Di e en ial Pa e n o Exp ession and Suga
Regula ion o A abidopsis haliana ADP-glucose
Py ophospho ylase-encoding Genes*
Recei ed o publica ion, Oc obe 14, 2004, and in e ised o m, No embe 26, 2004
Published, JBC Pape s in P ess, Decembe 14, 2004, DOI 10.1074/jbc.M411713200
Ped o C e ille´n‡, Tiziana Ven iglia‡, F ancisco Pin o§, Alicia O ea‡, A
´ngel Me´ ida‡,
and Jose´ M. Rome o‡¶
F om he ‡Ins i u o de Bioquı´mica Vege al y Fo osı´n esis and §Ins i u o de In es igaciones Quı´micas, Uni e sidad de
Se illa-Consejo Supe io de In es igaciones Cien i icas (CSIC), Ame´ ico Vespucio 49, 41092, Se ille, Spain
ADP-glucose py ophosho ylase (ADP-Glc PPase) ca a-
lyzes he i s and limi ing s ep in s a ch biosyn hesis.
In plan s, he enzyme is composed o wo ypes o sub-
uni s (small and la ge) and is allos e ically egula ed by
3-phosphoglyce a e and phospha e. The pa e n o ex-
p ession and suga egula ion o he six A abidopsis
haliana ADP-Glc PPase-encoding genes ( wo small sub-
uni s, ApS1 and ApS2; and ou la ge subuni s, ApL1–
ApL4) has been s udied. Based on mRNA exp ession,
ApS1 is he main small subuni o ca aly ic iso o m e-
sponsible o ADP-Glc PPase ac i i y in all issues o he
plan . La ge subuni s play a egula o y ole, and he
da a p esen ed de ine a clea unc ional dis inc ion
among hem. ApL1 is he main la ge subuni in sou ce
issues, whe eas ApL3 and, o a lesse ex en , ApL4 a e
he main iso o ms p esen in sink issues. Thus, in
sou ce issues, ADP-Glc PPase would be inely egula ed
by he 3-phosphoglyce a e/phospha e a io, whe eas in
sink issues, he enzyme would be dependen on he
a ailabili y o subs a es o s a ch syn hesis. Suga eg-
ula ion o ADP-Glc PPase genes is es ic ed o ApL3
and ApL4 in lea es. Suga induc ion o ApL3 and ApL4
ansc ip ion in lea es allows he es ablishmen o he -
e o e ame s less sensi i e o he allos e ic e ec o s,
esembling he si ua ion in sink issues. The esul s p e-
sen ed on he exp ession pa e n and suga egula ion
allow us o p opose a gene e olu ion model o he A a-
bidopsis ADP-Glc PPase gene amily.
S a ch is he majo s o age polysaccha ide in plan s and is
accumula ed as g anules in many di e en o gans such as
lea es, oo s, shoo s, ui s, o g ains, whe e i is used as a
ca bon and ene gy sou ce. The egula o y and a e-limi ing
s ep o s a ch biosyn hesis is he syn hesis o he glucosyl
dono , ADP-glucose, by ADP-glucose py ophospho ylase (ADP-
Glc PPase)
1
(EC 2.7.7.27) (1). ADP-Glc PPase is a he e o e -
ame (
␣
2
␤
2) composed o wo di e en ypes o subuni s (1, 2).
The small subuni (SS) is conside ed he ully ca aly ic one,
whe eas he la ge subuni (LS) plays a modula o y unc ion
egula ing he ac i i y o he enzyme (1, 3, 4). Ne e heless,
di e en s udies on ADP-Glc PPase om maize and po a o
ha e sugges ed ha bo h subuni s may ha e a egula o y ole
(5, 6). The ac i i y o mos plan ADP-Glc PPases is inely
egula ed by an allos e ic mechanism (7). The enzyme is ac i-
a ed by 3-phosphoglyce a e (3-PGA) and inhibi ed by P
i
. This
allos e ic modula ion is conside ed o ep esen he main con-
ol o he s a ch biosyn hesis a e in pho osyn he ic issues
and in many non-pho osyn he ic issues (1), and i is exe ed by
he ela i e a io be ween 3-PGA and P
i
. Howe e , in some
plan s, he enzyme om de eloping seeds does no espond o
hese egula o s (8–10). Besides he allos e ic egula ion o
ADP-Glc PPase ac i i y, a pos - ansla ional edox con ol ha
in ol es he ac ion o hio edoxins has been shown (11) and
ac s in esponse o suga s and ligh (12, 13).
Di e en iso o ms o ADP-Glc PPase ha e been desc ibed,
and many cDNAs and genomic DNAs ha e been isola ed om
bo h monoco and dico plan s. In mos cases, a single SS gene
and se e al LS genes a e p esen in a gi en species (1, 4). I has
been pos ula ed ha speci ic issues should p esen di e en
ADP-Glc PPases wi h dis inc egula o y p ope ies ha would
be dependen on he speci ic LS p esen in a gi en issue (3, 14).
Howe e , he e is no comp ehensi e s udy o he exp ession o
all he di e en ADP-Glc PPase iso o ms in a single plan
sys em. Six genes encode o p o eins wi h homology o ADP-
Glc PPase in he A abidopsis genome. Two o hese genes
encode o SS, ApS1 (A 5g48300) and ApS2 (A 1g05610); and
ou o hese genes encode o LS, ApL1 o ApL4 (A 15g19220,
A 1g27680, A 4g39210, and A 2g21590). We ha e shown e-
cen ly ha he A abidopsis la ge subuni s con e di e en eg-
ula o y p ope ies o ADP-Glc PPase in i o and would mod-
ula e he ac i i y o he ADP-Glc PPase enzyme acco ding o
he biosyn he ic s a ch necessi ies in a issue-speci ic manne .
Thus, acco ding o hei kine ic and egula o y p ope ies, he
la ge subuni s we e classi ied as sink and sou ce iso o ms (4).
Howe e , he dis inc ion be ween sink LS and sou ce LS needs
u he con i ma ion because in A. haliana only pa ial in o -
ma ion conce ning some genes has been epo ed (15–18). To
add ess his ques ion, a comple e s udy o he exp ession pa -
e n o all ADP-Glc PPase-encoding genes is needed.
Suga s play a basic ole as subs a es in ca bon and ene gy
me abolism as well as p ecu so s o s a ch biosyn hesis. They
a e also impo an signaling molecules con olling he g ow h
and de elopmen o plan s (19). Wi h ega d o he s a ch
syn hesis pa hway, i has been shown ha suga s media e he
induc ion o se e al genes o he pa hway (20–23), including
some genes encoding o ADP-Glc PPase (24–26), and also
igge he edox egula ion o ADP-Glc PPase ac i i y (12, 13).
Induc ion o ApL3 mRNA le els in esponse o suc ose (15, 27)
* This wo k was suppo ed in pa by G an s BMC2002-00984 and
BIO2003-00431 om he Minis e io de Ciencia y Tecnologı´a (Spain) and
by Jun a de Andalucı´a g oup CVI-281 (Spain). The cos s o publica ion
o his a icle we e de ayed in pa by he paymen o page cha ges.
This a icle mus he e o e be he eby ma ked “ad e isemen ”inac-
co dance wi h 18 U.S.C. Sec ion 1734 solely o indica e his ac .
¶To whom co espondence should be add essed. Tel.: 34-954-489526;
Fax: 34-954-486500; E-mail: [email p o ec ed].
1
The abb e ia ions used a e: ADP-Glc PPase, ADP-glucose py ophos-
pho ylase; SS, small subuni ; LS, la ge subuni ; 3-PGA, 3-phosphoglyc-
e a e; RT-PCR, e e se ansc ip ion-PCR.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 9, Issue o Ma ch 4, pp. 8143–8149, 2005
© 2005 by The Ame ican Socie y o Biochemis y and Molecula Biology, Inc. P in ed in U.S.A.
This pape is a ailable on line a h p://www.jbc.o g 8143
This is an Open Access a icle unde he CC BY license.
and ehalose (28) has been desc ibed in A abidopsis lea es
and seedlings. Howe e , in o ma ion conce ning he e ec o
suga s on he exp ession o he o he ADP-Glc PPase genes
om A abidopsis is sca ce.
The aim o his wo k is o unde s and he ole o he di e en
ADP-Glc PPase iso o ms in A abidopsis haliana, a model sys-
em o s udy s a ch biosyn hesis (29). We p esen no el da a
abou he exp ession pa e n o he six genes encoding o
ADP-Glc PPase iso o ms in di e en o gan and issues, and we
cha ac e ize he e ec o suga supply on he exp ession o
hose genes unde physiological condi ions. The da a p esen ed
p o ide solid e idence ha con i ms a wo king model p oposed
p e iously by us assigning speci ic oles o he di e en ADP-
Glc PPase subuni s (4).
EXPERIMENTAL PROCEDURES
Plan Ma e ial and G ow h Condi ions—A. haliana (Columbia
eco ype) was g own in g ow h cabine s unde a 16-h ligh /8-h da k
pho o- egime a 23 °C (day)/20 °C (nigh ), 70% humidi y, and a ligh
in ensi y a he plan le el o 100 mic oeins eins m
⫺2
s
⫺1
. Plan s we e
sown in soil o mRNA exp ession s udies assays o in pe li e o suga
induc ions assays. Suga induc ion ea men s consis ed o i iga ion o
he ma u e plan s wi h MS medium (30) supplemen ed wi h 100 mM
so bi ol, 100 mMglucose, 100 mMsuc ose, o 25 mM ehalose. We chose
suga concen a ions acco ding o p e ious epo s (15, 17). Du ing
suga ea men s, plan s we e main ained unde he same g owing
condi ions, and samples we e ha es ed a he middle poin o he
pho ope iod (8 h ligh ) du ing 4 consecu i e days, ozen in liquid
ni ogen, and s o ed a ⫺80 °C un il use.
RNA Ex ac ion and Re e se T ansc ip ion—To al RNA was isola ed
as desc ibed by P esco and Ma in (31). Be o e cDNA syn hesis, in
o de o emo e con amina ing genomic DNA, he RNA p epa a ions
we e incuba ed wi h 10 uni s o DNase I FPLC Pu e o 10 min a 37 °C,
ex ac ed wi h phenol and chlo o o m, p ecipi a ed, and dissol ed in
nuclease- ee MilliQ-wa e . Fi s -s and cDNA was syn hesized om
10
␮
g o o al RNA using Moloney mu ine leukemia i us e e se
ansc ip ase and oligo(dT)
12–18
p ime , acco ding o he manu ac u -
e ’s ins uc ions (Ame sham Biosciences). The eac ion was incuba ed
a 37 °C o 2 h and s opped by adding 1 ml o nuclease- ee MilliQ-
wa e . All eagen s we e om Ame sham Biosciences.
Real- ime Quan i a i e RT-PCR Analysis—Real- ime quan i a i e
RT-PCR assays we e achie ed using an iCycle ins umen (Bio-Rad).
The PCR mix u e con ained (in a o al olume o 25
␮
l) 5
␮
l o cDNA, 0.2
mMdeoxynucleo ide iphospha es, 2.5 mMMgCl
2
, a 1:100,000 dilu ion
o SYBR® G een I nucleic gel s ain (Molecula P obes)/ luo escein cal-
ib a ion dye (Bio-Rad), 0.3 uni o Taq polyme ase, 2.5
␮
lo 10⫻Taq
polyme ase bu e , and 0.2
␮
Mo each p ime . Speci ic oligonucleo ides
used we e as ollows: SA253 (5⬘-GAAAAACCAAAAGGGGAGCA-3⬘)
and SA254 (5⬘-CCTCAGTCCAAGGGAAGTGG-3⬘) o ApS1; SA255 (5⬘-
CTCGGTGTTTGCCTCCAAG-3⬘) and SA256 (5⬘-TCTCCTTCCCTTTT-
CTTCTCACA-3⬘) o ApS2; SA257 (5⬘-TCCCCACAGCAAACGACTT-3⬘)
and SA258 (5⬘-GGTGGCAGGTTTCTCCTTGA-3⬘) o ApL1; SA259 (5⬘-
TCGGAAAAACCAAAGGGAGA-3⬘) and SA260 (5⬘-GGCCAACGGGAT-
AATTTCAG-3⬘) o ApL2; SA261 (5⬘-AGATCGGGAAAAACGTGGTG-
3⬘) and SA262 (5⬘-CTCTTTTTAACTTCCGGCCAAAC-3⬘) o ApL3;
SA263 (5⬘-AGCAGATAGGCCAGAGGAAGG-3⬘) and SA264 (5⬘-GCGG-
GAAAGAAAAGATCGAAG-3⬘) o ApL4; and UBQF (5⬘-GATCTTTGC-
CGGAAAACAATTGGAGGATGGT-3⬘) and UBQR (5⬘-CGACTTGTCA-
TTAGAAAGAAAGAGATAACAG-3⬘) o UBQ10. The mal cycling
consis ed o 94 °C o 3 min, ollowed by 40 cycles o 10 s a 94 °C, 15 s
a 61 °C, and 15 s a 72 °C. A e ha , a mel ing cu e was gene a ed o
check he speci ici y o he ampli ied agmen . In he case o ApL4
(p ime s SA263 and SA264), he annealing empe a u e was 64 °C. The
e iciency o all he p ime s a he condi ions desc ibed abo e was
be ween 75% and 110% in all he es ed samples, and p oduc iden i y
was con i med by sequence analysis. A abidopsis Ubiqui in 10 (32) was
used as a housekeeping gene in he exp ession analysis. Absolu e Quan-
i ica ion (33) was pe o med by cloning he ampli ied p oduc s in
pGEM®-T ec o (P omega) and using hem as ex e nal calib a ion
s anda ds.
In Si u Hyb idiza ion Analysis—Fo mRNA in si u hyb idiza ions,
sense and an isense RNA p obes we e ob ained by ansc ip ion o he
3⬘-un ansla ed egion o he A abidopsis ADP-Glc PPase-encoding
genes cloned in o pBluesc ip SK(⫺) ec o (Clon ech) om he T7
p omo e . The digoxigenin-labeled ibop obes we e p epa ed ollowing
he manu ac u e ’s ins uc ions (Roche Applied Science). In si u hyb id-
iza ions we e pe o med as desc ibed by Me´ ida e al. (34), a 50 °C in
a solu ion con aining 2⫻SSC and 50% o mamide; signal was de ec ed,
and he sec ions we e moun ed in En ellan moun ing medium (Me ck).
The sec ions we e obse ed h ough an Olympus BX60 mic oscope by
No ma ski mic oscopy and pho og aphed using a JVC GC-X3E digi al
came a.
RESULTS
The A abidopsis ADP-Glc PPase Genes Show Di e en ial
mRNA Exp ession Le els—We ha e de e mined he absolu e
s eady-s a e mRNA le els o he six A abidopsis ADP-Glc
PPase-encoding genes by eal- ime quan i a i e RT-PCR. Spe-
ci ic p ime s o he six ADP-Glc PPase genes and o he
housekeeping gene, Ubiqui in 10 o A abidopsis (32), we e de-
signed, and hei e iciency and eliabili y we e checked. The
agmen s ampli ied by hese p ime s we e cloned and se-
quenced, and he esul ing plasmids we e used as ex e nal
calib a ion s anda ds (33).
All six mRNAs we e de ec ed in lea es, in lo escences, ui s,
and oo s o ma u e plan s, bu a e y di e en le els (Fig. 1).
ApS1,ApL1,ApL3, and ApL4 showed ele a ed mRNA s eady-
s a e le els, whe eas ApS2 and ApL2 mRNA we e accumula ed
a e y low le els. These da a a e in acco d wi h p e ious
obse a ions in ou labo a o y, in which ApS2 and ApL2 could
no be de ec ed by No he n blo .
2
ApS1 was he majo SS
iso o m in all o gans o he plan . A mo e complex si ua ion was
obse ed o ApL genes. ApL1 was he mos abundan LS in
lea es, wi h le els o mRNA up o h ee imes highe han ha
o ApL3 and mo e han 1 o de o magni ude highe le els o
ansc ip han ApL4. In in lo escences and ui s, ApL3 was
he mos abundan ADP-Glc PPase la ge subuni ansc ip . In
oo s, he le el o exp ession o all ADP-Glc PPase genes was
e y low compa ed wi h lea es, and ApL3 was he p edominan
LS wi h up o 3- old and 1 o de o magni ude highe le els o
ansc ip han ApL4 and ApL1, espec i ely. These da a show
ha he e is a issue-speci ic le el o exp ession o he di e en
ADP-Glc PPase subuni s in A. haliana.
Exp ession Pa e n a he Cellula Le el—The mRNA exp es-
sion pa e n o A abidopsis ADP-Glc PPase genes was s udied
by in si u hyb idiza ion in lea es, in lo escences, and ui s.
2
P. C e ille´n, unpublished obse a ions.
FIG.1.Exp ession p o ile o he A abidopsis ADP-Glc PPase-
encoding genes. The absolu e mRNA le els o all he A abidopsis
ADP-Glc PPase-encoding genes we e de e mined by eal- ime quan i-
a i e RT-PCR as desc ibed unde “Expe imen al P ocedu es.” In he
igu e, quan i ies a e ep esen ed in a loga i hmic plo in o de o
compa e he da a among he di e en genes. The alues a e he a e age
o he quan i ica ion o h ee cDNA p epa a ions om di e en expe -
imen s. The e o ba s in he plo ep esen he S.D.
ADP-Glc PPase Gene Exp ession and Suga Regula ion8144
Fig. 2 shows he exp ession pa e n o he SSs, ApS1 and ApS2.
Bo h genes a e de ec ed in he main s a ch-p oducing issues o
lea es, he mesophyll and he ascula companions cells (Fig.
2, a–d). In lowe s, a s ong signal is de ec ed in he s amens
and pis il, as well as in he ecep acle. This pa e n was ob-
se ed in bo h young (Fig. 2 o ApS1;ApS2 da a no shown)
and ma u e lowe s (Fig. 2, eand g). ApS1 and ApS2 a e also
clea ly exp essed in he emb yo (Fig. 2, hand i). A di e en
si ua ion was obse ed o he ADP-Glc PPase LSs. In lea es,
ApL1, ApL2, and ApL3 ansc ip s a e localized in he meso-
phyll and ascula companions cells (Fig. 3, a–e), bu ApL4
appea ed o be es ic ed only o he su ounding cells o he
ascula eins (Fig. 3, and g). Simila o he SS mRNAs, all
he LS mRNAs we e de ec ed in all issues o lowe s a di e -
en de elopmen al s ages (Fig. 3, h–k). In ui s, ApL1 is ex-
p essed uni o mly in he emb yo (Fig. 3, land m), whe eas
ApL2,ApL3, and ApL4 exp ession was es ic ed o he p o-
ascula cells o he emb yo (Fig. 3, n– ). An unspeci ic signal in
he seed coa ( es a) was obse ed in mos o he hyb idiza ions
wi h ui sec ions. No signal was de ec ed wi h he con ol
sense ibop obes o all he genes (Fig. 2, j–m o ApS1;ApS2
and LS con ol sense hyb idiza ions no shown).
The esul s shown indica e ha he e is no a di e en ial
pa e n o exp ession be ween he wo SSs. On he con a y, a
clea di e en ial exp ession pa e n o he LS genes was ob-
se ed in lea es and ui s. This ac , oge he wi h he di e en
mRNA exp ession le els (Fig. 1), sugges s a issue speci ici y ha
could be ela ed o he ole o each LS iso o m in egula ing he
ac i i y o he he e o e ame ic ADP-Glc PPase enzyme.
ApL3 and ApL4 A e Induced by Suga s in Lea es—Mos
s udies on suga - egula ed ansc ip ion o genes in ol ed in
he syn hesis o s a ch ha e been pe o med wi h seedlings o
de ached lea es (15, 24, 27). This ype o assay, al hough e y
in o ma i e, may no e lec he eal physiological esponse o
he plan , could magni y he di e ences in he mRNA le els,
and is es ic ed o speci ic issues. To o e come his p oblem,
we ha e designed a suga induc ion assay in which all he
di e en o gans o he plan could be ha es ed unde he same
expe imen al condi ions (see “Expe imen al P ocedu es”). A a-
bidopsis plan s i iga ed wi h MS medium we e g own o ma-
u i y. Then, plan s we e i iga ed wi h MS medium supple-
men ed wi h di e en suga s o se e al days. Samples we e
ha es ed a he middle poin o he pho ope iod, and he
mRNA le els we e de e mined by eal- ime quan i a i e RT-
PCR. This expe imen al design e lec s he ca bon s a us o
ma u e plan s in a be e way han in de ached lea es o
seedlings and a he same ime allows global analysis o di e -
en o gans.
Fig. 4 shows he s eady-s a e ADP-Glc PPase mRNA le els in
A abidopsis lea es a e i iga ion wi h MS medium supple-
men ed wi h 100 mMsuc ose. A ime-cou se analysis showed
ha only ApL3 and ApL4 mRNAs we e suc ose-induced and
ha , a e 72 h o ea men , a signi ican induc ion o bo h
genes could be obse ed (a 10- old and an 18- old inc ease in
ApL3 and ApL4 mRNA le els, espec i ely), and induc ion o
bo h genes was e en highe a 96 h. No a ia ion in he an-
sc ip le els was obse ed o ApS1,ApS2,ApL1, and ApL2
ansc ip s. A simila beha io was obse ed when A abidopsis
plan s we e supplemen ed wi h glucose (100 mM) o ehalose
(25 mM) (da a no shown). Resul s in Fig. 5 show he induc ion
o ApL3 and ApL4 a e 72 h o suga ea men . The mRNA
le els o ApL3 we e 4- old highe in he p esence o glucose (100
mM), 10- old highe in he p esence o suc ose (100 mM), and
6- old highe in he p esence o ehalose (25 mM) compa ed
wi h con ol plan s in he absence o added suga s. Induc ion o
ApL4 was e en highe , eaching 20- and 30- old he con ol
le els in he p esence o suc ose and ehalose, espec i ely. No
signi ican e ec was obse ed when plan s we e supplemen ed
wi h so bi ol, hus sugges ing ha suga induc ion o ApL3 and
ApL4 is no due o osmo ic s ess (Fig. 5). We also s udied he
e ec o suga ea men in o he o gans o ma u e A abidopsis
plan s, and no suga induc ion o ADP-Glc PPase-encoding
genes was obse ed in ui s, in lo escences, o oo s (da a no
shown). In summa y, ou da a clea ly show ha only wo genes
encoding o A abidopsis ADP-Glc PPase, ApL3 and ApL4, a e
suga - egula ed in sou ce issues (lea es), whe eas suga eg-
ula ion in sink issues does no con ol he le el o any ADP-Glc
PPase-encoding gene.
FIG.2.Pa e n o exp ession o he A abidopsis ADP-Glc PPase small subuni s. a–d, lea sec ions hyb idized wi h he an isense p obe
agains ApS1 (aand b) and ApS2 (cand d); e–g, lowe s a di e en de elopmen al s ages hyb idized wi h he an isense p obe agains ApS1 (eand
) and ApS2 (g); hand i, longi udinal sec ions o ui s showing he seed wi h he emb yo hyb idized wi h he an isense p obe agains ApS1 (h) and
ApS2 (i). j–m, con ol samples hyb idized wi h he ApS1 sense p obe; hyb idiza ions wi h he ApS2 sense p obe a e no shown. The mRNA in si u
hyb idiza ion expe imen s we e pe o med as desc ibed unde “Expe imen al P ocedu es” using speci ic digoxigenin-labeled ibop obes agains
each SS gene.
ADP-Glc PPase Gene Exp ession and Suga Regula ion 8145
DISCUSSION
We ha e cha ac e ized he exp ession o he A abidopsis
ADP-Glc PPase-encoding genes in di e en o gans and issues,
de e mining hei absolu e ansc ip le els by eal- ime quan-
i a i e RT-PCR and hei exp ession pa e n by in si u hyb id-
iza ions. Al hough he six ansc ip s we e de ec ed in all he
s udied o gans, ou esul s clea ly show ha he di e en
ADP-Glc PPase subuni s a e exp essed a di e en le els and
ha e speci ic exp ession p o iles among he di e en o gans o
he plan . Bo h SS-encoding genes a e exp essed in all he
s a ch-p oducing issues o he plan . Howe e , ApS1 exp es-
sion le el is up o 2 o de s o magni ude highe han ha o
ApS2. I has been shown p e iously ha ApS1 plays a c i ical
ole in s a ch biosyn hesis because adg1-1 mu an , a ec ed in
APS1, shows e y low le els o s a ch accumula ion (35). Be-
sides, in i o s udies wi h he wild ype (4) and a mu a ed
APS2 H137D p o ein
3
ha e sugges ed ha ApS2 is a non-ac i e
iso o m, wi h ApS1 being he only ca aly ic ADP-Glc PPase
subuni in A abidopsis. Ou esul s on he exp ession da a
3
T. Ven iglia, unpublished obse a ions.
FIG.3.Pa e n o exp ession o he A abidopsis ADP-Glc PPase la ge subuni s. a–g, lea sec ions hyb idized wi h he an isense p obe
agains ApL1 (aand b), ApL2 (c), ApL3 (dand e), and ApL4 ( and g); h–k, lowe s a di e en de elopmen al s ages hyb idized wi h he an isense
p obe agains ApL1 (h), ApL2 (i), ApL3 (j), and ApL4 (k); l– , magni ica ions o longi udinal sec ions o ui s showing he seed and he emb yo
hyb idized wi h he an isense p obe agains ApL1 (land m), ApL2 (n), ApL3 (oand p), and ApL4 (qand ). Con ol samples hyb idized agains he
sense p obes a e no shown. The mRNA in si u hyb idiza ion expe imen s we e pe o med as desc ibed unde “Expe imen al P ocedu es” using
speci ic digoxigenin-labeled ibop obes agains each LS gene.
FIG.4. E ec o suc ose on he
mRNA le el o ADP-Glc PPase-encod-
ing genes. The igu e shows a ime-
cou se mRNA exp ession analysis o he
six ADP-Glc PPase-encoding genes in
A abidopsis lea es o plan s induced wi h
100 mMsuc ose. The plan s we e ea ed
wi h suc ose, and he mRNA le els we e
de e mined by eal- ime quan i a i e RT-
PCR as desc ibed unde “Expe imen al
P ocedu es.” The da a in he plo a e no -
malized o he alue in he con ol sample
wi hou induc ion. The alues a e he a -
e age o h ee de e mina ions o wo
cDNA p epa a ions. The S.D. was less
han ⫾10% o each da a poin .
ADP-Glc PPase Gene Exp ession and Suga Regula ion8146
epo ed in his wo k con i m he ole o ApS1 as he main, i
no he sole, SS iso o m esponsible o ADP-Glc PPase ac i i y
in A. haliana.
The ADP-Glc PPase la ge subuni s a e conside ed o play a
egula o y ole, modula ing he ac i i y in esponse o allos-
e ic e ec o s (4). The mRNA le els o he ou genes encoding
o LS show signi ican di e ences. Whe eas ApL1 is he mos
highly exp essed ADP-Glc PPase LS in lea es (Fig. 1), ApL3
eaches he highes le els o exp ession in sink o gans (in lo-
escences, ui s, and oo s). Di e en ia ion is also obse ed a
he cellula le el (Fig. 3). In seeds, ApL1 appea ed o be ex-
p essed in all he emb yo cells, whe eas he o he s LSs a e
p e e en ially localized in he p o- ascula cells. In ma u e
lea es, we clea ly de ec he ApL1,ApL2, and ApL3 mRNAs in
he mesophyll cells as well as in he bundle shee , whe eas
ApL4 was es ic ed o he companion cells o he ascula
eins. We could no de e mine which subuni is exp essed in
he gua d cells o he s oma a, cells ha accumula e signi ican
amoun s o s a ch. Howe e , mic oa ay analysis has shown
ha ApL4 is he mos abundan LS in gua d cells, easse ing
he sink na u e o his gene (36). Thus, a clea dis inc ion
among he LSs can be es ablished. APL1 is he la ge subuni
showing he highes sensi i i y o 3-PGA and P
i
(4) and, by i s
le el o exp ession, would be he main iso o m in sou ce issues,
hus p o iding a ine modula ion o ADP-Glc PPase by he
3-PGA/P
i
a io o adjus s a ch syn hesis o ca bon ixa ion. In
sink issues, APL3 and, o a lesse ex en , APL4 would be he
main LS iso o ms pa icipa ing in he es ablishmen o he
ADP-Glc PPase he e o e ame . These wo LSs ha e been
shown o con e less sensi i i y o 3-PGA and P
i
o he enzyme
(4), and hus s a ch syn hesis would be mo e dependen on he
supply o subs a es om sou ce issues han on he allos e ic
e ec o s. The esul s suppo ou p e ious model in which a
unc ional dis inc ion among he di e en LSs was p oposed on
he basis o he kine ic and egula o y p ope ies ha hey
con e o he ADP-Glc PPase he e o e ame (4).
The specialized unc ion o he di e en ADP-Glc PPase iso-
o ms is also e lec ed in hei esponse o suga s. Coo dina ed
suga ansc ip ional egula ion o se e al s a ch biosyn he ic
genes has been sugges ed by nume ous wo ks (19). Induc ion o
se e al ADP-Glc PPase-encoding genes has been epo ed in
di e en species including A abidopsis (16, 25–28). We show
ha , unde physiological condi ions, only wo ADP-Glc PPase-
encoding genes, ApL3 and ApL4, a e con olled by suga s (Figs.
4 and 5). Suga induc ion o ApL3 and ApL4 genes is lea -
speci ic and does no ake place in sink issue. Induc ion was
s onge in esponse o disaccha ides han o glucose (Fig. 5),
sugges ing ha di e en signaling pa hways may be in ol ed
FIG.5.Suga induc ion o ApL3 and
ApL4 mRNA exp ession le el. A abi-
dopsis plan s we e ed wi h 100 mMglu-
cose, 100 mMsuc ose, 25 mM ehalose, o
100 mMso bi ol, and he ADP-Glc PPase-
encoding gene mRNA le els in lea es
we e de e mined by eal- ime quan i a-
i e RT-PCR as desc ibed unde “Expe i-
men al P ocedu es.” The igu e shows he
esul s o he ApL3 and ApL4 genes. The
da a a e no malized o he alue o he
con ol wi hou induc ion. The alues a e
he a e age o h ee de e mina ions o
wo cDNA p epa a ions. The S.D. was
less han ⫾10% o each da a poin .
ADP-Glc PPase Gene Exp ession and Suga Regula ion 8147

in he ansc ip ional con ol o ApL3 and ApL4 by suga s. To
da e, ehalose- egula ed exp ession o ApL3 has been shown
(28), bu no da a ega ding ApL4 egula ion ha e been pub-
lished. T ehalose eeding escues he pheno ype o he adg2-1
mu an lacking ApL1 (17), and i was p oposed ha unde
hese condi ions, APL3 would subs i u e o APL1. Ne e he-
less, ou esul s demons a e ha ehalose would al e he
subuni composi ion o ADP-Glc PPase by inducing bo h APL3
and APL4.
The low-s a ch mu an adg2-1 con i ms ou hypo hesis ha
in no mal si ua ions, APL1 is he main LS iso o m in he lea
mesophyll. In his issue, he e is also a signi ican amoun o
APL3 ansc ip (Figs. 1 and 3). Howe e , i is e y likely ha
in he me abolic en i onmen o he lea , mos o he s a ch
syn hesis was d i en by he APS1⫹APL1 he e o e ame . In
plan s ed wi h suga s, he in acellula le el o hexoses in-
c eases (27, 37). In his si ua ion, he change o he me abolic
condi ions in he mesophyll and he inc eased ApL3 and ApL4
exp ession would allow he es ablishmen o he e o e ame s
less sensi i e o he allos e ic e ec o s (4), so ha he enzyme
would espond o o be con olled by he supply o subs a es.
This would lead o a highe a e o s a ch syn hesis in esponse
o he highe ca bohyd a e a ailabili y, esembling he si ua-
ion in sink o gans. In ac , suga induc ion ea men s in-
c ease s a ch le els in ou plan s (da a no shown).
Se e al lines o e idence a e in appa en con adic ion wi h
he esul s ha we p esen on suga egula ion o ADP-Glc
PPase iso o ms. In p e ious s udies, i was epo ed ha he
ApL3 p omo e used o he GUS gene was only exp essed in
he bundle shee and ha i s exp ession was induced in he lea
mesophyll cells in esponse o suc ose (15). Howe e , acco ding
o he da a p esen ed in Fig. 2, ApL3 can be de ec ed in he
mesophyll cells o plan s g own in he absence o suga s. The
simples explana ion o his disc epancy is ha he ApL3
p omo e used o he GUS cons uc lacked elemen (s) needed
o i s no mal exp ession. Induc ion wi h suc ose would aise
he mRNA le els in he mesophyll cells, making i possible o
de ec GUS. On he o he hand, esul s ha e also been pub-
lished epo ing ha in A abidopsis de ached lea es, ApS1,
ApL3, and ApL2 genes we e induced by suc ose and glucose,
whe eas ApL1 was ep essed (ApS2 and ApL4 we e no known
a ha ime) (27). This disc epancy is explained by he p obes
and assays used in ha wo k. Sokolo e al. (27) used p obes
based on he a ailable sequences a ha ime, which co e-
sponded o a conse ed egion o he LS genes (38). Thus, hey
de ec ed ApL2, whe eas ou obse a ions using speci ic p obes
indica e ha his gene is unde ec able by No he n blo . Be-
sides, hey used de ached lea es main ained in he da k o he
induc ion assay. In ha case, he e ec o suga ea men
could be mo e se e e o unspeci ic due o highe wa e e apo-
a ion a es o dehyd a ion s ess (15). We ha e analyzed he
e ec o he suga s unde no mal physiological condi ions, and
he only ADP-Glc PPase-encoding genes egula ed by suga s a
he ansc ip ional le el a e ApL3 and ApL4.
The cha ac e iza ion o he exp ession pa e n and suga
egula ion o he ADP-Glc PPase gene amily allows us o
p opose a model o gene e olu ion o his amily in A abidopsis.
APS1 is essen ial o s a ch biosyn hesis and is he ully ca a-
ly ic subuni o he enzyme (4, 35), whe eas APS2 has se e al
amino acid subs i u ions and is no ac i e in i o (4, 39).
Thus, ou hypo hesis is ha he lone unc ional A abidopsis
SS is APS1. APS2, al hough i is s ill exp essed a e y low
le els in he plan , is a non unc ional ADP-Glc PPase subuni
ha could be accumula ing mu a ions h ough a p ocess o
pseudogeniza ion (40).
I is accep ed ha many genes appea by gene duplica ion
(40, 41), and i has been p oposed ha he di e en ADP-Glc
PPase LSs o a plan specie would esul by di e gence and
specializa ion (7). I is also accep ed ha he A abidopsis ge-
nome unde wen se e al ancien ounds o duplica ion (42).
Indeed, ApL3 and ApL4 genes a e loca ed in sis e genomic
egions ha ha e been desc ibed in di e en genomic analysis
(www. ig .o g/ db/e2k1/a h1/A abidopsis_genome_duplica ion.
sh ml). Ou biochemical s udies (4) showed ha bo h LSs con-
e simila egula o y p ope ies o he enzyme, and in he
p esen epo , we show ha bo h ApL3 and ApL4 genes a e
simila ly induced by suga s (Figs. 4 and 5). On he o he hand,
he exp ession pa e n o he wo genes is di e en (Figs. 1 and
3). These ac s suppo he hypo hesis ha hey a e ela ed
genes unde a p ocess o unc ional di e gence. Because i is
no usual o ind win genes so well cha ac e ized, we hink his
is a g ea example o sub unc ionaliza ion ha could be used in
u he genomic analysis (41).
In summa y, we ha e de e mined he exp ession pa e n
and suga egula ion o he six A abidopsis ADP-Glc PPase-
encoding genes. A clea unc ional dis inc ion can be made
be ween he di e en la ge subuni iso o ms ha could be
conside ed as sink (ApL2,ApL3, and ApL4)- o sou ce
(ApL1)-speci ic. Besides, a shi in he ADP-Glc PPase LS
composi ion in esponse o suga eeding allows he enzyme
o be e icien ly egula ed acco ding o he me abolic si ua-
ion and he s a ch necessi ies o a gi en issue. This hypo h-
esis is in acco dance wi h ou p e ious da a conce ning he
kine ic p ope ies o he enzyme (4).
Acknowledgmen s—We a e g a e ul o Te esa Ruı´z o c i ical ead-
ing o he manusc ip . The Plan Cul u e acili ies om he Cen o de
In es igaciones Cien ı´ icas Isla de la Ca uja (CICIC) a e also
acknowledged.
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