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.