Epigenetics mediated gene regulation in plants describes how epigenetic mechanisms like DNA methylation and histone modification can regulate gene expression without changing the underlying DNA sequence. The document discusses various epigenetic mechanisms in plants including DNA methylation, histone modifications, and RNA interference. It also outlines applications of epigenetics like improving plant stress tolerance and yield as well as evolutionary studies.

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Epigenetics mediated gene
regulation in plants
Centre for Plant Biotechnology and Molecular
Biology
Kerala Agricultural University

3.  Epigenetics – meaning and definition
 Mechanisms
 Implications
 Applications
 Conclusions
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4.  Changes in gene expression that occur by a
mechanism other than change to the DNA sequence
 Gene-regulatory information that is not expressed in
DNA sequences but transmitted from one generation
(of cells or organisms) to the next
 Coined by C. H. Waddington in 1942
 Epigenetics means ‘above’ or ‘on top of genetics’
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5. What Does “Epigenetics” Mean?
 Epigenetics describes phenomenon in which
genetically identical cells or organisms express their
genomes differently, causing phenotypic differences
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Genetically
identical cells or
individuals
Different epigenetic
modifications leading to
different expression patterns
Different
phenotypes

6. Molecular epigenetic mechanism
 Development of multicellular organisms occurs due to cells
differentiation by various programs of gene expression
 Cells have own epigenetic signatures like
1. Genotype
2. Developmental history
3. Environmental influences and it is ultimately reflected in the
phenotype of the cells and the organism
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cytosine 5-methylcytosine
DNA can be covalently
modified by cytosine
methylation.
TTCGCCGACTAA
Methyl-
cytosine

8. .... DNA Methylation
 DNA methylation in mammals mainly occurs on the
cytosine nucleotide in a CpG site
 In plants the cytosine can be methylated at CpG, CpHpG,
and CpHpH sites, where H represents any nucleotide
 DRM2, MET1, and CMT3: DNA methylases in plants
 CMT3 protein is unique to the plant kingdom
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9. .... DNA Methylation
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 Role in prokaryotes, as defence mechanism
•Escape from the restriction enzymes
•Protection from bacteriophages
 In eukaryotes, it controls the mechanism of transposable
elements in the genome

10. Classes of methyl transferase
 De novo class: Enzymes that create new methylation
mark on DNA
Eg. DNMT3a and DNMT3b
 Maintainance class: Recognizes the methylation
marks on the parental strand of DNA and transfers new
methylation to the daughter strands after DNA
replication
Eg. DNMT1
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(http://atlasgeneticsoncology.org/Educ/HeterochromEng.html)

13. 2. Histone modification
 Histone: Several small, basic proteins most commonly found in
association with the DNA in the chromatin of eukaryotes
 Packaging and ordering the DNA into structural unit called
nucleosomes
 Histone modifications also known as epigenetic modifiers
 Biological functions:
• In chromatin organization
• Gene expression
• DNA repair
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14.  Writers: enzymes that add a mark
 Erasers: enzymes that remove a mark
 Readers: proteins that bind to and “interpret” the mark
14(Tarakhovsky,. Nature Immunology, 2010)
Writers' introduce histone marks (circles), 'erasers' take them out and 'readers'
can recognize a particular form of histone modification.

15. Histone modifications occur primarily on histone tails
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(Zeng et al., 2007)

16. Histone Modifications and Modifiers
Residue on the
histone tail
Type of modification Modifying enzyme
Lysine Acetylation HAT, KAT
Deacetylation HDAC
Lysine Methylation, HMT, KMT
Demethylation HDM, KDM
Lysine Unbiquitylation Ub ligase
Deubiquitylation Ub protease
Serine/
Threonine
Phosphorylation Kinase
Dephosphorylation Phosphatase
Arginine Methylation PRMT
Demethylation Deiminase
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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

17.  Do not bind to DNA themselves
Can be recruited by:
 Histone modifications (through chromodomains,
bromodomains, etc.)
 Transcription factors
 DNA damage
 Act as transcriptional co-regulators
 Enhance activities of transcriptional repressors or activators
 Co-repressor: eg. HDACs
 Co-activator: eg. HATs
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18. General roles of histone modifications
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(Moggs and Orphanides, Toxicological Sciences, 2004)
During DNA DamageIn Gene Regulation
(Wade , Hum. Mol. Genet. 2001)
HDAC

19.  Also called post transcriptional gene silencing (PTGS)
 Is a biological process in which RNA molecules inhibit
gene action
 Andrew Fire and Craig C. Mello shared the Nobel Prize
in Physiology or Medicine - 2006
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(Long et al., 2011, Genetics, 189, 1093–1102)

21. Methods for studying epigenetic modifications
• DNA methylation– Bisulfite sequencing
• Methylation Sensitive Amplification Polymorphism
(MSAP)
• Histone modification
• Chromatin immunoprecipitation (ChIP)
• DNA adenosine methylation identification (DamID)
• RNAi – Deep sequencing
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22. Bisulfite treatment differentiates cytosine and
methylcytosine
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Bisulfite
treatment
TTCGCCGACTAA
No
treatment
TTCGCCGACTAA
TTCGCCGAuTAA
TTCGCCGACTAA TTCGCCGATTAA
Methyl-
cytosine
After bisulfite treatment,
unmethylated Cs are
read as T and so differ in
the treated and
untreated samples.
By contrast, methyl-C is
read as C and is the
same as the reference
sequence.

23. What is Stress?
 Any external factor that exerts disadvantageous
influence on organisms
Strategies to minimize stress influence:
 Tolerance, Resistance, Avoidance or Escape
 Physiological alteration in metabolic pathways
 Modification in gene expression pattern
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Plant materials and genotyping
Drought
tolerant
DK151
Drought
susceptible
IR64
1. Case study

25. Drought stress
treatment
Irrigated control Recovery
Irrigation was held at
55 d after transplanting
The stress was
maintained until leaves
of the treated plants
rolled completely and
their leaf relative water
content reached 70–
75%.
Field was irrigated at
weekly intervals
Stressed plants were
recovered by
rewatering
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Tissue sample for DNA isolation: Root and Leaf tissue collected from the drought-
stressed, well-watered, and recovered plants

26. Analysis of DNA Methylation-Sensitive Amplified
Polymorphisms (MSAPS)
 Double enzyme combinations:
• EcoRI/MspI and EcoRI/ HpaII, used to digest the DNA samples
 PCR is used to selectively amplify the DNA fragments
 A set of 26 randomly selected differentially amplified
fragments were isolated, re-amplified, and purified cloned
with TA-cloning vector
 The sequences obtained were analysed by NCBI BLAST
(http://blast.ncbi.nlm.nih.gov/Blast.cgi)
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27. type I Represents the band presence for both enzyme
combinations
type II Band presence only for EcoRI/HpaII
type III Band presence for EcoRI/MspI
type IV Represents the band absence for both enzyme
combinations
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28. Analysis of MSAP
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DNA methylation changes in leaves and roots of DK 151 and IR 64

29. Conclusion of experiment
 Drought - induced genome-wide changes in DNA
methylation/ demethylation.
 Large difference - DNA methylation/ demethylation
sites between drought tolerance DK151 and drought
sensitive IR64
 Resistant – Highly methylated, retained methylation
on stress
 Susceptible – Less methylated, lesser retention of
methylation
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30. MORPHOLOGICAL AND MOLECULAR
ANALYSIS OF GENETIC STABILITY IN
MICROPROPAGATED BANANA
(MUSSA SPP.) VAR. NENDAN
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2. Case study

31. m- Marker DNA (100 bp), 1- Mother plant, 2- SC 3rd, 3- SC 6th, 4- SC 8th, 5- SC10th,
6- SC 12th, 7- SC 14th, 8- SC16th 31

32. Subculture no. Hemi methylation
(%)
Internal full-
methylation
(%)
Total methylation
(%)
Mp 3.00 6.30 9.3
Sc3 3.20 9.20 12.4
Sc6 3.20 6.15 9.15
Sc8 3.42 9.36 12.78
Sc10 10.68 14.84 25.52
Sc12 16.89 26.79 43.68
Sc14 18.12 28.33 46.45
Sc16 18.27 30.43 48.7
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33.  Initial culture shows less methylation
 High variation occurs after 10th subculture and
onwards >48%
 Advantage of MSAP is direct identification of
methylated sequence in the genome
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A. Better understanding on the physiological mechanisms
 Epigenetic variation can causes heritable variation
 DNA methylation majorly involve in plant defence against herbivorous
and pathogens
 Heritable variation in plant growth responses to jasmonic acid and
salicylic acid
(Latzel et al., 2012)

35. B. Improving Plant Stress Tolerance
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 Stress tolerance can be improved by the controlling
transposable elements
 Plant phenotypic variation, improve long-term plant adaptation
to environmental challenges and, thus, increase productivity.
(Mirouze and Paszkowski, 2011)

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C. Evolutionary studies/ epigenetic diversity studies
 Variation of ecologically important plant traits, root
allocation, drought tolerance and nutrient plasticity
 Rapid evolution based on epigenetic variation alone should
thus be possible
(Zhang et.al., 2012)

37. D. Epigenetic mechanisms, yield, and heterosis
 Hybrids are in general, less methylated than their parental
inbreds
 Heterotic hybrids are less methylated than related nonheterotic
hybrids
 Low-yielding inbreds are more methylated
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38. F. Transient expression of transgene
 Single copy transgene into hypomethylated region
 Careful design of transgene construct
 Analysis of transformants at the molecular level
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 Plant genomes have a distinct range of target sites
 Ability to detect trait loci
 To identify additional significant QTL associated with
important agronomic traits
(Long et al., 2011)
E. Epigenetic QTL mapping

39. 39(Long et al., 2011, Genetics 189: 1093–1102)

40. CONCLUSIONS
 Epigenetics involves DNA methylation, histone
modification and RNAi
 DNA methylation effectively down-regulates/ up-
regulates gene activity by addition of a methyl group to
the five-carbon of a cytosine base
 Epigenetic changes can be studied by
• Bisulfite treatment, MSAP
• DNA adenine methylase identification
• Chromatin immunoprecipitation
• Deep sequencing for RNAi
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41. .... CONCLUSIONS
 Various application are:
• Better understanding of physiological mechanisms
• Improvement of plant stress tolerance
• Improvement of yield and heterosis
• Evolutionary and diversity studies
• QTL mapping
 Disadvantage – Transient expression of transgene
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