought
experience, allowing it to
achieve better resistance to
dehydration and improve its
survival prospects
Kinoshita and Seki, 2014
8

9. Friedrich et al., 2018
Temperature stress priming and memory gene expression. A mild cold stress (blue) or heat
stress (red) can act as a priming cue (P) and will trigger enhanced tolerance to cold or heat
stress in the primed state. This primed state is maintained over time in a memory phase
9

10. Stress memory is of two types
• Somatic stress memory
• Transgenerational or Intergenerational stress memory
Somatic Stress Memory- Stress memory whose duration is
limited to one generation of organisms.
• It may be mitotically heritable, but often lasts only a
fraction of the lifespan of the organism.
Intergenerational Stress Memory- only in the first stress-free
generation
Transgenerational Stress Memory- two or more stress-free
generations
Bhadouriya et al., 202110

11. Bhadouriya et al., 2021
• Intergenerational memory may be the product of cues introduced into the seed or embryo by
the mother plant or by environmental conditions during seed development.
• Transgenerational stress memory -is based on epigenetic mechanisms
11
Intergenerational Inheritance

12. Priming agents
Bhadouriya et al., 2021
Stress memory is induced by the priming agents. It keeps the plant in alert mode.
Under abiotic stress, a plant that is not primed shows normal tolerance, while a primed
plant shows enhanced tolerance by increasing molecular functions and inducing
tolerance mechanisms.
12

13. • Some of the Priming agents used in Biotic stress-
Salicylic acid (SA), β-aminobutyric acid (BABA),
methyl-jasmonic acid (MeJA), pipecolic acid
(Pip), and azelaic acid (AzA)
• Growth and Stress tolerance- choline, chitosan,
putrescine, ethanol, paclobutrazol, ZnSO4,
KH2PO4, CuSO4, Se and BABA
• Priming agents is of 2 types
Cis priming
Trans priming
13

14. Johnson et al., 2021
Diagrammatic representation of Cis and Trans priming, Cis- stimulus and the stress
are the same, and if stimulus is different from the stress it is known as “trans-priming
or cross tolerance.”
14

15. Epigenetic Mechanism
Plant epigenetic mechanisms include DNA methylation, histone modification, and
RNA-directed DNA methylation (RdDM).
Fedoroff, 2012 15

16. DNA Methylation Profile
• De novo methylation
• Maintenance Methylation
• Demethylation
• De novo methylation is mediated by the RNA-directed
DNA methylation pathway (RdDM), involving small
interfering RNAs (siRNAs), proteins
• DNA methylation usually occurs at the 5th C position of
cytosine base in the context of CG, CHG, and CHH (H=A,C
and T) to form 5-methylcytosine
• Methyl Donor - S-adenosyl-L-methionine
Zhi and Chang, 2021
16

17. De novo Methylation
Kumar and Mohapatra et al., 2021
17

18. Maintenance of DNA Methylation
Symmetric methylation can be maintained across mitotic and meiotic cell divisions.
18

19. Zhang et al., 2018
Methylated CHG recruits the H3K9-specific methyltransferases
SUVH4, SUVH5 and SUVH6. in turn, dimethylated H3K9
(H3K9me2) facilitates CMT3 and CMT2 function, thereby forming a
reinforcing loop between CHG methylation and H3K9 methylation
19

20. DNA Demethylation
DNA demethylation can be active or passive.
Passive DNA demethylation-
Lack of DNA methyltransferase activity or shortage of a methyl
donor following DNA replication results in failure to maintain
methylation.
Active DNA demethylation- DNA methylation can also be erased
enzymatically.
• In plants, a family of bifunctional 5-mC DNA glycosylases-
apurinic/apyrimidinic lyases initiates active DNA
demethylation through a base excision repair pathway
• Four bifunctional 5-mC DNA glycosylases includes
REPRESSOR OF SILENCING 1 (ROS1),
TRANSCRIPTIONAL ACTIVATOR DEMETER (DME),
DEMETER-LIKE PROTEIN 2 (DML2) and DML3
Zhang et al., 2018
20

21. • ROS1 is recruited to a subset of
demethylation target loci by the
INCREASED DNA METHYLATION (IDM)
complex.
• IDM1 catalyses acetylation of histone H3
lysine 18 (H3K18Ac) to create a permissive
chromatin environment for ROS1 function.
• After cleaving the glycosylic bond, ROS1
cuts the DNA backbone at the abasic site by
β-elimination or β,δ-elimination
• Resulting in a gap with a 3′-phosphor-α,β-
unsaturated aldehyde (3′-PUA) terminus or
with a 3′-phosphate (3′-P) terminus,
respectively.
• The 3′ terminus is subsequently processed by
DNA-(APURINIC OR APYRIMIDINIC
SITE) LYASE (APE1L) or by the DNA
phosphatase POLYNUCLEOTIDE 3′-
PHOSPHATASE ZDP.
• Then an unmethylated cytosine nucleotide is
inserted at the gap by a yet unidentified DNA
polymerase (POL?) and DNA LIGASE 1
(LIG1
Zhang et al., 2018
21
Active Demethylation

22. Histone Structure
Modifications such as acetylation, methylation, phosphorylation, ubiquitination,
ribosylation and biotinylation takes place at N-terminal end
Chen et al., 2010
22

23. Histone Modifiers
Liu et al., 2010
• Writer: an enzyme that is
responsible for adding a post-
translational modification(s)
into a given protein (e.g.,
HKMT)
• Reader: a protein or protein
complex that recognizes and
binds specifically to a
particular post-translationally
modified substrate
• Eraser: an enzyme that
removes a post-translational
modifications from a given
protein (e.g., HDM)
Schematic representation of the processes of writing,
reading, and erasing the histone post-translational
modifications. 23

24. Known writers, readers, and erasers of histone
methylation in Arabidopsis and rice
Liu et al., 2010
24

25. HAT – Histone Acetylase KAT- Lysine Acetyl Transferase HDAC –Histone de- acetylase
HMT- Histone methyl transferase KMT- Lysine methyl transferase HDM-Histone de –
methyl transferase, KDM – Lysine de-methyl transferase PRMT- Protein Arginine methyl
transferase
25

26. • Methylation at H3K9 and H3K27- Inactivation
• Methylation at H3K4 and H3K36- activation
• Methylation of histones can either increase or decrease
transcription of genes, depending on which amino acids in
the histones are methylated, and how many methyl groups
are attached.
• Gene expression is upregulated by acetylation,
phosphorylation and ubiquitination, while it is repressed by
dimethylation of H3K9 (histone H3 Lysine 9) and H3K27
(histone H3 Lysine 27), biotinylation and sumoylation.
• Jeddeloh et al., 1999 described DNA methylation and
histone modifications may share the same feedback loop
model to regulate gene activation or inactivation
Chen et al., 2010
26

27. Chromatin remodeling
Clapier and Carins, 2009
Remodelers are needed to pack the genome, to specialize chromatin regions, and to
provide regulated DNA accessibility in packaged region
Diagrammatic representation of different outcomes of chromatin remodeling
27

28. Techniques for Studying Epigenetic
Modifications
DNA methylation
• Bisulfite sequencing
• Methylation SensitiveAmplification Polymorphism(MSAP)
Histone Modification
• Chromatin immunoprecipitation (ChIP)
• DNAadenosine methylation identification (DamID)
RNAi
• Deep sequencing
28

29. Application of Epigenetics in Crop
Improvement
Differences in DNA methylation can be used as markers for QTL mapping, allowing the
phenotypic variation to be mapped to genomic regions with altered methylation (Cortijo et
al., 2014; Kooke et al., 2015).
Agarwal et al., 2010
Epialleles could be especially valuable in clonal populations for
varietal determination (Rodríguez López and Wilkinson, 2015)
A recent study in Arabidopsis showed histone demethylation of H3K4me2 mediated by
lysine-specific demethylase 1-like3 (LDL3) during root callus formation is important in
acquiring shoot regeneration competency (Ishihara et al., 2019).
Epialleles could serve as markers for use in crop breeding as a
complement to sequence-based markers, e.g. epigenomics assisted
breeding and epigenomic prediction (Pandey et al., 2016).
29

30. Epigenomic data and markers should be viewed as
complementary to sequence-based markers and a combination of
the two could potentially enhance predictive modeling or used in
fine-mapping of traits in regions where genetic variation is scarce
Agarwal et al., 2010
Epigenomic data can also be used to build models for phenotype
prediction.
Methylated DNA immunoprecipitation (MeDIP) chip data from Arabidopsis epiRILs
was used to build predictive models of plant height (Hu et al., 2015).
Fruit Ripening, Seed Development, and Germination
A correlation between DNA hypomethylation and the smaller size of the fruit has also
been reported (Daccord et al., 2017).
Anthocyanin content in apple fruits has been negatively correlated with DNA
methylation level at the promoter of MYB10 gene (Telias et al., 2011; El-Sharkawy
et al., 2015).
30

31. Construction of epiRILs for quantitative epigenetics.
Gahlaut et al., 2020
Scheme for construction of epiRILs with
stable inheritance by crossing of two parents
Hypomethylated Population
31

32. Genome wide Association analysis
Gahult et al., 2020
Schematic representation of epigenome-wide association mapping (EWAS) involving three
major steps: (A) epigenotyping to explore different epialleles, (B) precise phenotyping of
diverse germplasm, (C) Statistical analysis
32

33. Agarwal et al., 2010
Chemical inhibitors of DNA methylation, such as 5-azacytidine and zebularine
Schematic representation of epigenetic in Crop
improvement .
33

34. Abiotic Stress
Schematic representation of regulatory network governing epigenetic modifications in
response to abiotic stresses and during development in plants
Akhter et al., 2021
34

35. Epigenetics is used in developing Salt tolerance
Pharmacological inhibition of Cassava HDACs using suberoylanilide hydroxamic
acid (SAHA) also conferred salt tolerance (Patanun et al., 2017).
Enhances Heat tolerance in plants
Histone acetyltransferases (HATs) carried out acetylation of OsHAG702,
OsHAG704, OsHAC701. OsHAC704 loci in turn promoted heat stress tolerance in
rice (Liu et al., 2021)
Epigenetics in Vernalization
FLC is epigenetically silenced during the prolonged exposure to winter cold
(vernalization) which enables floral activation in the spring (Menon et al., 2021)
Epigenetics Memory will alter the transcript Splicing
mechanism
The exposure of Arabidopsis plants to a non-lethal heat stress results in de-repression
of splicing after a second heat stress, while plants that did not experience such heat
stress on first exposure.
35

36. Case Study
• HOS15 is a WD40-repeat protein that functions as a substrate receptor for the
CULLIN4 (CUL4)-based ubiquitin E3 ligase complex
• HOS15 mediates the proteasome-dependent degradation of histone deacetylase 2 C
(HD2C) under cold stress
• PWR contains two SANT domains
Abbrevations-HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 15
(HOS15), histone deacetylase 2 C (HD2C), POWERDRESS (PWR)
2021
36

37. Freezing phenotype of pwr-3, hos15-2, and pwr-3/hos15-2 double mutant plants. Two-
week-old plants of the indicated genotypes were exposed to −7°C and −8°C freezing
stress, followed by recovery at 23°C for 4 days.
37

38. Expression of COR genes is reduced in pwr, hos15-2, and pwr/hos15-2 double mutant plants.
The transcript levels of COR genes in Col-0, pwr, hos15-2, and pwr hos15-2 double mutants
were analyzed using qRT-PCR under cold stress. UBQ10 was used to normalize the
expression of COR genes. 38

39. Under normal conditions, the PWR-HOS15 complex negatively regulates the expression
of COR genes by interacting with HD2C and reducing histone acetylation. In response to
cold stress, proteasome dependent degradation of HD2C is mediated by the PWR-HOS15
complex, resulting in increased H3 acetylation on the chromatin in COR genes. Thus, the
structure of chromatin changes from closed to open
39

40. Developing Disease resistance
Zhi and Chang et al., 202140

41. Biotic Stress tolerance
Disease resistance
Arabidopsis locally primed to an avirulent strain of
Pseudomonas syringae or administion of β-aminobutyric
acid resulted in progenies with resistance to
Hyaloperonospora arabidopsidis (Slaughter et al., 2012).
Pest resistance
Worrall et al., 2012 demonstrated that priming by
preimbibition of seeds with 3.0 mM jasmonic acid (JA) in
tomato (Solanum lycopersicum cv. Carousel), which
conferred resistance against tobacco hornworms (Manduca
sexta), green peach aphids (Myzus persicae) and spider mites
(Tetranychus urticae). 41

42. Case study
Variety –GZ1
Priming agent- 20mM CaCl2
Infestation- 10 days old wingless
parthenogenetic female adult aphid
placed on the adaxial surface of the
second unfolding leaf in each plant
• Ca2+ signal is involved in the
regulation of callose synthesis.
• callose closure of the sieve pores and
callose coagulation on the sieve
plates serve as a physical barrier to
prevent insects from consuming the
phloem sap.
• β-1,3-glucan callose is synthesized by
glucan synthase-like (GSL) enzymes.
42

43. Populations of wheat aphids after
10 days of feeding on wheat plants
29.02%
Percentage of total time spent in the pathway. C-pathway
phase; F-derailed stylet mechanics; G-xylem ingestion;
E1-phloem salivation; E2-phloem ingestion. 43

44. Callose deposition in wheat leaves in response to wheat aphid. (a) 0 h, (b) 24 h, (c) 48 h
and (d) 72 h after leaves of control plants (seeds pretreated with water) were fed on by ten
wingless agamic adult aphids. (e) 0 h, (f) 24 h, (j) 48 h and (h) 72 h after the leaves of
plants grown from seeds pretreated with 20 mM calcium chloride (CaCl2) were fed on by
wheat aphids. 44

45. Expression levels of TaGSL8, TaGSL10, TaGSL19, TaGSL22 and TaGSL23 peaked at 48 h
after exposure to aphids, whereas those of TaGSL2 and TaGSL12 did not peak until 72 h after
aphid exposure
Seven TaGSL genes-TaGSL2, TaGSL8, TaGSL10, TaGSL12, TaGSL19, TaGSL22 and
TaGSL23
Relative expression of callose synthase genes (TaGSL) in wheat leaves fed on by wheat aphids.
qRT–PCR was used to detect the level of expression of seven callose synthase genes at 0, 24,
48 and 72 h after plants were exposed to ten wingless agamic adult aphids
45

46. (a) Determination of total calcium
(Ca2+) concentration in wheat
leaves in response to wheat aphid
attack. The test was conducted 20
days after planting (three-leaf
stage).
(b) Relative expression of TaCaM
genes in leaves exposed to aphids.
Conclusion- Seed pretreatment with CaCl2 primes the plant response against wheat aphid
attack, leading to modulation of callose deposition in the phloem.
46

47. Case study
47

48. Methodology of Drought Priming Stress
Both the drought priming and the drought stress were initiated at 10 days after anthesis. In total, T0C, no
former-generation priming + no offspring drought stress; T0D, no former-generation priming + offspring
drought stress; T1C, one-generation priming + no offspring drought stress; T1D, one-generation priming +
offspring drought stress; T2C, two-generation priming + no offspring drought stress; T2D, two-generation
priming + offspring drought stress; T3C, three-generation priming + no offspring drought stress; T3D,
three-generation priming + offspring drought stress.
48

49. Effects of parental drought-priming on
water potential (Ψw), osmotic potential
(Ψs) and relative water content (LRWC)
of flag leaves
Photosynthesis and Chlorophyll fluorescence
• T1D, T2D, T3D plants showed significantly
higher net photosynthetic rate, Stomatal
conductance, Transpiration rate, chlorophyll
fluorescence (qP) as compared with T0D.
• The maximum quantum efficiency of
photosystem II (Fv/Fm), CO2 concentration,
non-photochemical quenching of
chlorophyll fluorescence (NPQ) and the
actual photochemical efficiency (8PSII)
were also decreased by drought
significantly.
Effects of parental drought-priming on MDA
content, O2
-• release rate, and H2O2 content in
flag leaves
49

50. Effects of parental drought-priming on contents of
proline and glycine betaine in flag leaves
Effects of parental drought-priming on function of antioxidant system in flag leaves of
offspring plants under drought stress during grain filling in wheat
50

51. Mechanisms of parental drought-priming enhances tolerance to post-anthesis drought in
offspring of wheat. Red, green and black indicate being up-regulated, down-regulated and
not affected by the priming and stress
Conclusion-The results indicated improved drought tolerance of offspring plants by
priming parental plants
Effects of parental drought-priming on grain yield and yield components
51

52. Conclusion
52
Epigenetic changes in plants contribute to phenotypic variation at
multiple levels, from regulation of gene expression to development,
response to stress, etc.
Knowledge of epigenetics and epigenomics can help explaining the
mechanisms by which the environment affects plant phenotypes.
High-throughput profiling of epigenomes at the cellular level has the
potential to reveal genetically unexplained phenotypic variance.
However, more studies are to be carried out as epigenomics/epigenetics
literature is filled with contradicting reports.
A better understanding of the epigenome offers opportunities for better
practices for crop improvement.

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Decode it?
56