Epigenetic control of plant stress response

Review Article

Epigenetic Control of Plant Stress Response

Alex Boyko and Igor Kovalchuk*

Department of Biological Sciences, University of Lethbridge, Lethbridge,Alberta, Canada

Living organisms have the clearly defined strat-egies of stress response. These strategies are pre-defined by a genetic make-up of the organismand depend on a complex regulatory network ofmolecular interactions. Although in most cases,the plant response to stress based on the mecha-nisms of tolerance, resistance, and avoidance hasclearly defined metabolic pathways, the abilityto acclimate/adapt after a single generation ex-posure previously observed in several studies(Boyko A et al. [2007]: Nucleic Acids Res35:1714–1725; Boyko and Kovalchuk, unpub-lished data), represents an interesting phenom-enon that cannot be explained by Mendeliangenetics. The latest findings in the field of epige-

netics and the process of a reversible control overgene expression and inheritance lead to believethat organisms, especially plants, may have a flex-ible short-term strategy of the response to stress.Indeed, the organisms that can modify geneexpression reversibly have an advantage in evolu-tionary terms, since they can avoid unnecessaryexcessive rearrangements and population diversifi-cation. This review covers various epigenetic pro-cesses involved in plant stress response. We focuson the mechanisms of DNA methylation andhistone modifications responsible for the protec-tion of somatic cells and inheritance of stressmemories. Environ. Mol. Mutagen. 49:61–72,2008. VVC 2007 Wiley-Liss, Inc.

Key words: stress response; epigenetic and genetic regulation; genome stability; chromatin structure;Arabidopsis; tobacco

INTRODUCTION

Biological organisms are constantly exposed to environ-

mental stimuli named stresses, and they are capable of

establishing mechanisms of protection and adaptation.

Stress has the most significant and mainly negative effect

on organism growth, development, and reproduction [Arn-

holdt-Shmitt, 2004; Madlung and Comai, 2004]. It often

determines the distribution of species, and more impor-

tantly provides a selective evolutionary pressure on a

given population [Doroszuk et al., 2006]. Depending on

their character, stresses can be subdivided into internal

ones such as a production of free radicals during cell me-

tabolism, and external ones, which can be further subdi-

vided into two classes based on their abiotic or biotic na-

ture [Madlung and Comai, 2004].

Three different strategies can be applied to minimize

stress influence. They are tolerance, resistance, and avoid-

ance or ultimately escape. Because of their sedentary life

style, plants are restricted to tolerance, resistance, and

avoidance mechanisms only. Generation of new traits fol-

lowed by selection of the adaptive qualities represents a

long-term surviving strategy. In contrast to animals that

can compensate a slow evolution rate by escape or limited

exposure to stress, plants require efficient short-term strat-

egies based on the manipulation of the existing genetic in-

formation. These strategies include an alteration of plant

homeostasis during the somatic growth [Shinozaki et al.,

2003; Sung and Amasino, 2004], and heritable or transge-

nerational modifications of gene expression. [Whitelaw

and Whitelaw, 2006]. The latter modifications can occur

without changing original DNA sequence and are known

as epigenetic. They can be achieved on several interde-

pendent levels including reversible methylation of DNA

sequence, numerous histone modifications, and chromatin

remodeling [Wagner, 2003; Vanyushin, 2006]. All of

them may be regulated by a number of physiological and

*Correspondence to: Igor Kovalchuk, Department of Biological Sciences,

University of Lethbridge, University Drive 4401, Lethbridge, Alberta,

Canada T1K 3M4. E-mail: [email protected]

Grant sponsors: NSERC, HFSP, Alberta Ingenuity.

Received 31 May 2007; provisionally accepted 28 August 2007; and in

final form 7 September 2007

DOI 10.1002/em.20347

Published online 18 October 2007 in Wiley InterScience (www.interscience.

wiley.com).

VVC 2007Wiley-Liss, Inc.

Environmental andMolecular Mutagenesis 49:61^72 (2008)

developmental stimuli and ultimately stress. The spectrum

of external and internal influences experienced during the

life span of an organism may lead to generation of spe-

cific changes in gene expression that could be epigeneti-

cally (without changing DNA sequence) fixed and passed

into progeny forming epigenetic memories. Indeed, the

maintenance of changes in gene expression in prokaryotic

and eukaryotic organisms over several cell generations

was well documented [Bender, 2004]. In fact, the notions

that transgenerational changes in DNA methylation are

more frequently observed in plants than in animals

[Takeda and Paszkowski, 2006] is consistent with the sed-

entary life style of plants. In contrast to animals, plants

establish their germline late during the development,

therefore allowing the transmission of epigenetic memo-

ries accumulated during their life to the following genera-

tions. Factors and mechanisms involved in generation of

epigenetically-mediated changes in gene expression under

physiological and stress conditions, inheritance of epige-

netic memories and their impact on plant genome evolu-

tion are the focus of our review.

EPIGENETIC MODIFICATIONS REPRESENT COMPLEX ANDFLEXIBLE SYSTEM THATCONTROLS GENE EXPRESSION INPLANTS

DNAMethylation Is the Primary Epigenetic Mark

DNA methylation plays a crucial role in the regulation

of gene expression, in the activity of transposable ele-

ments, in the defense against foreign DNA, and even in

the inheritance of specific gene expression patterns [Ras-

soulzadegan et al., 2006]. The major difference in the

methylation patterns between plants and animals is that in

animals the percent of modified cytosines is substantially

higher, and CpNpG and asymmetrical cytosine methyla-

tion is absent [Finnegan et al., 1998b; Bender, 2004]. The

symmetrical CpG or CpNpG methylation is inherited dur-

ing the DNA replication in the form of hemimethylated

sequences. Hence, it provides the memory of methylation

imprint present in the parental DNA and also guides the

activity of methyltransferases [Bender, 2004]. On the con-

trary, the asymmetrical cytosine methylation must be rees-

tablished de novo after each replication cycle, since there

is no complementary methylated sequence available to

guide remethylation [Ramsahoye et al., 2000; Gowher

and Jeltsch, 2001]. Experimental evidences suggest the

existence of three distinct classes of enzymes responsible

for cytosine methylation.

The first class is represented by a plant homologue of

mammalian Dnmt1 methyltransferase, METHYLTRANS-

FERASE1 (MET1). The plants defective for MET1 show

the lack of wide spread CpG methylation [Lindroth et al.,

2001]. The second class of methyltransferases, CHRO-

MOMETHYLASE3, is unique to plants (Table I). A loss-

of-function cmt3 mutant is characterized by a genome

wide loss of CpNpG methylation, especially at centro-

meric repeats and transposons [Lindroth et al., 2001;

Tompa et al., 2002]. Recent studies by Kato et al. [2003]

on the activation of a normally silenced CACTA transpo-

son in the met1 and cmt3 single and double mutants indi-

cated redundancy in function of CMT3 with MET1 in

CpG and CpNpG methylation. The last known class, the

DOMAIN REARRANGED METHYLTRANSFERASES,

is composed of DRM1 and DRM, and shows homology to

mammalian Dnmt3 methyltransferase [Cao et al., 2000].

DRM1 and DRM2 are mainly directed on de novo methyl-

ation of asymmetric sites [Cao and Jacobsen, 2002b], and

they show some functional redundancy with CMT3 in

methylating CpNpG sites [Cao et al., 2003] (Table I).

While the presence of DNA methylating enzymes is

well proven, the existence of direct DNA demethylation

mechanisms remains controversial. The passive loss of

DNA methylation may occur due to the inhibition of de

novo methylation or inability to maintain the parental

imprint after DNA replication shown in met1 mutants

[Kankel et al., 2003]. Alternatively, active demethylation

may occur via the glycosylase activity by removing the

methylcytosines from DNA [Zhu et al., 2000; Zhu et al.,

2007; Morales-Ruiz et al., 2006; Agius et al., 2006]. It

may play a critical role in preventing the formation of sta-

ble hypermethylated epialleles in plant genome [Pen-

terman et al., 2007]. Indeed, the demethylation activity of

Arabidopsis DNA glycosylase DEMETER (DME) regu-

lates the gametophyte-specific activation of flowering time

(FWA) gene expression [Kinoshita et al., 2004]. It also

reverses the endosperm imprinting of the maternal copies

of MEDEA allele [Choi et al., 2002]. Similarly, Gong

et al. [2002] isolated REPRESSOR OF SILENCING 1

(ROS1), a DNA glycosylase/lyase, functioning on methyl-

ated rather than unmethylated DNA substrates (Table I).

Until recently, the link between stress exposure and

sequence-specific changes in DNA methylation was hypo-

thetical. It was demonstrated that prolong exposure to

cold triggers a stable transcriptional silencing of FLCleading to flowering inhibition [Henderson and Dean,

2004]. Moreover, flowering time directly correlates with

the level of DNA methylation in MET1 antisense knock-

outs [Finnegan et al., 1998a]. The met1 mutants do not

require cold treatment to initiate the flowering, which

proves that the developmental switch was epigenetically

controlled.

Exposure to cold of root tissues of maize seedlings

resulted in DNA demethylation of the nucleosome core

regions [Steward et al., 2000]. In fact, the DNA replica-

tion was strongly reduced in chilled tissues, thus allowing

speculations that genome hypomethylation was the result

of active rather than passive demethylation. The cold-

induced demethylation of a nucleosome core and relaxa-

tion of chromatin structure could serve as a stress-induced

Environmental and Molecular Mutagenesis. DOI 10.1002/em

62 Boyko and Kovalchuk

Environmental and Molecular Mutagenesis. DOI 10.1002/emTABLEI.PlantFactors

Invo

lved

inEpigenetic

Regulation

s

Nam

eandfunction

Effectonchromatin

Effectofmutationandinvolvem

ent

instress

response

Modification/

transcription

References

DNA

methylation

METHYLTRANSFERASE1

(MET1)/methyltransferase

Methylationofsymmetrical

CpG

sites;postreplicativedenovo

CpG

methylation;notrequired

forestablishingofnew

methylation

imprints

LackofCpG

methylation;passive

loss

ofDNA

methylation.MET1

isrepressed

inresponse

tostress,

leadingto

activationofrepressed

genes

Global/repression

Finnegan

etal.,1996;

Steward,et

al.,2000;

Kankel

etal.,2003;

Wadaet

al.,2004

CHROMOMETHYLASE3

(CMT3)/methyltransferase

CpNpG

methylation;functionally

redundantwithMET1andDRM

inmethylationofCpG

and

asymmetricalsites,respectively;

targetscentromeric

repeatsand

transposons

Loss

ofCpNpG

methylation

Global/repression

Barteeet

al.,2001;

Lindroth

etal.,2001;

Tompaet

al.,2002

DOMAIN

REARRANGED

METHYLTRANSFERASES

(DRM1,DRM2)/methyltransferase

Denovomethylationofasymmetric

sites;

functional

redundancy

with

CMT3in

CpNpG

methylation;possibly

reinforces

preexistingmethylation

Loss

ofdenovoasymmetric

methylationat

non-CpG

sites

Global/repression

Cao

etal.,2000,2003;

Cao

andJacobsen,2002a,b

DEMETER(D

ME)/DNA

glycosylase

Dem

ethylationofpreviouslysilenced

sequences,possibly

intissue-specific

manner

Inabilityto

activateim

printedgenes;

inheritance

ofmutantmaternal

allele

resultsin

seedsabortion

Local,promoters/

activation

Kinoshitaet

al.,2004;

Morales-Ruiz

etal.,2006;

Penterm

anet

al.,2007

REPRESSOROFSILENCIN

G1

(ROS1)/DNA

glycosylase/lyase

Dem

ethylationactivityonmethylated

butnotdem

ethylatedDNA

substrates

Hypermethylationandtranscriptional

silencingofspecificgenes;enhanced

sensitivityto

genotoxic

agents

Local,promoters/

activation

Gonget

al.,2002;

Agiuset

al.,2006;

Penterm

anet

al.,2007;

Zhuet

al.,2007

Histonemodifications

SUVH1/histonemethyltransferase

MethylationofhistoneH3K9;has

a

minorim

pactonheterochromatin

reinforcem

ent

Loss

ofH3K9methylation

Global/repression

Naumannet

al.,2005

SUVH2/histonemethyltransferase

Methylationofhistones

H3K9,H3K27,

H4K20;heterochromatin

reinforcem

ent;

SUVH2mediatedgenesilencingdepends

onMET1andDDM1

Loss

ofH3K9;H3K27andH4K20

methylation;reductionofDNA

methylationin

heterochromatin

Global/repression

Naumannet

al.,2005

SUVH4(K

RYPTONITE)

(SUVH4/KYP)/histone

methyltransferase

MethylationofhistoneH3K9;activity

isdependentonCpG

DNA

methylation

ingiven

loci;has

aminorim

pacton

heterochromatin

reinforcem

ent

Loss

ofH3K9methylation;negative

effect

onCpNpG

methylation

Global/repression

Jacksonet

al.,2002;

Johnsonet

al.,2002;

Jasencakovaet

al.,

2003;Naumannet

al.,

2005

HISTONEDEACETYLASE6

(HDA6)/histonedeacetylase

ReinforcingCpNpG

methylationinduced

byRNA-directedtranscriptional

silencing

Reactivationofpreviouslysilenced

transgenes

Local/repression

Aufsatzet

al.,2002

Chromatin

remodeling

METHYL-CpG-BIN

DIN

GDOMAIN

PROTEIN

S

(AtM

BDs)/5-m

ethylcytosin

bindingproteins

BindmethylatedCpG

andchangelocal

chromatin

structure

via

modification

ofcore

histoneproteins;promote

heterochromatin

form

ationandrepeat

silencing

Latefloweringandreducedfertility

(mbd

11);shootbranchingandearly

floweringdueto

transcriptional

repression

ofFLC(m

bd9)

Local/repression,

activation

Ben-Porath

andCedar,

2001;Zem

achandGrafi,

2007andreferenceswithin

(continued)

Epigenetic Control of Plant Stress Response 63


TABLEI.Continued

Nam

eandfunction

Effectonchromatin

Effectofmutationandinvolvem

ent

instress

response

Modification/

transcription

References

LIK

EHETEROCHROMATIN

PROTEIN

1(LHP1)/

Chromodomainprotein.Bindsto

histone

H3K9;chromatin

condensationand

coating

Inabilityto

repress

expressionof

euchromatic

genes

associated

with

specificdevelopmentalstage

Global/repression

Gaudin

etal.,2001;

Mylneet

al.,2006

DECREASED

DNA

METHYLATIO

N

(DDM1)/SWI2/SNF2DNA

helicase

ControlofDNA

methylation,possibly

throughthebindingmethyl-CpG

binding

domainproteinsandaffectingtheir

subnuclearlocalization

Decondensationofcentromeric

heterochromatin,redistributionof

remainingDNA

methylation,changes

inhistonemethylation.Silencingof

Rgenes

andretrotransposons;DNA

dam

ageresponse

Global/repression

Singer

etal.,2001;

Johnsonet

al.,2002;

Soppeet

al.,2002;Stokes,

2002;Zem

achet

al.,2005;

Shaked,et

al.,2006

DRD1/SWI/SNF-likeprotein

Directingnon-CpG

DNA

methylation

inresponse

toRNA

signal;interacts

withDNA

methyltransferases

andDNA

glycosylases;targetspromoterandLTRs

ineuchromatin

Nosignificantdefects

inCpG

methylation;loss

ofnon-CpG

methylationonpreviouslysilenced

promoters

andtransposons;down

regulationofROS1

andDME

Local,promoters/

repression,activation

Kannoet

al.,2004,2005a;

Matzkeet

al.,2006

RNA

POLYMERASEIV

b(polIV

b)

(subunitsNRPD2a

andNRPD1b)/RNA

polymerase

Guides

cytosinemethylationusing

smRNA

signals;NRPD1bpossibly

recruitsDNA

methyltransferases

to

asymmetricsites;workstogether

withDRD1

Donotshow

significantdefects

inCpG

methylationbutexhibitloss

ofnon-CpG

methylationon

previouslysilencedeuchromatic

promoters

andtransposons

Local,promoters/

repression,possibly

activation

Kannoet

al.,2005b;

Matzkeet

al.,2006

MAIN

TENANCEOFMETHYLATIO

N

1(M

OM1)/similar

toSWI2/SNF2

Regulationoftranscriptionofsilent

heterochromatic

regions;transgene

silencing;preventingtranscriptionof

180-bpsatelliterepeatsbutnotof

transposons

Release

oftranscriptional

gene

silencingandof5Srepeatsilencing;

noeffect

onheterochromatin

organization,andDNA

methylation.

Global/repression

Amedeo

etal.2000;

Vaillantet

al.,2006


transcriptional switch for many stress-regulated genes

[Steward et al., 2002].

Several other papers suggest that changes in DNA meth-

ylation are required for stress protection. Dyachenko et al.

[2006] demonstrated a two-fold increase in CpNpG methyl-

ation level in nuclear genome of M. crystallinum plants

exposed to high salinity conditions. The increase in methyl-

ation was associated with switching from C3-photosynthe-

sis to CAM metabolism. Similarly, Sha et al. [2005]

reported that the age-dependent increase in methylation

confers resistance to blight pathogen X. oryzae in rice.

Methylation contributes greatly to the plants ability to

respond to stress. Hypomethylation found in met1 results

in specific expression of 31 genes, many of which being

related to stress response [Wada et al., 2004]. Demethyla-

tion of one of the genes, NtAlix1, also occurs under the

viral infection, showing that the induction of this gene

under natural stress requires sequence demethylation.

Steward et al. [2000] also showed that transcriptional acti-

vation of ZmMI1 gene in maize seedlings was dependent

on the cold stress-mediated sequence demethylation.

ZmMI1 gene contains a retrotransposon-like sequence,

and its activation is mirrored by the cold-induced root-

specific demethylation of Ac/Ds transposon regions fol-

lowed by their activation [Steward et al., 2000].

Activation of transposons in response to stress is a com-

mon phenomenon. Low temperature treatment decreases

methylation and increases the excision rate of Tam3 trans-

poson by binding its transposase to GCHCG (H 5 not G)

sites immediately after DNA replication, thus preventing

de novo sequence methylation [Hashida et al., 2003,2006].

Stress-mediated induction was shown for Tos17 (rice) [Hir-

ochika et al., 1996], Tto1 (tobacco) [Takeda et al., 1999],

Tnt1 (tobacco) [Beguiristain et al., 2001], and BARE-1(barley) [Kalendar et al., 2000] retrotransposons. An

intriguing hypothesis that stress-activated transposons could

positively contribute to genome adaptation to growth in

colder climates was supported by the detection of mPingtransposition into a rice homologue of flowering time gene

CONSTANS in stressed cultivars [Jiang et al., 2003].

Indeed, Song et al. [1997] suggested that a number of

transposable elements and their derivatives present within

the resistance gene (R-gene) loci played a significant role

in a rapid diversification of this gene family. These publi-

cations support the long-standing hypothesis proposed by

Barbara McClintock. She suggested that all kinds of

stresses could potentially reshape a plant genome via trans-

poson activation [McClintock, 1984].

HistoneModifications: ReinforcingDNA-Methylation Imprints

Since gene transcription occurs within the nucleosome

consisting of DNA wrapped around an octamer histone

core, modifications of histone proteins via (de)methyla-

tion, and (de)acetylation also regulate gene expression. It

was demonstrated that the euchromatin state is dependent

on hyperacetylation of histones H3 and H4 along with

methylation of H3 in lysine K4 position [Bender, 2004].

In contrast, the formation of heterochromatin structure

requires underacetylation of H3 and H4, methylation of

K9, and demethylation of K4 residues in H3 [Bender,

2004] (Fig. 1). It was suggested, however, that different

methylation states of histone H3 might also result from

the deposition of two independent H3 variants differing in

sequence and posttranslational modifications, particularly

in the enrichment with methylated K9 and K27 [Zil-

berman and Henikoff, 2005].

There are several experimental evidences suggesting

the interdependence of DNA and histone methylation. It

was shown that the loss of CpG methylation in met1 mu-

tant results in the loss of H3K9 methylation [Soppe et al.,

2002; Tariq et al., 2003]. In contrast, the loss of H3K9

methylation in KRYPTONITE [KYP] histone methyl-

transferase kyp mutant does not affect the CpG methyla-

tion [Jasencakova et al., 2003], suggesting that H3K9

methylation acts downstream of CpG methylation and

reinforces heterochromatin. On the contrary, DNA meth-

ylation at CpNpG sites appears to be partially dependent

on the activity of KYP [Jackson et al., 2002].

Histone methylation can recruit other proteins such as

HETEROCHROMATIN PROTEIN 1 (HP1), that binds to

methylated H3K9 [Lachner et al., 2001] and helps propa-

gate heterochromatin to the adjacent regions on chromo-

some [Grewal and Moazed, 2003]. In parallel, Arabidop-

sis homologue of HP1, LIKE HETEROCHROMATIN

PROTEIN 1 (LHP1) is involved in regulating flowering

time in response to the environmental stimuli [Gaudin

et al., 2001; Mylne et al., 2006].

Methylated DNA serves as a substrate for binding nu-

clear proteins named methyl-CpG-binding domain pro-

teins or MBDs. These proteins bind to 5-methylcytosins,

recruit the enzymes modifying core histone proteins and

change local chromatin structure [Ben-Porath and Cedar,

2001] (Table I; Fig. 1). It must be noted however, that

not all of MBDs are able to bind methylated CpG in

vitro, and therefore can be possibly involved in control of

chromatin structure through other mechanisms [reviewed

in [Zemach and Grafi, 2007].

Overall, histone modifications represent another stress

responsive element in the system of epigenetic control

over gene expression. Chua et al. [2003] established a

link between the light-dependent transcriptional induction

of a pea plastocyanin gene and histone acetylation. It was

suggested that binding an enhancer to nuclear matrix acti-

vates the transcription through alteration of the local chro-

matin structure, thus increasing acetylation of the pro-

moter and 50 coding region [Chua et al., 2003]. Tsuji

et al. [2006] demonstrated that transcriptional activation

of submergence-inducible genes in rice, ADH1 and PDC1



was reversibly mediated through histone H3K4 methyla-

tion and H3 acetylation.

Chromatin Remodeling Proteins: Shaping andMaintaining Chromatin Structure

Control of gene expression through DNA methylation

and histone modifications is complemented by the activity

of chromatin remodeling proteins. Among them are the

members of SWI2/SNF2 DNA helicase family that are of

crucial importance in DNA repair, recombination, gene

expression, and replication [Havas et al., 2001]. SWI2/

SNF2 family proteins alter the structure of chromatin

through the disruption of DNA-histone interaction [Geiman

and Robertson, 2002].

Among the first described members of this family is

DECREASED DNA METHYLATION 1 (DDM1) protein

that controls methylation directly and indirectly by chang-

ing histone methylation [Johnson et al., 2002] (Table I).

Recently, Zemach et al. [2005] demonstrated that

AtMBDs bind DDM1. They also reported a disrupted

localization of AtMBDs to chromocenters in the ddm1

mutant. It suggests that DDM1 may facilitate localization

of MBDs at specific nuclear domains (Fig. 1). The ddm1

mutant shows 70% reduction of global genome methyla-

tion [Jeddeloh et al., 1999], activation of transposable

elements [Miura et al., 2001; Singer et al., 2001], and

phenotypical instability [Kakutani et al., 1996]. The

ddm1-induced hypomethylation also results in transcrip-

tional activation of a previously silent disease resistance


Fig. 1. Possible mechanisms of response to stress via various chromatin

modifications. Epigenetic modifications allow the reversible generation

of new heritable states of gene expression without modifying coding

DNA sequence, and can be introduced on three distinct levels that

include DNA methylation, histone modifications, and chromatin remodel-

ing. Transition of chromatin from eu- to heterochromatin can be initiated

by a number of external and internal stimuli that direct methylation of

specific DNA sequences through the action of DNA methyltransferases

MET1, CMT3, and DRM guided by smRNAs and DRD1/polIVb com-

plex. The reversible character of these changes is mediated either by a

passive loss of DNA methylation during DNA synthesis or by active

demethylation of previously methylated sequences by DNA glycosylases

DME and ROS1. Methylation of DNA facilitates the reversible modifica-

tions of histone proteins by SUVH class histone methyltransferases and

HDA6 histone deacetylase leading to the reinforcement of heterochroma-

tin structure. In parallel, the methylated cytosines serve as a substrate for

a DDM1-controled binding of MBD proteins. This further promotes the

heterochromatin formation and gene silencing. Similarly, LHP1 protein

binds to histone H3K9 and promotes the chromatin condensation and

coating. Other proteins, like MOM1, may control gene silencing in a

DNA methylation-independent manner by unknown mechanisms. Epige-

netic modification of DNA sequence represents a flexible and sensitive

mechanism involved in plant adaptation to new environments and ge-

nome evolution.


gene array [Stokes, 2002], and activates a number of ret-

rotransposons [Kato et al., 2004].

DDM1 deficient plants are more sensitive to UV-C and

g-radiation than wild type and met1 mutant plants

[Shaked et al., 2006]. This indicates that the increased

radiation sensitivity can be mediated by disrupting chro-

matin remodeling functions rather than cytosine methyla-

tion. It should be noted, however, that Shaked et al.

[2006] did not analyze the double met1cmt3 mutant. If

they considered the functional redundancy of MET1 and

CMT3 [Kato et al., 2003], it could have affected their

conclusions.

Other reports also indicated the link between the chro-

matin maintenance and stress response. Mutants of nuclear

protein BRU1 involved in the maintenance of chromatin

structure were highly sensitive to genotoxic stress, and

were characterized by the increased intrachromosomal ho-

mologous recombination [Takeda et al., 2004]. Similarly,

the expression of another gene, MIM1, involved in the

maintenance of chromosome structure and required for the

efficient homologous recombination, was significantly

induced by DNA-damaging treatments [Hanin et al., 2000].

Another SWI/SNF like protein, DRD1, represents a

novel plant-specific chromatin remodeling protein that is

required for RNA-directed de novo methylation of target

promoters [Kanno et al., 2004]. It is also necessary for the

full loss of induced de novo DNA methylation after a

silencing RNA trigger is withdrawn [Kanno et al., 2005a].

DRD1 interacts with two other factors, NRPD1b and

NRPD2a, that represent subunits of a novel plant-specific

RNA polymerase, pol IVb [Kanno et al., 2005b]. Together,

DRD1 and pol IVb complex act downstream of a small

RNA (smRNA) biogenesis pathway, directing reversible

silencing of euchromatic promoters in response to RNA

signals, possibly, through recruiting of DNA methyltrans-

ferases to homologous DNA sequences [Matzke et al.,

2006] (Table I; Fig. 1). Interestingly, among the putative

DRD1 targets, there are DNA glycosylases, ROS1 and

DME, that are involved in active DNA demethylation

[Morales-Ruiz et al., 2006; Pentermanet al., 2007]. The

downregulation of ROS1 in the drd1 and pol IVb mutants

supports the importance of the DRD1/pol IVb pathway for

the active loss of induced de novo DNA methylation

[Kanno et al., 2005a].

Another mechanism of gene expression control is repre-

sented by the nuclear protein MAINTENANCE OF

METHYLATION (MOM1) that has limited homology to

DDM1 [Amedeo et al., 2000], and is involved in DNA-

methylation-independent silencing of repetitive sequences

in Arabidopsis [Vaillant et al., 2006]. MOM1 prevents

transcription of 180 bp satellite repeats of transposons

[Vaillant et al., 2006]. In mom1 mutants, the release of

transgene silencing [Amedeo et al., 2000] and 5S repeat

repression [Vaillant et al., 2006] occurs without reducing/

alternating their DNA and histone methylation patterns

(Table I). This suggests the existence of two distinct epi-

genetic silencing pathways, DNA methylation dependent

and DNA methylation independent.

smRNAs AS A SENSITIVE ANDSELECTIVE TRIGGER THATDIRECTS EPIGENETIC MODIFICATION

The main advantage of epigenetic regulation is a fast

stimulus-directed generation of new transcriptional states

that are heritable and reversible. What guides the epige-

netic regulations and determines the specificity of epige-

netic changes in response to particular environmental

stimuli?

One of the key mechanisms involved in targeting chro-

matin structure and modifying the gene expression pattern

in response to environmental stimuli is based on the activ-

ity of smRNAs, which were shown to guide transcription

repression through the modifications of DNA and histones

[Matzke et al., 2004; Bender 2004; Chan et al., 2006].

The production of smRNA and epigenetic DNA modifica-

tion represents two separate aspects of the RNA directed

DNA methylation (RdDM) pathway [Pikaard, 2006]. The

details of smRNA biogenesis are beyond the scope of our

review.

There are two current models explaining how smRNAs

interact with a target locus in DNA, suggesting a homol-

ogy-based pairing of smRNAs with either genomic DNA

sequences (the DNA-recognition model) or nascent RNA

transcribed from the target locus (the RNA-recognition

model) [reviewed in Matzke and Birchler, 2005]. In the

light of the DNA-recognition model, the interaction of

smRNAs with genomic DNA sequences could provide an

attractive substrate for cytosine methyltransferases. The

ability of DRD1/pol IVb complex to interact with DNA

methyltransferases and DNA glycosylases suggests the

involvement of this complex in the maintenance of revers-

ible epigenetic states of euchromatic promoters in

response to RNA signals [Matzke et al., 2006].

The initial methylation imprint on DNA in response to

the stress-induced RNA signal can be created by DRMs

at asymmetric sites and then perpetuated at symmetric

CpG and CpNpG by MET1 and CMT3, respectively [Cao

and Jacobsen, 2002a,b]. In contrast to non-CpG methyla-

tion that is substantially reduced when the signal is

removed, CpG methylation can be maintained through sev-

eral generations [Bender, 2004] (Fig. 1). These findings are

intriguing, since non-CpG methylation is very unique to

plants. It may represent an additional degree of complexity

added to the gene expression control system to insure its

fast and reversible response to environmental stimuli.

The small RNAs reported up to date were involved in

the regulation of plant development [Chen, 2004; Juarez

et al., 2004; Kidner and Martienssen, 2004]. They were

tissues-and organ-specific [Sunkar and Zhu, 2004; Lu



et al., 2005], and were regulated by a number of abiotic

stresses, including mechanical stress, dehydration, salinity,

cold, abscisic acid, and nutrient deprivation [Sunkar and

Zhu, 2004; Borsani et al., 2005; Lu et al., 2005; reviewed

in [Sunkar et al., 2007]. A high number of nonconserva-

tive miRNAs, available in some and absent in other spe-

cies, might support the hypothesis that the development

of a specialized miRNAs network was driven by the phys-

iological and stress conditions specific for each species

[reviewed in Lu et al., 2005].

Identifying 22 miRNAs from the developing secondary

xylem of P. thrichocarpa, Lu et al. [2005] further con-

firmed that species-specific miRNAs contribute to the reg-

ulation of gene expression associated with the specific

growth/stress conditions. The expression of many ptr-

miRNAs was induced in developing xylem of stems in

the presence of gravitropism-mediated mechanical stress

[Lu et al., 2005]. This stress triggers the upregulation of

ptr-miR408 expression, and regulates the plastocyanin-

like protein mediating lignin polymerization. In addition,

mechanical stress downregulates the expression of ptr-

miR164 and ptr-miR171 that target genes involved in cell

division and elongation in response to gravitropism.

Recent data by Sunkar and Zhu [2004] demonstrated

the existence of stress-inducible changes in the Arabidop-

sis miRNA pool. Among the most interesting candidates

were miRNA402 and miRNA407 regulated by dehydra-

tion, salinity, cold, and abscisic acid. Whereas miRNA402

targets ROS-like DNA glycosylase, miR407 targets a SET

domain protein functioning in histone Lys methylation

[Sunkar and Zhu, 2004]. Some of miRNAs were shown

to have multiple target sites within the same gene, imply-

ing that different levels of gene repression might be

achieved through a various number of miRNAs bound to

the target [Doench et al., 2003].

The stress-induced miRNAs have a tissue-specific

expression pattern, indicating the requirements in the

organ-specific functional and metabolic differences in

response to stress. Indeed, miR393 downregulates TIR1, apositive regulator of auxin signaling, and has the strongest

expression in the inflorescence under physiological condi-

tions. Hence, the strong induction of miR393 by stress is

consistent with the inhibition of plant growth under the

stress conditions [Sunkar and Zhu, 2004]. Consistently

with the role of miRNAs in the establishment of a stress-

induced gene expression pattern, Arabidopsis mutants

hen1-1 and dcl1-9 that are partially impaired in the pro-

duction of miRNAs, were shown to be hypersensitive to

abiotic stresses [Sunkar and Zhu, 2004].

Recently, a new class of siRNA, nat-RNAs, (for siR-

NAs derived from the natural antisense transcripts) has

been reported [Borsani, et al., 2005]. They are involved

in stress-mediated regulation of genes located in the anti-

sense overlapping pairs, and are capable of generating

complimentary transcripts. The authors showed that the

induction of one of the genes in such an antisense pair by

stress results in the production of nat-siRNA, which

guides the cleavage of other gene transcript followed by

the downregulation of gene activity. Similarly, the studies

of Katiyar-Agarwal et al. [2006] demonstrated the induc-

tion of another specific nat-siRNA during the Pseudomo-nas syringae infection that conferred resistance to patho-

gen. This mechanism may play an important role, since

there are thousands of genes that are localized in the anti-

sense overlapping pairs [Borsani, et al., 2005].

INDUCIBLE EPIGENETIC CHANGESMAYGUIDE GENOMEEVOLUTIONANDSHAPE PLANTGENOME

Modification of DNA sequence via selective cytosine

methylation plays a crucial role as a stable epigenetic

mark during inheritance in plants. Despite their reversibil-

ity, the changes in DNA methylation are quite stable

modifications that are not easily reset and are frequently

transmitted throughout several generations. Indeed, back-

crosses of ddm1 mutant to wild type plants did not revert

the phenotype, demonstrating that the decreased DNA

methylation can be stably transmitted during meiosis,

gametogenesis, and mitosis, irregardless the presence of a

functional DDM1 gene [Kakutani et al., 1999]. In parallel,

the progeny of MET1 antisense plants exhibit DNA hypo-

methylation, independent of the presence of the transgene

locus that previously triggered inhibition of MET1 [Fin-

negan et al., 1996].

Perhaps, the best example of the methylation-mediated

heritable changes are the epialleles, representing different

forms of the same gene regulated epigenetically. The epi-

alleles can be formed in response to a number of stimuli,

and may play an important role in acclimation. The excel-

lent examples of such epialleles are methylated and deme-

thylated forms of FWA gene. Both of them are equally

stable, and can be inherited as a true Mendelian trait

based on methylation but not on the sequence difference

[Zilberman and Henikoff, 2005]. Targeting of DNA meth-

ylation to FWA gene was triggered by positioning of its

promoter and transcription start site within two pairs of

direct repeats [Soppe et al., 2000]. In parallel, two Arabi-

dopsis ecotypes, Ler and Da (1)212, carry the transposon

insertions in the first intron of FLC gene, representing the

independent adaptive events that lead to the establishment

of cold independent flowering initiation by preventing

high expression of FLC gene [Michaelis et al., 2003; Liu

et al., 2004].

Another class of heritable epigenetic traits is repre-

sented by paramutations, when silencing by a trigger al-

lele occurs in trans and results in the generation of a

silenced state of a target allele that remains stable after

the target allele segregates out [Chandler et al., 2000].

Recent studies by Alleman et al. [2006] confirmed the



critical role of siRNAs in the generation and maintenance

of chromatin states in maize and in a characterized MOP1(MEDIATOR OF PARAMUTATION 1), an RNA-depend-

ent RNA polymerase gene required for the generation of

paramutations. It can be predicted that the mi-/si-RNA-

induced transgenerational response to stress exists in

plants, and soon there will be many reports describing

this phenomenon.

The adaptive role of heritable stress memories was

recently supported by the observations that exposure of

plants to biotic (pathogen) or abiotic (salt) stresses leads

to the global and loci-specific changes in the genome sta-

bility and methylation; it also results in the elevated toler-

ance of the immediate progeny to the previously applied

stress [Boyko et al., 2007].

Genome rearrangements represent another epigenetic-

sensitive mechanism affecting a genome stability. The

dual role of DNA homologous recombination (HR), as a

DNA repair pathway and as a putative evolutionary tool,

was intensively discussed over the past several years

[Puchta, 2005; Schuermann et al., 2005 and references

within]. It has been suggested that HR can be involved in

genome evolution through the rearrangements of existing

sequences, frequently resulting in gene duplication or de-

letion events. Indeed, the degree of the HR-mediated

V(D)J rearrangements is dependent on DNA methylation

[Bassing et al., 2002]. It can be suggested that the stress-

directed changes in DNA methylation can either stimulate

or prevent the rearrangements in different genomic loci

[Rizwana and Hahn, 1999]. The highly conserved gene

families located in clusters could possibly increase their

diversity using HR. Actually, it has been suggested that

the evolution of plant R genes involved gene duplication

and recombination events [Meyers et al., 2005]. The fact

that the meiotic and somatic HR can be induced by a va-

riety of biotic and abiotic stresses could suggest that

changes in the HR represent one of the mechanisms of

stress adaptation [Kovalchuk et al., 2003a,b,2004; Molin-

ier et al., 2005; Boyko et al., 2006,2007]. In fact, in sev-

eral studies, the induced HR was inherited by the progeny

of stressed plants as an epigenetic trait persisting in the

population of one or several non-stressed generations

[Kovalchuk et al., 2003b; Molinier et al., 2006; Boyko

et al., 2007].

Our recent studies established a defined correlation

between exposure to stress, loci-specific epigenetic

changes, and genome stability of exposed plants and their

progeny, using a well-studied model of TMV infection

[Boyko et al., 2007]. We have demonstrated that the prog-

eny of tobacco plants treated with TMV inherited the ele-

vated rates of HR that coincided with the increased fre-

quency of rearrangements in R-gene like loci. We have

shown that the progeny of stressed plants increased levels

of global genome methylation and exhibited loci-specific

hypomethylation. Namely, the R-gene loci that carry

homology to the N-gene, gene of resistance to TMV were

found to be hypomethylated, and as a consequence rear-

ranged more frequently [Boyko et al., 2007]. Since the

plants used for infection (SR1 cultivar) did not have the

N-gene, it would be correct to assume that the locus-spe-

cific changes in methylation and rearrangements could be

a plant strategy of creating an active R-gene. Our studiesindicated that the similar transgenerational changes in

recombination and DNA methylation can be generated by

a number of abiotic stresses, suggesting the existence of a

general epigenetically controlled mechanism directed to

the selective relaxation of DNA sequences. Such a mech-

anism possibly allows faster evolution of these genome

regions [unpublished data]. It might somewhat resemble a

phenomenon reported in flax, where a number of heritable

changes could be rapidly induced by environmental

changes [Chen et al., 2005; Cullis, 2005].

CONCLUSION

In summary, we want to emphasise that the epigenetic

control over plant response to stress is a complex phe-

nomenon. Epigenetic modifications do not only occur dur-

ing plant exposure to stress, but also establish numerous

changes in gene expression that can persist over several

generations. A number of epigenetically-mediated changes

generated in response to stress may result in the formation

of epialleles and paramutations that can remain in the

population over many generations, resulting in the diver-

gence of plant ecotypes. The epigenetic marks thus may

regulate gene evolution in a stress-directed manner, allow-

ing a rapid generation of new adaptive alleles on genetic

and epigenetic levels. In Figure 1 we have integrated the

current knowledge of how the developmental program-

ming, physiological stimuli and exposure to stress modify

the DNA and histone methylation/acetylation, chromatin

structure and potentially (re)establish new patterns of epi-

genetic inheritance leading to the modified gene expres-

sion, establishment of new epialleles, regulation of ge-

nome rearrangements and adaptation to new environmen-

tal pressures. Some links in this model are still

hypothetical and lack the direct experimental support.

However, a number of supportive evidences are being

published every year, and the continuously increasing in-

terest of researchers in this problem provides us with

promising expectations of new bright discoveries in this

field.

ACKNOWLEDGMENTS

We would like to thank Mikhail Pooggin for critical

reading of the manuscript and Valentina Titova for its

proofreading.



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Epigenetic control of plant stress response