Chapter 2

Review of Literature

E ngineering salt tolerance in sensitive varieties of crop plants,

requires a complete understanding of the molecular mechanisms

involved in transduction of signal of stress perception and expression of

stress responsive genes. The efficiency and the time of expression of

these genes are critical in bringing stress tolerance in plants. The last

decade has seen significant advance in gaining knowledge about the

transcriptional regulation of the genes are responsive to environmental

stimuli. The present chapter describes, in brief, about the cis-elements,

transcription factors and modulation of chromatin structure that are

involved in regulation of plant genes and plays a major role in generating

abiotic stress response.

Section 1: Cis-elements and trans-factors

The stress regulated change in gene expression is mediated by several

pathways guided by different signaling molecules. The molecular

pathways that regulate the abiotic stress responsive genes are stimulated

by factors like ABA , ethylene, jasmonic acid, salicylic acid, Ca2+ ion and

have been broadly divided into ABA-dependent and ABA-independent

pathways.

The degree of transcriptional expression of a gene under a particular set

of conditions is mainly dependent on various cis-elements and their

organization in the promoter region of the gene. The trans-factors that

binds to the cis-elements, form a large complex that ultimately effect the

initiation and/ or elongation of the transcripts by RNA polymerase. The

change in expression pattern of a gene results when the group of

transcription factors covering a particular set of cis-elements changes

and the new set of transcription factors binds to new group of cis-

3

elements and/ or one or more cofactor of the transcriptional complex is

altered.

Some of the transcription factors and their respective cis-elements,

functioning in abiotic stress response signal pathway have been listed in

Table LR_l. From the analysis of the literature in Table LR_1 and other

related papers, one can infer a few important points as given in the

following pages.

The expression of a gene requires the function of more than one cis­

element simultaneously

By virtue of the immense effort, put together worldwide, to understand

the stress responsive gene regulation, it has became evident that a single

cis-element or transcription factor may not be sufficient enough to

modulate the gene activity.

ABA responsive expression of genes is guided mainly by the presence of

ABRE element in the promoter of these genes, but unless a second

elements like CEl.CE3, DRE or a second ABRE-element is present

nearby, the existing ABRE element cannot contribute to the expression of

the respective genes (Shen and Ho, 1995; Shen et. al., 1996; Hobo et. al.,

1999; Narusaka et. al., 2003).

The co-operative activity of transcription factors like Myc and Myb have

also been found to be necessary for the expression of many stress

responsive genes (Abe et. al., 1997). The Myb and Myc transcription

factors have been found to interact with each other through a WD-40

domain containing proteins (Ramsay and Glover, 2005).

Promoters of many stress responsive genes contains DRE element in

Arabidopsis within near vicinity to the transcription start site but all

these genes are not regulated by DREB transcription factors as detected

by microarray analysis in DREB over-expressing transgenic lines.

4

Table LR_l :-A summary of major classes of abiotic stress responsive transcription factors (TFs) as reported from different plant systems

Class of Transcript ion Fal'to.-s

HOMEODOMAIN (liD)

:\ 6 I amin•• acid mllt.i!l that loi<h 111111

thn ,. f.,jd, ink thr···· h,·hn, ~'"~ ·>utd.'

D:'\A. lh•lh.ll anJ lll form., • lwlix turn

lwli" 'tructun· that .u·•· f~.und in m.lll'

prokaryotk TF~. I J,•lix lii I its into major

groc"•· t.fthe 1>:'\.\.

Suhdass, Examplt•s ,,nd Organism

HD-Zip famil)' llonh·od<>mJ.in ·~~O<'t.ltt·d with a

lt·ul'inl' :npp•·r eg. At! IB 12 ,md AtHR7 (Arabidopsis thaliana)

PHD fingt!r family Plant hom.·ndom~in a,.,n,·io~wd

with a llng•·r domain

cg. Allin I (.!lrJ•(ago sa/11'<1)

Bt>ll family Assol'iatcd with .1 chstinctiw Bt>ll domain

eg.

lh.BIIlD I (0~'7" <ati>·a)

Cis-Elem(•nt

CAATNATTG

GTAAT(G/C)ATTAC

TAAATG(C/T)A

GI':Gl;n; or

GTG<;."\G

T~TCA

Function

Upr"gulatt·d in water

ddldt ,,tr<'SS in a ABA

dep,·nd manner;

Regul,He growth of root

and inllorl'sccnn· shoot

under ~tress

Cau~rs induvtion of

salt inducibl,· g•·n<

MsPRP/ and b ,lightly

clownn·gulat•·d in rot>ts nf

sen .... itin: \ an~ttt"~

(),•,n·xpn·s~ion hrings

tolt'ranc-,. to hiot i< 'tr .. ss

Olsson ct. dl., (2004)

Luo l't.al. (W05;

Class ofTranscription fa<·tors Subdass, txamplt•s ami Organism Cis-Eit>mtmt Fund inn Reference

hut make:. plant• mort·

s..n~itivr to :,alt and oxi

dative stre~s

ZF-HD family /.mr fing.-r .t'sodakd to a hom .. odomJ.in

eg. ZFHI> I (Arabidop:,i~ thah.\na) t '.\CTI\.A.ATTGTCAC ovt'r-t·xpret'sstion indun·~ Phan Tran et.al.

the ··xprC'ssion of Sl'Vcral (2007)

stn·:,~ rdakd gcn<'-'

including ,·rd I; transg,·nic

plant> havt· impro,·,·d

drought tolerann·;

inh'racts with :\Al' TFs

WOX family W u ... ch, l re l.ltNI hom .. nhux

eg. H0~9 (Arabidopsis th.,JianJ} A '• .nstit uti\ l'ly <'pn-~s- Zhu t•t.aL 1~004)

ill)! prntdn th.11 j, n,·,·d.

,·d lor thl· indu,·tinn of

man~ ,·uld n·•P'"ht• g~:nt·;

l )oe.s not fwl<·tiolh in

rcspons,· t<J salinity

dnd ABA

Cl.1ss ofTranscription Factun;

AP2/EREBP Al'2 (APFTAll A?J.t FRf.BJ> <· th_,·I.-n. n­

sponsiv•· ,.J,·rn .. nt binding prott·in) .1rt> •.tnl<JUt'

to plant> J.nd an· l'haractt·ri:z,•d l.y tht·· f,•,Jt .

ur''' ',f th .. ir .\,]'} d<>mdin that indudt·, ,1 )U

amino .Kid long'\ ll'rminal YRG .-l,·m.·nt

trich in bask and hnirophi:Jk ,m1ino ,.,·id~;

prnh~l·l) invon•d in Dr'\,-\ hin.hng) and a

C-t..rmina! RA YD dt·m··nt (,·ap.,hk nf

forming an JmphipathJC a-helix; ma) lw

rc-spun~ibl fnr protein ·prntdn in!t'rat1inn.')

Subclass, Examples and Organism

K:'I:OX family Knottt·d t·.-latrd homeobox

:--:o transcription factor is yet rrportt.'d

from plants that functions 111 abiotic stn·"

n·ponsc.

eg. Knotted-! (Arabidupsis)

FRF familv ( \mtains tWt> AI'} domam

cg.

Cis-Hement

DREB {d<>hydration rl'spon~t.' t•lement binding) (C/ A)CCGAC

truns..rwtion fat'tnrs- nnC' th•· mosl wi<lly studit·d

plant tr,mscTiptinn fac:tor studi<'d m pl.mts from a

wid.- varieties of plant sp .... ks lik<' Ar .. bidopsis,

Oryza 'urn·,z. Pcnm.,,·ram. Phj·comzrr.·llu. Gus~rpzum

t'k. In .-ac:h spedn,, there ,lrt> many class<•s of

DREB g•·m·s that dilf,·r in tlwir function>. Abo

known Js CBF •Jr !JBf in mJn)· •p•·t'i(·s.

lund ion

runct!on~ in plant

dt'Yt> lopment

DREB genes functiom in ,1

Rd'erenn:

l)imosthcnts Kizis

et .a! (200 I)

Riechmann and

;''1c)('I'O\\'it/. (I ';l9X)

Wang t't.al. <2008),

wid,· \arit~ of'Stress(bintic anc l'nng •·t.al. (W08),

abitJt'it) dnrl in ABA d(·pmdcnt Schram ··t.al(/008),

.111d indcp,.ndmt mann,·r. It Liu •·t.al. (2008),

causes upngulation of many

Important grl>Ufl> uf ge.tws likt­

COR ,RD2';l, Rab, ERD eh.

Agat·wal •·t.al. (200'/)

Shen t>t.al. (2003)

Chm et.al. (200~)

l'RF I in .'\rahin<~psis ,·asuc-s hyper Agarwal •·t .a! (J006)

acetYlation ot hist•>tll'> \'ia its inter l.iu <'! .al. ( 1998)

action with ADA! and GC~5

Class ofTransc.:ription factors Suhdass, Examples and Organism

Hnt·ntly. m~ny DREB-Like genes hav" b<.:< n

rt>port.-d from Arahidop'i~. Dt>ndr.mtha.

So,·ah,·an etc.

ERF (Fthylc•nc• n .. ,pon,,- •·l..rr'lt'nl hinding

factor or Ethyknl' Reposivl' Factt)rs)

F.RFs consists of a large group c>f protc-ins

m 1 arious plant system~. ERh h,lvt· he en

dn·ided into I) groups in Arabidopsi' and

l S groups in ri<',·. Wnhin a svstem, the

sam,· F.RF ~ct]U<'nt'<' ,·an b< r .... ognis,·d l·~·

difkrl'nt type' of ERFs undn clilfcrcnt

st't' of conditions. Somt• c·xampk an·­

.'\tERI'l, AtERF/ & .\tf.RI-'5 (Ar.lbidopslsJ

AtERH, AtF.Rl4 & Atf.Rf·7 IArahidopsis)

Cis-F.lt•ment

:\GCCGCC

(GCC-box)

Fundinn

Perfom1s their !unction

undt>r various str"""-~

lik<· DRI-'lk TINY, a I >RFB

likl' prot.,in !rom arahid

psis can bind to both

DIU: and ERf "''lU<'ll<<'S.

ERh respond to extrace­

llular signals to morlulatc

the ,wtivit v of GCl' box­

mediated g<'IW expn·s~i­

on>,po>itiVl'l)' or negativ­

vlv. Rl'prc-ssnrs show stro­

ng•-r activity than al·tiv

atnrs.

lnvolwd in 'iin 3 & sap 18

ml'diall'd gl'm· 1'1 pression

Sun t>t.al. t200!l)

l.iu et.al. I 200X)

Xu,ng ,mrll'~• \ '001\)

l'ujimoto .-1. al. (2000)

1\;akann l'l. al. (200&)

1-ujimot<><'f .• 1 (.'000)

Snng and (.;Jlbraith!2006)

Song •·t. •1. (}00~)

nmstitulivc ly ·~xpr<·~•ed root Hu et.al. (200~)

abundant prott>.in; uprt>gulatt'd

bv .-th~·],•nt> and upr .. ~tlat.-d hy ,u]d str.·ss

Class ofTranscription fa<·tors

CA.\1TA (CGI-domain nmtaining pmtt'in}

l'A:vllAs form <1 nn,·h· rlisn•,,·r• d n .. ,,.j da~• of

Calmodulin hinding lrJnsniptiPn i'Jt tnr• (in

,•ukaryott'') that ho~, tlw Idle owing ch,n·o~, t.·risti,·

domJII". ·

Suhd.azos, Examples ann Organism

AP2 family l'nntains one AI') domain

t>g. ,1tABR I ( Arahidopsis thJ!iana)

RAV faml)·

Cnnt:Ains a B3 dnrnain (a O'IJA-binding

domain found in pldnL') in addition tr>

ont AP7 domain

eg.

CaRAVI

a "\l s (nud.:ar lnt db/dlinn "ignal), J u;.)

domam (J uniqu,· Jnd nov.-!,, <jLt,•nct· •pcdti,·

t>:-.;A hi11Jing Jomain), TIG rlunl.lin dnr prot..in

dinwrizatiou and n•Jil·>f'"'·iti, binriing 111 ll'-..\i,l . '11\;K (anl..vrin r.-p<'abJ and a \'Jriah!t- numb.-r '' eg.

IQ motilt 'requin·.! i;>r ('aM binding;. RnCAMTA I (llra.<st• a narus)

Cis-Elem<mt

CAACA &

cAccn;

CCCG-containing

s('qtl('l1lT

<<;; .·\IC)c<;cc(T ICICJ.

CG(C/T)G-cort',

G-HOX AHRF

CA,CGTG(T /G/C),

AHRF-CF

(CI A)Ai,:G<-:._({(T IGIC) .

~undion

Func-tions as a rqm·ssnr of

g<'ll<' ··xpresswn undPr

ahiotic stress and extt·mal

ABA treatment. Mutant'

Plant; are hypersen~itin·

to 'l.trt::~s

Provides l"motk tolt·ran,·.­

and db,.ase resbt,mc~

Rc..·f't.-rence

Pandt•Y t•t.JI. noos)

Sohn t .'OOb \

Finklrr ctJ\. (2007)

Mitsud,1 l't.al.(200l)

Kaplan l't.al.(2006)

Choi ctal.(200S)

Scrl'l'llt'd l>Ut of a CO!\A libran Bouch~ t'Lll.()00?)

Clas~ ofTranscriptiun factor~

~Al (l\.'\\1, A I At. Cllq '! ~

!".\C •umsist• of a gnlUp of plant spt•dfk

TFs th.lt ar< •hara<t,•rh··d iJ, a um.,cn.-d

'\!.t,•rminal :\AC rlomain .md a mun

din·r~ifi~d C ·l<·nnnal domain, urig_inallv

idmtUkd in !':\M, .\I .\.1- and CUC

protdns.

Subclass, lixamplt:'s and Organism Cis-Elt:'ment

OsCBTI

CGT(G/A)

<'g. ~ti\:AC rs.,Junum cul>ervsumi

A'\ACOI'I, AI\Al'OSS & A]'.;Al'072 (Arahidopsb!

1:-unl't iun

..:•mstru<"t•·•lli·om RNA of ahiotit·

:,tresseJ ti~:'\Ut'

Llprcgulatcd i11 rl':<ponst·

to v ariow' stn ·~:;cs likC'

b.:at, cold, UV aml \:al 'I

A tSR4 IS salt sp<,cilk

A tS!{6 is cJ,,,., not ,·xpr

,•;so under salt ,·,mditi·

ons AtSR ~-6 arC' alw

indtKed in rt>spun,t• to

ABA and mt·th) I jasmonat~

f.unction in .~u·,·ss j,; vt·t

not rrported

Induc·,·d hy drought, cold

and wounding

Provid<'~ drought tnlt"r.mcl'

Yang t·t.al. (.?U02)

Kikuchi t·t, al, (2000)

Du•·al ct a!., (2002)

Obm t>l al (200S)

Collinge and Boller

( ~Olll )_

H~gedus. <'tal. (2001)

Class ofTranscription Factm·s

Ht'lix-Turn-Helix

l·lt-hx-Tum-Hdix motiff is c·omp"'"d ol two (1 hdin·> joint>d ll\· ,, ~hort 'tran<! <>I

am.ill<> adds <\nd i' lnund in man~ pn>h m'

that r('gulat•· gt·ne, xpn,ssion. J,; pl.mh

two major groups of protl'in that contain

llc>lixTurn lldix MotifTart> ~\YB, & ll'ih

Subdass, Examples .1nd Organism

MYB transcription fal'tor family MYB pratdn' g•·n,·J·ally 'ontain• tw<>

(R) I R ~; cnor,· common in plant>) or thr<·<·

(R I /1{7 t R ~)]):"<A binding d••main kno" n as

!V1 Y B motif!~ .md ,.,,ch suJ1 motiflpn."'"

a I klix -turn hdix ~tru<·tun· with thrt'\'

ngularl.\· ·'P<J,·cd tr~·ptophan n·,idu, .,,

cg. HOSIO (Arahidopsis thaliana)

AtMYB44 (Aralmlopsis)

JAmyb (Oryza sativa)

Ht·at Shock Factor ( HSh) famil)" (' lln,ists of a '\ \l'rrnm.1 ·. I)'\'\ bindi"'g docn,un

Cis-Element

TGGTTAG

(M;AAn)(nTJ"CT)

pallindromic consensus

lnduc·.·d in rC'pon.<(' tn biotic

dnd &J.biutic !<itrt·s!'i. ( )vcn•xpr··

ssion incrvJ"'' drought and

'"It stn·"' tukrJnn·.

Lipstc:k (1996)

Manm & l'.w.t\rrs (I 'Jn)

Abc I! ct al, 2001

Nccckd for thC' C'Xpn•ssion of g<'lh' Zhu et. al., (2005) likc '\CED3 in order to iucr,·a."'

tul,·ran<'(' to i'n·<'zing .md salt str'·"

Stomatal closure undn str<'ss Jung ct.al. (2008)

UprcgulatC'cl in rC'spons<· to lee ct.a1.(2001;

jasmonic acid & wounding

No\'t'r t·t. al., (1996)

Prancll ct. al., ( 1998)

Clasli ofTranM:ription factorli Suh~..-lalis, fxamplt"li and Organism Cis-Eltlmt"n1 Function Rt>fert"nn·

nmtaining antiparal!.-1 fnur-strandv<l 11-slwt't

packed against a bundle of three helices,

th,· last two ol whi,·h linn.' a llclix turn l..·hx

mot ill and the llrst om- is f(''-)Uir('(ll l'\'.'\ tin·"''-)-

U<'lll c sp.-dllr~- _ Adj.Kent to thi> i> lwpt.td

hydrophobi<- r.:pt>at~ tliR-A/Bl invnh-·,-cl in

oligumerit.allfln and tlw C--t.-rmn.1l ,ontain' tlw

a<th·aticm clr,main an 1 1 -atninn acid d,·Jetinn

in J soh<·nt-cxpo,,od loop h,·tw<·,·n t""

Jl-sb~:l't' is • p,·,·uliarit y .. r plant l!Sf,

H~h .1rc- divid .. d into A l .. -\}, Band C das~.-s.

eg. HSFAl and HSFA2 (TomatnJ F-untions in heat ~tn·'s _ Srharl' ct. al. ,( 1998)

HSFA2 'w"d' to intaa<-1

\\ ith HSI-'A I for nudt>Jr

import.

Spl7 (oryt:a sativa) lndurcd in hl'Jt strcs; Yamanourhi <'\. al., (?00))

HSI-AS (Arabidopsis) Acts as a rt>pn·ssor;funl'tiom in llaniwal ct. al., (2007)

HSFA l (Arabidopsis) lwat and dmught Stress.

Basi( Hdix-loop-Hdix a bill II domaiu ,·omi•b nl a otrvtcb 11! about CA!'\:"--:TG (E-hox) .r..1urr<' C <'t.al. ( 1 'Jil9)

1 X h~·drophJli( and ba~k amino a~•d, at th.- R.lll".1'' .md (;\"' .-r (200S;

:\ termm,>l ,·nd n! th·· dumam, f(,!l,lwt·d bv ('0 :::-

tw•· •mph>J•-llhi< a -h.-lj,·,., s•-pdr4tt'd h,- d iuop M)T transcription factors (a hlll.ll-ZII' TF) likt· CACATG

AtMYC2 AI3A-mediated drought Al>c 11 ct..ll. (2002)

g<'nl' <'Xprcssion

Clas~ ofTranscription fat·tol·!> Suhdass, F.xample!i and Organism

bZip Tilt' lv!p Th ol.ll' W!d..lv found in .ill <>r:<.m In all plants r many groups of hZip Th are

ism; and contdill> a basic !):\A bmding found. In Ar.1hidopsis therl' arr 10 groups of

domJin .md a ku<'ill<'· t.ipJwr fi>r <Lnlt'rit.ati<•ll hZip Th.

SonH· of' th~· l\·u~·inc n·.'\idth' arc !'I.e •nH· tinH''\

r<'ph~<· .. d by iwl.·ud1w, ,,din,·, m.·thionim·

or plwny: cg.

OsBZ8 (O~yza sativa)

TRAB I (O~rza sativa)

Zim·-fingt•r /.in< -hng<>r n>lltnin~ a ><'qu,•n( ,. nwlilt' in

whkh, ;~tdn and/or histidin,· ··" "rdin.1ll • Cysl/ Hb2 (CC'HH) family :till I .ItO Ill fnTrliiii;! J [ot'a) p• ·pt id1 ,,] IU< 1 UTI'

ncn-~>.lr~· for l>'-\ or protein bmding

Tlh· motJit' con tam tw• > ,vst.·in and t wn histidin.·

r~·sidu,·s. There an· mam· typl's under tl1h l~uud:,

like TFIIIA type cunscrn·d scqucncl' motiff­

C'X2-4FX 1LX1l !X3->I1

TACC;TA (A-hox)

GACGTC (C-hox)

CACGTG (G-hox)

G-box is l<n111d in many

constitutively expressing

genes.

Functiun

Uprcgul('s und"r salinity str•·ss, Mukhcrji ct.al. (2006)

more in tolrrant varit'ties than

s'·.nsitivc nncs; i{,:prntcd as a n·pr-

essor tl1at can nvcrcom .. the Lc<· l't.al.(200'1)

activatin)! capadtv of Mvb transcrip-

tion fa,·~,r and :luwnl:"gulatt-d I a-am;·la.l' gene in n·spom1· t<) sugar

com:entr ation; ABA .. rcsponsi v,·

ABA indu('ib!,- activator Hobo l't.al. ( 1999)

Tabtsup (1999)

Class ofTran~cription Fa(·tol'!> Suhdas~. Fxamplt•s and Org<~nism Cis-£1~men1 Function Rt'l't>renn·

cg. ZPT? · 2 (!\rabid< >psi') :\Gt '1 1')/ CAGT Functirlll~ as act in>lor in various Ynshik. et.al. (.~001;

ahioti< st n·.~s,•s

ZA T 1 I ( Arabidopsi~) D.-vl .. tova et. al. :_1005

WRKYtypc The DNA binding domain of tlwsc TFs have one

or two 60 amino acid motiff with a highly conse- C/T)TC;AC(T/C:

nsus sequ<>nct> WRKYGQK which is adjacent to (W-Box)

a novel zinc-finger domain. Thert> are 74 mem-

hers in Arabidopsis and more> than 90 in ric<'.

eg Lt vVRKY 21 (Larrea rndcntaca) llpregul,ned m Cold, s.ll:nJt; ,md Zou ct.dl.(2007)

wuund111g stJTSS<.:~. Suggested to

"urk .l~ .111 Jct:,·atur m ,\B:\ :-.ig.ud-

lllg .md rqJre::..,ur 111 G:\ sJgn.llmg.

CysJ/His (CCCH) family consensus ~e<luenct' (( 'XxCX 1CX lll)

eg. AtSZFl and AtS/.1-:! 1Arabidop~is) Nt•gativt·ly n·gulat<' gene !:>un et.al. (2007)

c:xpression undt·r salt str.-ss

Cys2/Cy:;2(CCCC) familJ eg. OsiSAPX ( 'onf,'rs n·si~tanc<" to n>ld KJnnrganu and

<lrought and ~alinil} stress. Guptd ()00!1)

Hence, other associated cis-elements are necessary for active functioning

of the DREB binding elements. Moreover, genes containing same DRE

element have been reported to be controlled either by DREB1A or

DREB2A. Also, some of these genes are controlled by both DREB 1A and

DREB2A. The other cis-elements present on the promoters of such genes

have been suggested to determine the functionality of the DRE elements

(Yamaguchi-Shinozaki and Shinozaki, 1994; Baker et.al. 1994; Jiang

et.al. 1996; Stockinger et. al., 1997).

ABA-independent expressiOn of ERD1 (EARLY RESPONSE TO

DEHYDRATION 1), encoding ClpD (a Clp protease regulatory subunit) in

Arabidopsis requires a Myc-like cis-element (binds to certain NAC

transcription factors) and a 14-bp rps1 site 1-like sequence (binding site

for ZFHD factors). In transgenic studies it has been found that ERD 1

expression is not enhanced until both these DNA binding proteins are

overexpressed together. Also, it has been found that group of cis­

elements controlling the expression of ERD 1 under dehydration is

different from those responsible for dark-induced expression of ERD 1

(Simpson et.al, 2003; Tran et.al. 2004).

All such evidences points to the fact that modulation of gene activity

under a specified condition comes from a set of cis-element on its

promoter and is not entirely due to the presence of a particular cis­

element. Infact, some recent in silico genome wide studies, have also

indicated the presence of different types of cis-element that are present

in the different classes of stress responsive genes (Ma and Bohnert,

2007).

Change in stress responsive gene expression is coupled to a change

in transcription factor or cofactors in the regulating complex

The regulatory complex responsible for a certain kind of expression

pattern of a gene involves a large number of interacting proteins and is

5

generally tethered to the DNA by the transcription factors. A change in

pattern of expression of a gene with altered conditions is a result a

change in the set of transcription factors of the complex or in any of its

co-factors.

In response to stress, various transcription factors are either induced or

post-translationally activated by addition or removal of moieties like a

phosphoryl group or are regulated by degradation through proteasome

complex and change of its localization within the cell. For example in

Pennisetum glaucum, phosphorylation of DREB negatively regulates its

DNA binding activity (Aggarwal et. al., 2007).

Most transcription factor resposible for the expression/repression of

stress responsive target genes are induced under stress conditions and

bind its respective cis-elements leading to expressiOn of the

corresponding gene. For example HSF3 in Arabidopsis is induced under

heat stress and causes the expression of HSPs. Overexpression of HSF3

also causes expression of the target HSPs under control conditions

(Prandl et. al., 1998). Over the years many transcription factors (like

DREBs, Myc, Myb etc.) have been found to cause the induction of target

genes specifically under stress condions (see Table LR_l for references)

A transcription factor, under certain conditions, can occupy a previously

unoccupied cis-element and modulate the degree of transcription by

suppressing the activity of previously positioned transcription factors.

The new transcription factor can draw a new set of proteins in the

existing complex and thus changes its mode of regulation. A typical

example is the repressor OsBz8 in rice which represses a-amylase

synthesis by binding to the ABRE cis-element and overcoming the

activity of Myc transcription factors. OsBz8 has been found to be

upregulated and activated by phosphorylation under abiotic stress

conditions (see Table LR_l).

6

The gene expression can also be modulated when the occupancy of a cis­

element is swapped by a similar transcription factor but with a different

activity. This is most commonly found in case of ERFs in Arabidopsis,

some of which are activators while others are repressors of gene

expression (see Table LR_1).

In plants DNA methylation 1s a common phenomenon reported to

regulate gene activity. 5-methyl cytosine is treated as a separate base in

context to gene regulation. Most sequence specific DNA binding protein

have been reported which cannot bind to methylated DNA. Some of these

proteins have been reported to bind only to methylated DNA while others

are insensitive to DNA methylation (Inamdar et. al., 1991; He et. al.,

2001)

As mentioned, transcription factors are a part of large regulating

complexes. Hence a change in gene expression could be an outcome of a

change in the cofactors of the regulating complex. The DELLA nuclear

growth repressors which are suggested to be transcriptional regulators

function in GA and ethylene signaling pathways (Pysh et. al., 1999;

Achard et. al., 2003). DELLA protein has been reported to accumulate

and cause induction of proteins responsible for lowering the level of

reactive oxygen species (ROS) under stress conditions (Achard et.al.

2008).The transcription regulating complex has often been reported to

contain modifiers of chromatin structure. CBFl in Arabidopsis was

shown to interact with a GCNS like histone acetyltransferase via ADA

adapter protein (see Table LR_l). ERF repressors have been reported to

function through a SIN3 and SAP18 containing complex which also

includes histone deacetylase (see Table LR_l). A change in any such

components will result in a change in gene expression. CAMTA are a

recently discovered transcription factors that are widely found in various

groups of eukaryotes (see Table LR_1). The transcriptional activation

domain has been mapped for AtCAMTAl and HsCAMTA2 (Bouche et.al.

7

2002; Song et.al. 2006). While the mechanism of CAMTA functioning is

not clear, some knowledge has come from its mammalian counterpart.

The HsCAMTA2 which is involved in transcriptional regulation of cardiac

genes interacts with the homeodomain of another transcription factor

Nkx2.5 through its CG-1 domain. The ANK domain interacts with histone

deacetylase HDAC5 (Song et.al. 2006). When HDAC is present in the

complex and interacts with CAMTA2, interaction with Nkx2.5 is

abolished and transcription is not activated. The interaction between

CAMTA and HDAC is restricted by phosphorylation of HDAC that results

in active transcription of the respective genes (Song et. al., 2006).

From the brief description given above and the specific examples cited in

Table LR_l, it follows that the organization of cis-elements, the

transcription factors and cofactors functions in a co-ordinated fashion

and provide a tight regulation on gene transcription in response to an

upstream signal.

Section II: Chromatin remodeling

The naked DNA of prokaryotic system is readily accessible to all the

activators in the cytoplasm and hence the presence of a repressor is

generally highly required to regulate a gene transcriptionally and

overcome the activity of various activators. On the other hand, the

nuclear membrane enclosed eukaryotic DNA 1s assembled into

nucleosome (a complex of histone octamer) and needs an activator,

generally, to overcome the general negative effect of the nucleosome over

transcription initiation and elongation, the activity of a transcriptional

activator is an absolute requirement for altering the interaction between

DNA and nucleosome. The activity of a repressor is still required to have

a tight regulation over transcription. The term chromatin remodeling is

generally refers to a discernable change in histone-DNA interactions in a

nucleosome (Lusser and Kadonaga, 2003)

8

The interaction between DNA and nucleosome can be altered by two

ways;-

• Through covalent modification of theN-terminal tail of the histones

• Through alteration of nucleosome structure that include changing

the path of the DNA over the nucleosome or mobilizing the

nucleosome in trans or in cis or interchanging a existing type of

histone with one of its variants.

Chromatin remodeling through alteration of nucleosome structure

The enzyme complexes involved in displacement of a nucleosome over a

particular stretch of DNA sequence, consumes the energy of ATP

hydrolysis and hence, are called ATP-dependent chromatin remodeling

factors (Imbalzano, 1996; Cairns, 1998; Varga-weisz and Becker, 1998).

These multi subunit ATP-dependent complexes have a Snf-2 like family

of DEAD/H ATPase as the catalytic centre (sharing homology to

helicases) as their catalytic centre (Gorbaleya, 1993; Eisen et. al., 1995;

Peterson, 2000). Based on the type of Snf-2 like ATPase, the chromatin

remodeling complexes are grouped into several classes of which some of

the most characterized ones are listed in the Table LR_2.

Homologues of SNF2 like ATPases are found almost in all plants for

which genomic and eDNA sequences are available. In recent times many

of such ATPase homologue had been studied with varied degree of detail

in Arabidopsis. Table LR_2 lists some of these chromatin remodeling

factors along with their functional aspects.

Arabidopsis proteome contains 42 ATPases that have the seven

diagnostic SWI2/SNF2 motif (http://www.chromdb.org; Kwon et. al.,

2005), four of which are classified as SNF2 subfamily of transcriptional

coregulator (Verbsky and Richards, 2001). In rice, three such genes has

been given the same category while poplar has six such genes

9

Table LR_2: Different classes of ATP-dependent chromatin remodeling complex on the basis of the type of ATPase subunit

Type> and Stuctural Example> and func:tional Aspt"cts

lmportancl' of Organi!im

S\Vl/SNf complex

Tlw A.l Pa~t· con tams a :vSWI/ Si\F (yea:;!) Reauitcd to the .l p.u-llnJlnr lo<.u;< Ow.-.. l!u/-!'-"" ··• .. J.\ I '''-6J

bronwhotlldin yRSC (yc.tSt) by mtt.:ractin~ with tran>tnption l.urd1 ct.al. ( l "'J'I;

ISWI complex The ATPa.se t·ont,un a

SA "iT domain

CHDt complt>x

hBAr (hum.m) f.I...:tors and dn.· inYo, ... d in tr·dJt~n ipt Pt·kNm & \Vorkm.m

I!BR.\1 (hum.IJl) ion regulation. t'.J.U.:i{·~ m tra.r•" (20CXI.>

dRR:\l{J)T,.>s>uphilu) mobihzation ,,f nudt..·u;o!llt: a11d h.1ve Pelt·r<on ( lOOS)

At~YD (.~rabtdop;IS) D:\A bindmg ability. yRSC ha.< he,·n Suwiars~ll.tlll P (.!000)

:\tBRM (BRAH\1.<\) found to 11111h1hzt· nud,·osom,·~

( clraf.idup.,j., ) 1n ci:'.

y!SVV 1 a & h (yea~ I) Int..:rat.t~ w1th D:'\A as wdl a:; lu~tonc l.d{o\ C 1 t a!. \! 'J'IIi 1

y!Sv\/2 (yt:a>t) t<uk Tb~:~e complexes appt'ars to I ,·Ro~ C, l.dl. ilO!I(I)

dASF fDro:.svrh•luJ .ts~,·ntbk nud(·o:;omt-~ dJlO enhance B .. ,·har I l.\, t .... : .< 2U0fl)

dCHRAC :<t<.~hllttv ol chromatm structure Ho.dt,·n,•k I .-1 . .IL( 2•;021

( Drossophilaj

d:\URf (Drossophilu)

h('HR,\C (humdJI)

h.:" LIRr (htm•.tn 1

Poot RA d . al. (21100 1

Strohn.~r R .-1 •1.\llnJ l i

Vary J (' t·t.al. (200 J)

l..!n>t & l:ln k.·r I '<XIl 1

The :\TPa;c contains a d:V1i2 (Drv~.\vphilu;

Chromodum.till ,md a h:S URD (human)

0;\;A binding mot iff

Po~st.~t.·:; a hhtonc dcan~tylast' :;uburut \\ Jd, & /.hdn;,: (I 'I'JXi

and i:; ,·;tunated to ch.mgt· th(~ Zhang Y t'l .AI. 1 llJ'l4)

nudeosunte .u-dliw,·hrun· to facilitate v\/ang HB {200 I!

IN080 Compkx yl\!080.COM

Lu-ge compl.,x •·ont.tin (yt-.l:;t)

mg prott:in:< 'mnla~·

to Bao:tn-i,ll Ru" B

hdicast· .. \TP,ls<· dom

am is :;pill into two by

an inst·rt ron

SWRl

A TP ae dc,main 1~ split

into two by dll insation.

SWR-C <ye.t.~t)

hi:stonr cit: acct vl.1U1 •n .tU ivi t).

A general reprr:-so1 c.omplex

( )nly <·hromatin n:mooeling <·omplex Jon"on /.( J t't a!.( ?00 I l

po~:;cs..~in~ hd~ea~e al'tivity

(Ill <I)' be ducc to Ruv protnn~)

1NORO.C0~1 is mvolved in 0!\:A

rcp.ur ami many gene transtTiption

Swaps H2A w1th H2Az in the nude- Krogan .-t . .tl.(20011

osomes.Coutdins At..11 and Apr4 ~·1•zuguLhl t; t:r.al 1 )0041

proteins tlldt .u-t.· a part of acctyldtion

l·omplex ;\uA4. Also l·ontaim

Rvb I and Rvbl hclil'.l't'S. Involved in

transcription r<'gulation uf many gnws.

(http: //www.chromodb.org; Kwon et. al., 2005) In contrast, metazoan

have only one or two SNF2 genes. This could be a indication that these

ATPases may have more specific roles in plants than their metazoan

counterparts. This also supports the observation that mutation in one of

the SNF2 ATPase is lethal in Drosophila but viable in Arabidopsis like

mutants of AtBRM and AtSYD. This could also be an outcome of

functional redundancy between two or more ATPases as found between

AtSYD and AtBRM (Bezhani et. al., 2007). It has been found that AtSYD

and AtBRM regulates very small number of genes ("' 1 (Yo each of which

20% is common between these two types of ATPases). Regulation of genes

by these ATPases also shows some growth stage preference. The only

chromatin remodeling complex which has been shown to be involved in

abiotic stress is AtCHR12 (see Table LR_3)

Apart from the ATPase subunit, there are many other components in the

ATP-dependent chromatin remodeling complex that have indispensible

roles. The SWI/SNF complexes contain nearly ten components of which

the SWI2/SNF2 ATPase, SNFS and SWI3 have been observed to form the

core complex in yeast, Drosophila and human. These three components

have been shown to remodel chromatin in vitro (Phelan et. al., 1999)

Homologues of these subunits have been reported in plants as well.

In Arabidopsis BUSHY (BSH) has been found to the only homologue of

yeast SNF5p, human INI 1 and Drosophila SNR 1. The ubiquitously

expressed Arabidopsis BSH gene has been observed to partially

complement ySNFSp mutation in yeast but was unable to activate

transcription like ySNFSp (Brzeski et. al., 1999). More recently

homologues of SNFS have been reported from Pisum sativum which has

been found to be functionally similar to BSH gene from Arabidopsis in

molecular level interactions. This PsSNFS gene was found to be

accumulating during last stages of embryo development and in response

to ABA and drought stress in germinating seeds and vegetative tissues

10

Table LR_3:- Different types of ATP-dependent Chromatin Remodeling Factor from plants and their functions

fador Namt· and Type, Fum tion and ffi(xlt~ of at·tion Interacting Rt·l'<"rt·nn'

Organism Prott"'in!<

Splayed (AtSyd) SWIISNF2 like remodeling factor AtSwi3A(strongly) Wagner and

Arabidopsis Functions in vegetative and reproductive A tSwi3B(strongly) ;\1t·ycrowll.l ( 2002)

developmental pathways; AtSwi3C(wt"akly) Su et .al. ( 1006)

Mutant~ shows precautious floral tran~ibons Kwon d.al. (200!))

mainly under nun-mductive short-days does not interact Bezhani d .a I. ( 2007)

conditions; with AtSwi 3D

Required for maintenance of stem apical

meriskms (S.\.\1) during reproductiw phase;

Contains a partial Bromodomain which

appears to be required for stabk binding to

acetylated histones;

Syd Expn:ssion is highest in dividing cells;

During Floral tram11:lons ~yd ,;eems to

function by modulating the activity of LFY

transcription fador;

R~:gulatcs stt'm cdl fate by directly regula-

ting the transcription ofWUSCHEL.

Contams the expression of about I 01(, genes

of Arabidopsis.

N-terminal ATPasc AT-hook containing

region is sufficient for biological dctiVIty;

In ,·itro ATP-dependent chromatin remodel-

ing not yet shown.

Brahma ( AtBR.'\1.) SWI/SNF2 I like rcmodt'!ing factor AtSwi3B (weakly) 1-arrond ..-t.al.( 20(.1+)

Arabidopsis Function:; in vegetabve and reproductive Atswi K(strongly) Hurtado et.dl.( 2006)

developmental pathw.!ys; (via N-terminal H.-zham et. al. (2007)

Functional homologue of dBR.\-1; portion)

Suppo~ed to be a repres~or of photoperiod

dependent llowering pathway and !unctions

in the genetic pdthway of downregulation of does not inter acts

gen··~ like CO:--.iSTA;\;S, FLOWERI~G with AtSwi3A

LOCUS T and SUPPRESSOR OF CO I; .md AtSwdD

FwlCtions as a part of h1gh molct.ular rn~s

complex (determined by gel filtration

chromatography);

net:essary for the transcription of many

homeotic genes like AP2, AP3, PISTILLA,

:--.lAC-liKE and ACTIVATED BY AP3/Pl;

As a whole is rt>;punsiblc for the t>xpression

of about I~;. genes ( espedally thuS<! re<Jmred

at early developmental stage of seedlings);

Involved in locus specific regulation (not a

global regulator).

In vitro Nucleosome mobilization assays

not yet reported.

Fa{:tor ~arne anu Typt>, Function and mode of a(~t.i()n l nh'racting Rt"lt-n•nc ·e

Org.anism Protl"ins

Of tht> total number of gt>nes controllt>d by Bt·zhani et .. d.(2007)

AtSYD and AtBR.\1, about 20% are common.

,V\utants of these hvo genes also sho'" many

common ft>atures (definitely not all). Also

thcy have some common interacting protein

Henc·e, AtBRM and AtSYD havt> many

redundant functions

AtCHRt2 A Brahma type SWI/SNF2 rt:rnodding not yet reported \1lynarova .-t.al.{2007)

Arabidopsis f'.!ctor

Re>ponsible for Growth arn·~t of .-\rabidop~is

pl,mts under environmental ~trt·>s conditions

by bringing changes to the cxprc,:sion of

dormancy related gene~.

Mutant plants are similar to wild types,

indicating the chromatin remodeling factor

$pc·dfkally operates under ~tress condition:<

DDM 1 (Dec:rcase in SWJ/S:'-JF2 Like n·modding Factor :\'ot yet reported l:lr.nski and

DNA Methylation 1 Re:<pomible lor D:'-.IA meythylation of Jnmanow,k1(200 3)

Arabidopsis tran~posons and repeats; J~dddoh <'l.al. (199'1)

Also responsible for maintaining demt·th:vl· Saje et .al. ( 200~)

ated conditions of certain gymc loci like

BONSAI;

In vitro ATP-dept>ndent chromatin n·modd-

ing assay~ shows it can mobilize a nuclt>os·-

orne from tht> end to a mort> <:t>ntral position.

Binds to DNA and nudeo~orne~ nonspedfi.-

cally but preference is mor~· for nud,·osome'

PICKI.E CHDJ type remo<..lcling factor; :'-lot yet n:ported Oga~ t·t.al. ( 199'-;1)

Arabidopsis Involved in gene repre~sion; Fukaki et.al. (2006)

:\'ecessary for repres~ion of LE( 'I (critical

activator of embryo development);

Shows ub1quitou~ expn::::sion pattern;

~egatively regulates auxin-indu~.:ed pericyde

cell div1sion which is neccs~ary for :ateral

root initi.1tion.;

Nc·ces~ary lor miAA medi.1ted repression

of ARF7 and ARF 19

CHRll ISWI-like chromatin remodeling factor; :\'ot yet reported Huanca-Mamani

Arabi<..lopsis Expressed dunng gametogenesis and et.al. (200))

embryogenesis;

requires for plant growth, cotyledon and

female gametophyte development.

indicating its role in ABA-dependent abiotic stress response in Pisum

sativum (Rios et. al., 2007).

SWI3 proteins have also been reported to operate in Arabidopsis.

Arabidopsis genome has been found to quote four SWI3 genes which

have been named as SWI3A, SWI3B, SWI3C and SWI3D. All these

proteins show differential interaction with different SWI/SNF2 ATPases

(see Table LR_3). SWI3B has been reported to interact with FCA (a RNA

binding protein involved in floral development) and is involved in

vegetative and reproductive growth and developmental regulation

(Sarnovski et. al., 2002; Zhou et. al., 2003). The multiplicity of SWI3

genes in Arabidopsis supports that the SWI/SNF complexes are more

specific in function in plants than their metazoan counterparts.

Second messengers and nucleosome remodeling

Different second messengers are known to play vital roles in different

signal transduction networks but their involvement in chromatin

remodeling was not known till recently when the inositol phosphates and

their derivatives were shown to play a role in nucleosome displacement

directly (Shen et. al., 2002; Stegar et. al., 2002)

Via in vitro nucleosome reconstitution studies, it was shown that the

signaling messenger IP6 inhibits the nucleosome mobilization by yeast

ISW2 containing NURF chromatin remodeling complex whereas IP4

stimulates SWI/SNF mediated nucleosome displacement (Shen et.al.

2002).

In vivo studies in yeast mutants defective for genes of inositol phosphate

pathway and different chromatin remodeling complexes revealed that IP4

and IPS are necessary for nucleosome mo?ilization by IN080 and

SWI/SNF complexes on promoter of PH05 gene whose expression

depends on extracellular phosphate level (Stegar et.al. 2002).

11

Various inositol phosphates and its derivatives are known to get

upregulated in response to osmotic and salinity stress (Meijer et.al. 1999;

Dewald et. al., 2001). Hence, a high possibility of occurrence of such

signaling molecule mediated chromatin remodeling at stress responsive

loci within the genome is always there. More recently, in Arabidopsis, it

has been found that ATX1, a functional homologue of Drosophila

trithorax protein, which regulates many homeotic genes during

development, binds to phosphoinositol-5-phosphates (Alvarez-Venegas

et.al. 2006). It was also observed that binding to these PI-5-P relocalizes

ATX1 from cytoplasm to nucleus. Hence, by binding to specific

transcription factors or cofactors, the inositol phosphates can be targeted

to specific locations in the genome where the chromatin remodeling

complexes can be modulated. Role of other signaling molecules also

remained to be seen.

Chromatin remodeling through histone modifications

Post-translational modifications of histones had been the centre of

attraction of transcriptional regulation of genes in all eukaryotes since

the discovery and elucidation of the role of histone acetylation in gene

regulation (Allfrey et. al., 1964). Histones are now known to get modified

in a variety of ways including acetylation, methylation, phosphorylation,

ubiquitination, glycosylation, ADP-ribosylation, carbonylation and

sumoylation. Histone modification in plants regulate gene expression in

response to diverse exogenous stimuli including stress (abiotic and

biotic}, temperature, and light and also to endogenous signals operating

in pathways of growth, development and differentiation (Fuchs et. al.,

2006; Pfluger and Wagner, 2007). Most of the studies of histone

modification in plants have been done on Arabidopsis but recently some

work has been done in rice, maize and few other crop plants.

12

The acetylation (which is an undisputed mark of active genes in all

eukaryotes) of lysine residues histones are known to be mediated by

bromodomain containing HATs (histone acetyltransferases), the

mammalian homologues of which, like GCN5/HAG 1 (belonging to GNAT

family), HAC1 and HAC12 (belonging to CBP/p300), and HAF2/TAF1

(belonging to TAFII family), have been discovered in plants (Bharti et. al.,

2004; Benhamed et. al., 2006; Mao et. al., 2006; Long et. al., 2006; Han

et. al., 2007). Similarly the acetyl group removing HDACs (histone

deacetylase) like HDA19 and HDA6 (belonging to RPD3 family), and

HD2A and HD2B (belonging to plant specific HD2 family) have also been

reported from plant systems (Benhamed et. al., 2006; Ueno et. al., 2007).

The methylations of lysine and arginine residues are achieved mainly by

SET domain containing histone methylatransferases (HMTs). SuvH group

of proteins are responsible for H3K9 and H4K20 methylations in

Arabidopsis (Jackson et. al., 2004; Naumann et. al., 2005; Ebbs and

Bender, 2006) while the arginine methylations are mediated by PRMTs

(protein arginine methyltransferases) (Pei et. al., 2007). The mono­

ubiqitination of H2BK143 (the only known site m plant for this

modification) is established by Ring-type E3 ligase like HUB 1 and are

removed deubiquitinases like SUP32/26(Liu et. al., 2007; Sridhar et. al.,

2007). The methylations from histone residues are removed by JmjC­

domain and LSD1-type HDMs (histone demethylases) or by a process

involving deimination (Berger, 2007; Kouzarides, 2007). Apart from

phosphorylation at H3S 10 and H3S28 which are found in other

eukaryotes also), H2T11 is also phosphorylated in plant nucleosomes

(Pfluger and Wagner, 2007). A particular modification exists in different

variant forms. The methylation on specific lysine residues could be

present in mono-, di- or trimethylation form. The dimethylation of

arginine could have symmetric or asymmetric orientations. All these

variants imparts different set of information on gene regulation. The

various combination of modification within a nucleosome carries specific

13

information about the regulation of genes and has been termed as

"Histone code" and the effect also depends on the position of nucleosome

in the gene (Jenuwein and Allis, 2001).

The histone modifications are one of the important determinants that

differentiate euchromatin from heterochromatin. In Arabidopsis,

H3K9me2 and H3K27me2 have been found to be the specific marker of

heterochromatin in Arabidopsis and the presence of both has been found

to be necessary for the action of chromomethylase3 (Lindroth et. al.,

2004; Jackson et. al., 2004). A particular modification may have different

meaning and occurrence within the genome of different species. In maize,

H3K9me2 is also found in euchromatin (Shi and Dave, 2006).

Reversible dynamic changes of histone modification have been observed

in plants upon subjection to stress. HAC 1 has been found to be

necessary for transcriptional upregulation of heat shock gene HSP17

(Bharti et. al., 2004). Prolonged exposure to cold silences the flowering

time repressor· FLC (Sung and Amasino, 2006; Schmitz and Amasino,

2007). The cold induCible CBF1 has been found to recruit GCNS

containing complex via ADA adapters (Mao et. al., 2006). AtERF7

function in ABA signaling has been suggested to recruit HDA19 via its

interaction with HDAC complex subunit SIN3 (Song et. al., 2005).

Induction of genes in response to abiotic stresses in the tobacco BY2 cell

cultures and in Arabidopsis Cells has been observed to be associated

with rapid increase in H3S 10 phosphorylation and immediately followed

by increase in H3 phosphorylation and H4 acetylation. Changes in H3K4

methylation and H3 acetylation has been observed in submergence

inducible genes m rice also (Tsuji et. al., 2006). Thus histone

·modifications are a major switch in regulation of stress responsive genes.

14

Section III: DNA methylation

DNA is composed of four bases- adinine, guanine, cytosine and thymine.

The cytosine residue is found to be methylated at 5th position in fungi,

plants .and mammals but not in Drosophila, yeast and Coenarhabitis

elgans. This 5- methyl cytosine (5mC) is not synthesized as a separate

base. A set of enzyme play a crucial role in establishing, maintaining and

removing the methyl group on and from cytosine residue. The impact of

cytosine methylation on gene repression and heterochromatin formation

is known for decades but still mystery of mechanism of its establishment

and removal, and way to control gene expression is not solved

completely. Owing to its huge impact on gene regulation, 5-methyl

cytosine is, sometimes, coined as "5th base of DNA" (Pennings et. al.,

2005)

Patterns of DNA methylation and its preservation

In animals the cytosine methylation is particularly found 1n symmetric

CpG residues while in plants it is present on symmetric CpG and CpNpG

residues and asymmetric CpNpN residue (Finnegan et. al., 1998; Bendor,

2004). Asymmetric methylation from human has been reported for very

few locus (Vu et. al., 2000). The CpG and CpNpG methylation are

inherited in the form of hemimethylated sequences. This hemimethylated

strands are then methylated completely by different types of DNA

methyltransferase or methylase(Holliday and Pugh, 1975; Riggs, 1975).

Hence, certain pattern of gene expression is imprinted from parental

DNA to newly synthesized ones (genomic imprinting) (Bender, 2004). On

the other hand the asymmetric DNA methylation, which mainly occurs

as a result of RNA-directed DNA methylation, has to be re-established de

novo after each cycle of replication (Ramsahoye et. al., 2000; Gowher and

Jeltsch, 2001).

15

DNA methylation has been found to impart its negative gene regulatory

mechanisms by the following ways:-

• Changing the chromatin structure and histone modification

(discussed later)

• By bringing a change in DNA bending capacity

• By creating sites for 5mC binding protein (sequence Specific and

Non-Specific) and by removing the non-5mC DNA binding

transcription factors (He et. al., 2001)

• Inhibiting RNA polymerase elongation as reported from Neurospora

(Rountree and Selker, 1997)

DNA methyltransferases

Plant DNA methyltransferases (DNA mtase) are categorized broadly into

three groups (Boyko and Kovalchuk, 2008)

Group I: Dnmt/ Met Class of DNA mtase: This group is exemplified by

plant homologue of mammalian Dnmtl and is responsible for CG

methylation.

Group II: Chromomethylases (CMTs): The plant specific chromo­

methylases are responsible for CNG methylation.

Group III: Domain Rearranged Methyltransferases (DRMs): This group

is responsible for asymmetric .CNN methylation.

Demethylation of DNA is mediated by DNA glycosylasejlyase. These

enzymes generally cause excision of 5-methylcytocine base and

introduction of a nick in the DNA backbone (Zhu et. al., 2000; 2007). The

DNA repair mechanism then add an unmethylated cytosine. Of the

different DNA glycosylasejlyase studied so far, DME has been found to

remove cytosine methylation from maternal alleles of MEA and FW A and

16

hence is involved in genomic imprinting in Arabidopsis (Henderson and

Dean, 2004).

Functions of CpG, CpNpG and CpNpN methylations

The CG methylation has been found to have a larger effect on global

methylation and works in silencing of the heterochromatic region and

transposons of the plants. A global loss of CG methylation along with

release of transcriptional silencing of a number of transposons and

heterochromatic repeats (centromere and pericentromeric sequences)

was observed in mutants affecting CG methylations in Arabidopsis

(Mathieu et. al., 2003; Lippman et. al., 2003, 2004; Kato et. al., 2003;

Zhang et. al., 2006: Zibberman et. al., 2007).

The CNG methylation has been found to be associated with many

transposable elements (Zibberman et. al., 2007; Zhang et. al., 2006) but

only a few transposons were transcriptionally activated in mutants

affecting CNG methylation though there was a significant decrease in

CNG methylation globally (Matheu et. al., 2005; May et. al., 2005;

Vaillant et. al., 2006; Tompa et. al. 2002). It has been concluded that

probably CNG methylation is involved in fine-tuning the regulation on

transposable elements (Vaillant and Paszkowski, 2007)

Similar genome wide and mutant studies have revealed that asymmetric

CNN methylation is involved in locus specific regulation of transposons,

transgenes and endogenous genes (Agius et. al., 2006; Gong et. al., 2002;

Penterman et. al., 2007; Morales-Ruiz et. al., 2006).

DNA methylation and environmental stress

Though being hypothesized for a long time, the first concrete evidence of

modulation of DNA methylation by an external stimuli was obtained by

the observation of cold induced demethylation of nucleosomal core DNA

17

In roots and hypomethylation of ZmM 11 gene (that contain a

retrotransposon like sequence) in maize (Steward et. al., 2000, 2002).

Hypomethylation is also observed for stress related genes in tobacco

plants mutant for MET1 (Wada et. al., 2004). Activation of transposons

and retrotransposons due to loss of DNA methylation like Tos17 in rice,

Ttol and Tntl in tobacco, Tam3 in Antirrhinum etc. in response to abiotic

stress due to hypomethylation are also well documented (Hashida et. al.,

2003, 2006; Hirochika et. al., 1996; Takeda et. al., 1999; Beguiristain et.

al., 2000)

The hypomethylation and demethylation events may be needed for

reshaping the genome (mainly via the activation of transposons) of the

organisms in order to adapt to the changing environment as suggested

by Barbara McClintock decades ago (McClintock 1984) and pointed out

in some recent reviews (Boyko and Kovalchul, 2008)

Hypermethylation of certain regions of the genome have also been

reported in response to many environmental stresses. Inhibition of

flowering in Arabidopsis by transcriptional silencing of FLC by cold

(Henderson and Dean, 2004) was found to be due to DNA

hypermethylation. Recently CpNpG-hypermethylation of CCWGG

sequences in a Satellite DNA in response to salt stress has been reported

in facultative halophyte Mesembryanthemum crystallinum which

switches from C3-photosynthesis to CAM metabolism (Dyachenko et. al.

2006). In this case, CCWGG sequence was not found to be

hypermethylated in the promoter region of CAM pathway enzyme

phosphoenolpyruvate carboxylase and hence, it was suggested the

specific hypermethylation of the Satellite DNA may be needed to form

specialized chromatin structure to regulate a large set of gene and to

adapt to the changed environment and the metabolism pattern.

18

Section IV: Inter-relation between DNA methylation, histone modification, nucleosome positioning and chromatin remodeling

Cytosine methylation has been reported to cause structural transition of

DNA from B- to Z- and A-form (Behe and Felsenfeld, 1981; Tippin et. al.,

1997; Rich and Zhang, 2003;). Z-form of the DNA is incompatible with

nucleosome formation (Nickol et. al., 1982; Garner and Felsenfeld, 1987).

Nucleosome assembly on human FMR1 gene indicates that CpG

methylation significantly lowers histone octamer affinity for longer

repeats like CGG77/76 and CGGNN4s (Wang and Griffith, 1996; Godde et.

al., 1996). While nucleosome that include shorter repeats CGG13, were

more precisely positioned in case of methylated DNA (Godde et. al.,

1996), yet it was found that CpG methylation did not affect widespread

repositioning of nucleosome. Of the tv20 moderate to very strong

positions in which a nucleosome could assemble on human H 19 ICR

(Imprint Control Region), occupancy of two prominent positions were

very much reduced on methylated templates (Davey et. al., 2003). From

various evidences it is now suggested that DNA methylation can affect

nucleosome formation and positioning but it depends on whether 5mC

exocyclic group must come in such a position that it can cause steric

interference with the path of DNA in the nucleosome (Pennings et. al.,

2004).

Many ATP-dependent Chromatin remodeling factors have been shown to

control DNA methylation in plants. The most studied of them is DDM 1.

DDM 1 is suggested to control DNA methylation directly or indirectly by

bringing changes in the histone modifications at transposons and repeat

regions of the genome in Arabidopsis (Johnson et. al., 2002). Apart from

global hypomethylation resulting in ddml mutants of Arabidopsis, .

hypermethylation of certain genic loci is also reported. ddml plants have

been found to be more sensitive to UV-C andy-irradiation. It is suggested

19

that DDM 1 may facilitate localization of MBDs at specific nuclear

domains (Zemach et. al., 2005).

Like ddml plants, mutants for ROS 1 (DNA demethylating protein) also

shows hypermethylation at certain genic loci like BONSAI (BNS),

SUPERMAN (SUP) and AGAMOUS (AG) in Arabidopsis (Jacobson et. al.,

2000; Saze and Kakutani, 2007). In fact, frequent occurrence of ectopic

DNA hypermethylation in global hypomethylation background has been

observed m many other instances. metl, mutant for a DNA

methyltransferase, also shows the same phenomenon (Mathieu et. al.,

2007; Reinders et. al., 2008).

The BNS loci, which is flanked by non-LTR type retrotransposons (LINE),

get hypermethylated while the LINE sequence get hypomethylated upon

repeated self-pollination of ddml mutant Arabidopsis plants. Arabidopsis

plants lacking this LINE insertion at BNS locus does not show

hypermethylation in ddml background. Hence, it was predicted that the

flanking transposons controls the methylation of BNS locus. However,

SUP and AG doesn't contain any transposons near to them, but still gets

hypermethylated in ddml and metl plants (Saze and Kakutani, 2007).

Based on some recent evidences, it been hypothesized that global

hypomethylation triggers both inhibition of DNA methylation and de novo

methylation by RdDM (RNA dependent DNA methylation) pathway which

leads to local hypermethylation of a number of loci (Mathieu et. al., 2007;

Saze and Kakutani, 2007; Saze et. al., 2008).

MAINTENANCE OF METHYLATION! (MOMl) that shares limited

homology to DDMl has been found to be involved in DNA methylation

independent silencing of repetitive DNA sequences in Arabidopsis

(Vaillant et. al., 2006). The release of transgene silencing and 58 repeat

repression without alteration of DNA and histone methylation pattems

(Amedo et. al., 2000; Vaillant et. al., 2006), clearly depicts the existence

20

of methylation-dependent as well as methylation-independent pathways

of epigenetic silencing.

DNA methylation is closely linked to heterochromatization and gene

silencing. In heterochromatin the histones H3 and H4 are found to be

hypoacetylated, dimethylated at K9 and K27 positions of H3 and

hypomethylated at K4 position of H3 (Bender, 2004). It was shown that

loss of methylation in metl mutant Arabidopsis plants is associated with

loss of H3K9 dimethylation. But in mutants for KRYTONITE (KYP;

histone methyl transferase), loss of H3K9 methylation is not associated

with loss of CpO DNA methylation, indicating, H3K9 methylation occurs

downstream to CpO DNA methylation (Jasencakova et. al., 2003). Some

loss of CpNpO methylation is observed in kyp mutants (Jackson et. al.,

2002). Also, proteins like HP1 binds to H3K9 methylations and helps in

spreading of the DNA methylation (Lachner et. al., 2001; Grewal and

Maozed, 2003). Again, the 5mC-Binding Domain Proteins (MBDs), have

been shown to recruit enzymes that modify the histones (Ben-Porath and

Cedar, 2001). Recently, deubiqitination of H2B at K143 has been shown

to be mandatory for RdDM induced H3K9 dimethylation and DNA

methylation at transgenes and transposons (Sridhar et. al., 2007).

In Arabidopsis, COPIA elements are rich in CO-methylation while SN1 is

rich in non-methylation mainly. Mutants with affected CO-methylation

shows reduced H3K9 dimethylation at AtCOPIA loci only whereas

reduction in H3K9 methylation was observed at AtSN 1 loci in mutants

with affected non-CG methylation (although CO- and non-CO

methylations were affected at both the loci respectively). It was

demonstrated that SRA domains of KYP and SUVH6 (both involved in

maintenance of dimethylation of H3K9) shows differential binding to CO­

and Non-CO-methylated DNA. Thus, the maintenance of H3K9

dimethylation could be maintained by different sets of protein at different

//~~~~~ .. TH- I 6 44~ ' ' .. :::: !1!,~- \! >'1 '2-.-~ b t-1\. ~., b% ~~·-

\ . I

<:·::c::>/ 21

locii, suggesting that the functional relationship of DNA and histone

methylations are locus specific (Johnson et. al., 2007).

The control of gene silencing and H3K9 dimethylation by TOUSLED and

RPA2 in a DNA-methylation independent manner in Arabidopsis, suggest

that DNA methylation and histone modifications could be functionally

distinguished also (Kapoor et., al., 2005; Wang et. al., 2007).

Section V: 'Epigenetic memory of stress' can be passed to the next generation

In order to adapt, upon exposure to stress, plants try to reorganize their

genome and pattern of gene expression that involves changes in the

pattern of DNA methylation and chromatin structure. As these changes

were mainly reported from somatic cells, it was not clear if such changes

could be inherited as stress memory to the next generation.

It is known in plants, that rate of homologous recombination is enhanced

by heat and UV-B stress (Ries et. al., 2000; Lebel et. al., 1993). Recently it

was found that the rate of homologous recombination remained at an

elevated level in unstressed progenies of stressed transgenic plant

harboring a construct containing two partial but overlapping fragment of

gene encoding GUS such that a complete functional GUS encoding gene

could only arise by homologous recombination. Hence, the number of

cells undergoing homologous recombination was detected by GUS

histochemical staining (Molinier et. al., 2006). In the same work, it was

inferred that this stress memory is passed to the progenies through

gametes of both male and female stressed plants. This hyper­

recombination memory was further found to be independent of the

presence of transgenic allele in the treated plants. It was concluded that

stress leads to increase genomic flexibility even in successive untreated

generation and may increase the potential for adaptation.

22

Abiotic Stress

Alteration in

Transcription Factor Binding

Alteration in

Nucleosome

positioning

and Histone

Variants

Alteration in

CG and N'on­

CGDNA

Methylation

Alteration in

Histone

Modifications

Genome Reorganization and

Reshaping of Gene Expression ...._

----------- Patt ern

Epigenetic memory of

stress

•

Figure R 1: A schematic representation showing the modulation of transcriptional regulatoin in response to stress and its aftereffects. Stress can trigger any one or more of the transcriptional regulatory swiches in the plant system as shown above. A change in any one of these regulatory modules could induce an alteration in other transcriptional phenomenons which in turn can lead to reorganization of the genome of the stressed plants and a change in gene expression pattern. Such a genome w ide alteration can print the 'epigenetic memory" of the

experienced stress within the genome of plants of subsequent generations.