ORIGINAL RESEARCH
T ansc ip omic analysis e eals he pa icipa ion o NTRC
in i on homeos asis in A abidopsis
Fe nando Rod íguez-Ma ín
1,2
| Juan M. Pé ez-Ruiz
1,2
| F ancisco J. Cejudo
1,2
1
Ins i u o de Bioquímica Vege al y
Fo osín esis, Uni e sidad de Se illa and CSIC,
Se illa, Spain
2
Depa amen o de Bioquímica Vege al y
Biología Molecula , Facul ad de Biología,
Uni e sidad de Se illa, Se illa, Spain
Co espondence
F ancisco J. Cejudo,
Email: [email p o ec ed]
Juan M. Pé ez-Ruiz,
Email: [email p o ec ed]
Funding in o ma ion
Minis e io de Ciencia e Inno ación, y
Uni e sidades (MICIU)/Agencia Es a al de
In es igación (AEI), G an /Awa d Numbe s:
PID2020-115156GB-I00,
PID2023-146573NB-I00
Edi ed by Y. Allah e diye a
Abs ac
NADPH-dependen hio edoxin educ ase C (NTRC) plays a cen al ole in edox
egula ion o chlo oplas pho osyn he ic me abolism. Acco dingly, A abidopsis (A abi-
dopsis haliana) NTRC-null mu an s show de ec i e pho osyn he ic pe o mance and
g ow h inhibi ion. Rema kably, hese mu an s show almos a wild- ype pheno ype a
he seedling s age, which aises he ques ion o whe he NTRC plays di e en unc-
ions h oughou plan de elopmen . In his wo k, we ha e add essed his issue by
pe o ming ansc ip ome compa isons o A abidopsis wild- ype and n c mu an lines
a seedling and adul s ages o de elopmen . In con as wi h he high impac o
NTRC on lea es om adul plan s, he low ansc ip omic di e ences in seedlings
sugges ed a less ele an unc ion o NTRC a his s age o plan de elopmen . No a-
bly, he n c mu an showed ansc ip omic changes esembling he esponse o Fe
excess h oughou plan de elopmen , hough his esponse was almos unique a
he seedling s age. The lack o NTRC caused al e ed le els o Mn, Zn, Cu, S, P, K and
Na, bu no signi ican di e ences in he con en o Fe, as compa ed wi h he wild
ype. Mo eo e , a he seedling s age, he lack o NTRC caused hype sensi i i y o Fe
de ici bu a p o ec i e e ec in esponse o Fe excess, mos likely due o lowe ROS
accumula ion in he mu an seedlings. Ou esul s e eal he di e en impac s o
NTRC h oughou plan de elopmen and iden i y Fe homeos asis as a p ocess highly
a ec ed by NTRC, mos no ably a he seedling s age.
1|INTRODUCTION
Plan chlo oplas s, he o ganelles ha pe o m pho osyn hesis, a e
he sou ce o me abolic in e media es ha suppo plan g ow h.
Fu he mo e, hese o ganelles ha e an impo an senso ac i i y able
o moni o changes in en i onmen al condi ions, which in luence pho-
osyn hesis pe o mance (Schwenke e al. 2022; Tano and Wood-
son 2022). A cen al egula o y mechanism ha allows he apid
esponse o chlo oplas pho osyn he ic pe o mance o en i onmen al
cues, such as ligh in ensi y and da kness, is based on he p o ein
di hiol-disul ide exchange, which is he basis o hiol-dependen edox
egula ion (Mi le and Jones 2024; Sies e al. 2024). This egula o y
mechanism, which la gely elies on he p o ein disul ide educ ase
ac i i y o hio edoxins (TRXs), is uni e sally ound in any ype o
o ganism, om bac e ia and ungi o animals and plan s.
In con as wi h he e o ophic o ganisms, which ha bou wo, a
mos h ee, TRXs ha use educing powe om NADPH ia he pa -
icipa ion o an NADPH-dependen TRX educ ase (NTR), he TRX
gene amily in plan s is ema kably complex (Meye e al. 2012).
Among plan cell compa men s, chlo oplas s a e he o ganelles wi h
Recei ed: 10 Janua y 2025 Accep ed: 18 Ma ch 2025
DOI: 10.1111/ppl.70203
Physiologia Plan a um
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Physiologia Plan a um. 2025;177:e70203. wileyonlinelib a y.com/jou nal/ppl 1o 14
h ps://doi.o g/10.1111/ppl.70203
he highes con en o TRXs (Za agnini e al. 2019; Geigenbe ge
e al. 2017), which a e g ouped in o ypical and a ypical iso o ms
(Chibani e al. 2021). Though i emains o be es ablished he sou ce
o educing powe o all chlo oplas TRXs, i is known ha ypical
TRXs, such as hose o he ypes mand , which a e he mos abun-
dan in plan chlo oplas s (Okegawa and Mo ohashi 2015), a e
educed by e edoxin (FDX), he inal accep o o he pho osyn he ic
elec on anspo chain ia he ac ion o a FDX-dependen TRX
educ ase (FTR) (Schü mann and Buchanan 2008). Thus, he FDX-
FTR-TRXs edox sys em links he edox egula ion o chlo oplas
enzymes o ligh . Chlo oplas s ha bou an addi ional edox sys em,
NTRC, an NTR wi h a TRX domain a he C- e minus (Se a o
e al. 2004), which has a high a ini y o NADPH (Be nal-Baya d
e al. 2012). The inding ha he hyd ogen pe oxide sca enging
enzyme 2-Cys pe oxi edoxin (PRX) is e icien ly educed by NTRC
(Alkhal ioui e al. 2007; Moon e al. 2006; Pé ez-Ruiz e al. 2006), led
o he p oposal o an an ioxidan unc ion o his enzyme. Howe e ,
he A abidopsis n c mu an , which is de oid o NTRC, shows a a he
se e e g ow h inhibi ion pheno ype (Lepis ö e al. 2009; Pé ez-Ruiz
e al. 2006; Se a o e al. 2004), and impai men o di e en edox-
egula ed p ocesses suppo ing he p oposal ha NTRC ac s as a cen-
al hub in chlo oplas edox egula ion, as ecen ly e iewed (Cejudo
e al. 2021).
The inding ha A abidopsis mu an s combining he de iciencies
o NTRC and 2-Cys PRXs eco e a WT-like pheno ype unco e ed
he essen ial unc ion o 2-Cys PRXs in con olling he educing
capaci y o chlo oplas TRXs, which allows op imiza ion o he pho o-
syn he ic pe o mance o he o ganelle in esponse o changes in ligh
in ensi y (Pé ez-Ruiz e al. 2017; Yokochi e al. 2021). Mo eo e ,
NTRC is essen ial o he ac i i y o TRXs o he ypes xand du ing
he ea ly s ages o plan de elopmen (Ojeda e al. 2017). Howe e ,
n c seedlings de elop no mal g een co yledons (Lepis ö e al. 2009;
Ojeda e al. 2021), and nei he he lack no he o e exp ession o
NTRC has any e ec on emb yogenesis in de eloping seeds (Galla do-
Ma ínez e al. 2023) in con as wi h i s ele an ole a la e s ages
o plan de elopmen . Hence, NTRC has di e en ial unc ions
h oughou plan de elopmen : a poo con ibu ion a ea ly s ages o
plan de elopmen when chlo oplas biogenesis occu s, whe eas in
ully di e en ia ed chlo oplas s om ma u e lea es, his enzyme plays
a key ole in he edox egula ion o pho osyn hesis in esponse o
changes in ligh in ensi y.
Despi e he ex ensi e analysis o he unc ion o NTRC in adul
lea es, hence, wi h ully di e en ia ed chlo oplas s, he unc ion o
his enzyme a ea lie s ages o plan de elopmen emains poo ly
known. In his wo k, we ha e add essed his issue by pe o ming a
compa a i e analysis o he ansc ip omes o he A abidopsis n c
mu an , as compa ed o WT, in seedlings and lea es om adul plan s.
Ou esul s show a low ansc ip omic impac o he lack o NTRC a
he seedling s age, in con as o he se e e impac on adul lea es.
Rema kably, hese analyses e ealed ha he lack o NTRC a ec s he
exp ession o genes in ol ed in Fe homeos asis, igge ing ansc ip-
omic changes esembling he esponse o Fe excess, which is mo e
ele an a he seedling s age.
2|MATERIALS AND METHODS
2.1 |Biological ma e ial, g ow h condi ions, and Fe
ea men s
A abidopsis haliana WT (eco ype Columbia) and n c mu an
(Se a o e al. 2004) we e ou inely g own in soil in g ow h chambe s
unde sho -day (8/16 h ligh / da k) pho ope iod a 22 and 20C
du ing ligh and da k pe iods, espec i ely, and ligh in ensi y o
125 μmol m
2
s
1
. WT and n c seedlings we e g own on Mu ashige
and Skoog (MS) medium con aining 0.35% (w/ ) Gel i e (Duche a)
unde con inuous ligh a 22C and 125 μmol m
2
s
1
.
Fe ea men s we e pe o med in syn he ic media (pH 5.5, 0.7%
aga ) con aining 5 mM KNO
3
, 2.5 mM KH
2
PO
4
, 2 mM MgSO
4
,2mM
Ca(NO
3
)
2
,70μMH
3
BO
3
,14μM MnCl
2
,10μM NaCl, 1 μM ZnSO
4
,
0.5 μM CuSO
4
, 0.2 μMNa
2
MoO
4
, 4.7 mM MES (Rod íguez-Celma
e al. 2013) and a a iable concen a ion o Fe-EDTA as sou ce o Fe.
Fo Fe ea men s a he seedling s age, seeds we e ge mina ed in
syn he ic media con aining ei he 50 μM Fe-EDTA (Fe-su icien , con-
ol condi ion), 500 μM Fe-EDTA (Fe-excess), o medium wi hou Fe-
EDTA and supplemen ed wi h 100 μM e ozine (sodium 4-[3-(py idin-
2-yl)-6-(4-sul ophenyl)-1,2,4- iazin-5-yl]benzene-1-sul ona e) o che-
la e esidual Fe (Fe-de icien ). Fe ea men s a he adul s age we e
pe o med on plan s g own in Fe-su icien medium o 21 days and
ans e ed o Fe-su icien (con ol condi ions), Fe-excess o
Fe-de icien media o 9 days.
2.2 |RNA ex ac ion, RNA-Seq and RT-qPCR
analyses
Two di e en ansc ip omics expe imen s, a he adul and seedling
s ages o plan de elopmen , we e ca ied ou . Fo adul samples,
eigh -week-old sho -day g own WT and n c plan s we e andomly
selec ed, and young ose e lea es we e collec ed du ing he day
pe iod (a e 2 h o illumina ion a 125 μmol m
2
s
1
). The expe imen-
al design consis ed o h ee biological eplica es o each geno ype,
each o hem con aining lea es om h ee indi idual plan s. WT aw
RNA-Seq da a om he adul s age can be ound in Gene Exp ession
Omnibus, iden i ied wi h accession numbe GSE147793. RNA
ex ac ion was pe o med om pooled lea samples using he Su e
P ep ki (Fishe ), ollowing he manu ac u e 's ins uc ions. RNA con-
cen a ion and pu i y we e es ed by an Agilen 2100 Bioanalyze , a
mic o luidics-based pla o m ha pe o ms quali y con ol o DNA
and RNA samples be o e sequencing. Lib a y cons uc ion o cDNA
molecules was ca ied ou ollowing he manu ac u e 's ins uc ions
using he T uSeq S anded To al RNA wi h Ribo-Ze o ki (Illumina).
The gene a ed DNA agmen s we e sequenced wi h he Illumina
HiSeq 4000 pla o m a STAB-VIDA (Capa ica, Po ugal), yielding
app oxima ely 60–80 million 150 base pai s long pai ed-end eads o
each sample. Fo seedling samples, six-day-old WT and n c mu an
seedlings, g own unde con inuous ligh in MS medium and lacking
ue lea es, we e collec ed. The expe imen al design consis ed o
2o 14 RODRÍGUEZ-MARÍN ET AL.
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h ee biological eplica es o each geno ype. RNA ex ac ion and
quali y con ol we e ca ied ou ollowing he abo e indica ed p oce-
du e o adul samples. The cDNA lib a ies we e cons uc ed acco d-
ing o he manu ac u e 's ins uc ions using he Illumina S anded
mRNA p ep liga ion ki (Illumina). Sequencing o he DNA agmen s
was pe o med wi h he Illumina Nex Seq 500 pla o m a Cen o
Andaluz de Biología Molecula y Medicina Regene a i a (CABIMER,
Se ille, Spain), yielding app oxima ely 18–26 million 75 base pai s long
single-end eads o each sample.
Fo all samples, he Fas QC so wa e package was used o con ol
he quali y (h p://www.bioin o ma ics.bab aham.ac.uk/p ojec s/ as qc/).
Once he good quali y o all samples had been de e mined, ead mapping
o The A abidopsis Col-0 TAIR 10 e e ence genome (h ps://plan s.
ensembl.o g/) and ansc ip assembly we e pe o medwi h heso wa e
ools HISAT2 and S ingTie (Pe ea e al. 2016) using de aul pa ame-
e s. Di e en ial exp ession analysis was ca ied ou wi h he Ball-
gown (Pe ea e al. 2016) and LIMMA (Ri chie e al. 2015) R packages.
Di e en ially exp essed genes (DEGs) we e selec ed using a old
change o ± log
2
(2) and P- alue <0.05 compu ed acco ding o a mod-
e a ed - es . Volcano plo s and Venn diag ams we e gene a ed using
he R packages ggplo 2 and eule . Gene On ology (GO) e m en ich-
men analysis o e he di e en gene se s was pe o med wi h he
en ichGO unc ion o he Bioconduc o R package Clus e P o ile
(Yu e al. 2012). F om he same R package, he compa eClus e unc-
ion was used o compa e he di e en gene on ology e ms o adul
and seedling samples. P incipal Componen analysis was ca ied ou
using he p comp unc ion om he R package s a s o he R
Co e Team.
Fo Real- ime quan i a i e PCR (RT-qPCR) analysis, o al RNA
was ex ac ed using he T izol eagen (In i ogen) and cDNA
syn hesis was pe o med wi h 1 μg o o al RNA using he Maxima
i s -s and cDNA syn hesis ki (The mo Scien i ic) acco ding o manu-
ac u e 's ins uc ions. RT-qPCR was pe o med using an IQ5 eal- ime
PCR de ec ion sys em (Bio-Rad) wi h oligonucleo ides lis ed
in Supplemen al Table S1. Exp ession le els we e no malized
using ACTIN2,UBIQUITIN10 and UBIQUITIN-CONJUGATING ENZYME9
as e e ence genes.
2.3 |Measu emen s o chlo ophyll le els and
de e mina ion o pho osyn he ic pa ame e s
Chlo ophyll le els we e measu ed as p e iously desc ibed (Pé ez-Ruiz
e al. 2006). Room empe a u e chlo ophyll a luo escence was mea-
su ed using a pulse-ampli ude modula ion luo ome e IMAGING-
PAM M-Se ies (Walz). The maximum quan um yield o PSII was
assayed a e incuba ion o plan s in he da k o 30 min by calcula ing
he a io o he a iable luo escence, F
, o maximal luo escence,
F
m
(F
/F
m
). Induc ion- eco e y cu es we e pe o med using ed
(635 nm) ac inic ligh a 81 μmol m
2
s
1
o 10 min. Sa u a ing
pulses o ed ligh a 10,000 μmol m
2
s
1
in ensi y and 0.6 s du a-
ion we e applied e e y 60 s, and eco e y in da kness was eco ded
o up o 10 min. The pa ame e Y(II), co esponding o he espec i e
quan um yield o PSII pho ochemis y, was calcula ed acco ding o
epo ed equa ions (K ame e al. 2004).
2.4 |De e mina ion o anion supe oxide con en
The de ec ion o supe oxide anion con en was pe o med wi h ni o-
blue e azolium (NBT) s aining, as p e iously desc ibed (Co doba
e al. 2016), wi h some modi ica ions. B ie ly, a leas six seedlings o
WT and n c mu an g own in Fe-de icien , Fe-su icien (Fe-con ol)
and Fe-excess media we e andomly selec ed and incuba ed in s ain-
ing bu e (10 mM phospha e pH 7.6, 10 mM NaN
3
and 0.1% NBT
(Sigma)) o 2.5 h a oom empe a u e in agi a ion. Then, he
NBT solu ion was emo ed, and a bleaching solu ion (e hanol: ace ic
acid: glyce ol; 3:1:1) was added. Once he chlo ophyll had been
emo ed, a new bleaching solu ion was added o he seedlings, which
we e s o ed a 4C un il images we e aken wi h an Olympus Mic o-
scope. Fo he quan i ica ion o he a ea o co yledons s ained wi h
NBT, colou h esholds we e used in he ImageJ so wa e.
2.5 |Ionomics de e mina ions
Fo WT and he n c mu an , whole ose e samples o 8-week-old
plan s g own in soil unde sho -day condi ions and 4-day-old seed-
lings g own unde con inuous ligh in MS medium we e lyophilized o
de e mina ion o ion con en by induc i ely coupled plasma mass
spec ome y (ICP-MS) a he Se icio de Análisis, Ins i u o de Recu -
sos Na u ales y Ag obiología de Se illa (IRNASE, CSIC), Se ille, Spain.
In b ie , 4 mL o HNO
3
(65%) was added o he weighed lyophilized
ma e ial. The diges ion was ca ied ou unde p essu e in a mic owa e
o en (MARS ONE CEM). The ea ed samples we e dilu ed wi h ul a-
pu e wa e and passed h ough nylon il e s. Ion de e mina ion was
pe o med using an AGILENT 7800 ICP-MS. P incipal Componen
analysis was ca ied ou using he p comp unc ion om he R pack-
age s a s o he R Co e Team.
2.6 |S a is ical analysis
S a is ical analyses we e pe o med using G aphPad P ism e sion
6.01 (G aphPad So wa e, Inc.), and he expe imen al esul s we e
analyzed using wo- ailed S uden 's - es s.
3|RESULTS
3.1 |Compa a i e ansc ip omic analyses e eal a
poo impac o NTRC a he seedling s age o plan
de elopmen
Wi h he aim o de e mining he impac o NTRC a di e en s ages
o plan de elopmen , we ha e pe o med a compa a i e
RODRÍGUEZ-MARÍN ET AL.3o 14
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ansc ip omic analysis o A abidopsis WT and he n c mu an a
seedling and adul s ages. To ha end, genome-wide ansc ip omes
we e de e mined by RNA-Seq analysis o h ee independen biological
samples ha es ed om young lea es o 8-week-old A abidopsis WT
and n c mu an plan s g own unde sho -day pho ope iod (adul
s age) and om seedlings ha had been g own o 6 days on MS
medium unde con inuous ligh (seedling s age) (Figu e 1A, C). Though
he n c mu an con ained lowe chlo ophyll le els a bo h de elop-
men al s ages, he di e ence was mo e signi ican a he adul s age
(Figu e 1B, D). The sequencing quali y o he samples was assessed by
he high pe cen age o mapped eads and co ela ion be ween biolog-
ical eplica es (a ound 99%) (Figu e S1A-D). A p incipal componen
analysis showed ha he n c and WT seedling eplica es clus e ed
oge he , whe eas eplica es om adul n c clus e ed sepa a ely om
he WT (Figu e S2), hus indica ing a mo e ele an impac o NTRC
on he ansc ip omes a adul han a seedling s ages.
The compa ison o he ansc ip omes om lea es o he adul
plan s o he WT and he n c mu an , using a old change o gene
exp ession o ±log
2
(2) and p< 0.05, iden i ied a la ge se o 535 DEGs
in he n c mu an , 358 up egula ed and 177 down egula ed
(Figu e 2A). S ikingly, a ela i ely low numbe o DEGs (94), o which
35 we e up egula ed and 59 down egula ed (Figu e 2B), we e iden i-
ied in he compa ison o he WT and he n c mu an ansc ip omes
a he seedling s age. The lis s o up egula ed and down egula ed
genes in he n c mu an a bo h de elopmen al s ages a e a ailable
as supplemen al in o ma ion (Supplemen al Appendix S1). A Venn
diag am ep esen a ion o DEGs a adul and seedling s ages iden i-
ied an o e lapping se o 24 DEGs common o bo h s ages
(Figu e 2C). Thus, he highe numbe o DEGs be ween he n c
mu an and he WT a he adul s age con i ms he deep impac o
NTRC on plan g ow h, in line wi h he se e e g ow h inhibi ion phe-
no ype o he n c mu an (Figu e 1A, B), whe eas he modes an-
sc ip omic changes shown by he n c seedlings e eals he poo
impac o NTRC a his ea ly de elopmen al s age, which is in line wi h
he almos WT-like pheno ype o he n c seedlings (Figu e 1C, D).
Mo eo e , he low numbe o DEGs commonly de ec ed a seedling
and adul s ages indica es ha he pa icipa ion o NTRC h oughou
plan de elopmen is es ic ed o a ew biological p ocesses.
3.2 |The n c mu an shows ansc ip omic
changes esembling he esponse o Fe excess
To iden i y he p ocesses a ec ed by he lack o NTRC a adul and
seedling s ages, a gene on ology (GO) e m en ichmen analysis was
pe o med (Supplemen al Appendix S2). In line wi h he se e e g ow h
inhibi ion pheno ype o he n c mu an , he compa ison o he WT
and n c mu an ansc ip omes a he adul s age showed he pleio-
opic e ec s caused by he absence o NTRC, a ec ing a wide ange
o biological p ocesses, as p e iously epo ed (Lepis ö e al. 2009).
Up egula ed genes in he n c mu an a his de elopmen al s age a e
en iched in GO e ms in ol ed in me abolism (ca bohyd a es, amino
FIGURE 1 G ow h
pheno ypes o A abidopsis WT
and n c mu an a adul and
seedling s ages o de elopmen .
Rep esen a i e pho og aphs o
WT and n c mu an plan s g own
unde sho -day pho ope iod o
8 weeks (A) and seedlings g own
in MS unde con inuous ligh o
6 days (C). Chlo ophyll le els in
WT and n c adul lea es (B) and
seedlings (D), de e mined om a
leas 10 eplica es, ep esen ed as
a e age alues ± SD. As e isks
ep esen signi ican di e ences
compa ed wi h he WT (*,
p< 0.05, ***p< 0.001;
S uden 's - es ).
4o 14 RODRÍGUEZ-MARÍN ET AL.
Physiologia Plan a um
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acids and e apy oles), abio ic s ess esponse (hypoxia, nu ien
le el, ni a e, ligh , hea , me al ion), bio ic s ess esponse (insec , sal-
icylic acid), and de oxi ica ion (Figu e 3A). In con as , GO e ms
ela ed o glucosinola e and glyce olipid me abolisms, cell wall o gani-
za ion, cellula homeos asis, esponse o s a a ion and ion anspo
we e he biological p ocesses iden i ied among he down egula ed
genes in he n c mu an (Figu e 3B). A he seedling s age, mos
en iched GO e ms among he up egula ed genes in he n c mu an
we e ela ed o me al ion homeos asis (Figu e 3C), whe eas GO e ms
associa ed wi h cellula homeos asis, esponse o s a a ion and ion
anspo , as well as esponse o me al ion and jasmonic acid biosyn-
hesis, we e iden i ied among he down egula ed genes (Figu e 3D).
To u he analyze he impac o NTRC a di e en s ages o plan
de elopmen , Venn diag ams we e cons uc ed iden i ying common
GO e ms among up egula ed and down egula ed genes in he n c
mu an a adul and seedling s ages (Figu e 4A, B and Supplemen al
Appendix S3). This analysis iden i ied GO e ms ela ed o de oxi ica-
ion and homeos asis o Fe en iched a bo h s ages o de elopmen
(Figu e 4C). The ac ha many o he DEGs be ween he n c mu an
and he WT om adul and seedling s ages iden i ied e ms such as
“In acellula i on homeos asis”,“I on ion homeos asis”,“Mul icellula
o ganismal-le el i on homeos asis”,“Response o i on ion”and
“Response o i on s a a ion”(Figu e 4C) e ealed he e ec o NTRC
on he ansc ip ion o genes in ol ed in Fe homeos asis a bo h
de elopmen al s ages he e analyzed, a p e iously un ecognized unc-
ion o NTRC, which seems mo e ele an in seedlings han in lea es
om adul plan s.
In plan s, Fe homeos asis in ol es di e en o gans and issues
including oo s and he ascula sys em and, in lea pho osyn he ic
cells, di e en cell compa men s (Liang, 2022). Thus, o igu e ou he
e ec o NTRC on Fe homeos asis a di e en de elopmen al s ages,
DEGs in ol ed in Fe homeos asis o esponse o Fe in he ansc ip-
omic analyses o seedling and adul samples we e selec ed, and hei
old-changes in he le el o exp ession we e ep esen ed in di e en
o gans and cell compa men s (Figu e 5A). Genes encoding pep ides
(FEP1, FEP2, IMA4, IMA6) ha posi i ely egula e he ansc ip ion
ac o s bHLH38, bHLH39, bHLH100 and bHLH101, all o hem
in ol ed in ac i a ion o he exp ession o genes esponsi e o Fe
de iciency, we e ound among he mos down egula ed genes in seed-
lings and, a lowe ex en , in adul lea es o he n c mu an
(Figu e 5A), hence indica ing ha he lack o NTRC igge s ansc ip-
omic changes esembling he esponse o Fe excess. This no ion was
u he suppo ed by he iden i ica ion o genes in ol ed in he ans-
po o Fe h ough he phloem and xylem, such as OLIGO PEPTIDE
TRANSPORTER 3 (OPT3) among he down egula ed genes a bo h
s ages. Fu he mo e, genes encoding he Fe anspo e in oo cells
IRON REGULATED TRANSPORTER 1 (IRT1) and enzymes o he side e-
in biosyn he ic pa hway we e also down egula ed in seedlings o he
n c mu an (Figu e 5A). These genes showed di e ences only a
he seedling s age due o he na u e o he adul samples, which
excluded oo issues. In lea es cells, he compa a i e ansc ip omic
analyses e ealed up egula ion o genes encoding anspo o Fe o
he acuoles, VACUOLAR IRON TRANSPORTER-LIKE 1,2and 5(VTL1,
FIGURE 2 Compa a i e ansc ip omic analysis be ween he WT
and he n c mu an a seedling and adul s ages o de elopmen . RNA-
Seq analyses we e ca ied ou wi h RNA samples ex ac ed om young
lea es o 8-week-old A abidopsis WT and n c mu an plan s ha had
been g own unde sho -day pho ope iod (125 μEm
2
s
1
)(adul s age).
Fo he seedling s age, he WT and he n c mu an we e g own o
6 days on MS medium unde con inuous ligh (125 μEm
2
s
1
). Volcano
plo s ep esen ing di e en ially up egula ed ( ed), down egula ed (blue)
and unal e ed (black) genes o n c e sus WT compa isons a adul (A)
and seedling (B) s ages. (C) Venn diag am indica ing he common DEGs
be ween adul and seedlings s ages in he n c mu an .
RODRÍGUEZ-MARÍN ET AL.5o 14
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VTL2, VTL5) and he chlo oplas PERMEASE IN CHLOROPLASTS 1
(PIC1), bo h a adul and seedling s ages (Figu e 5A). Fu he mo e,
genes in ol ed in Fe-S assembly (NEET) and e apy ole biosyn hesis
(HEMA1) in o he chlo oplas and, ema kably, genes encoding e i-
ins (FER1,FER3 and FER4) we e up egula ed in adul and seedlings
samples o he n c mu an , u he suppo ing he no ion ha he lack
o NTRC igge s a Fe excess ansc ip omic esponse. On he con-
a y, CONSERVED IN THE GREEN LINEAGE AND DIATOMS 27
(CGLD27), which is equi ed o g ow h unde Fe s a a ion (U zica
e al. 2012), was down egula ed in he n c mu an a bo h de elop-
men al s ages. Among genes encoding enzymes in ol ed in he sca -
enging o eac i e oxygen species (ROS), o med in he p esence o Fe
ia he Fen on eac ion, s omal APX (sAPX) was up egula ed in he
n c mu an , whe eas FeSOD1-3 genes we e down egula ed in mu an
seedlings and FeSOD1 up egula ed in adul lea es. Finally, se e al o
he DEGs be ween he n c mu an and he WT we e chosen o ali-
da e he esul s o he RNA-Seq da a by RT-qPCR analysis. The lowe
le el o ansc ip s o he bHLH38,bHLH39,FEP1 and OPT3 genes
and he highe le els o he FER1 and NEET genes in adul lea es
(Figu e 5B) and seedlings (Figu e 5C) o he n c mu an alida ed he
esul s o he ansc ip omic analysis.
3.3 |E ec o Fe excess and de ici ea men s on
pho osyn he ic pe o mance
To gain a deepe insigh in o he unc ion o NTRC in me al ion
homeos asis, we sough o analyze he e ec o Fe de iciency and
excess ea men s on he n c mu an pe o mance a he wo de el-
opmen al s ages analyzed. Fo seedling ea men s, WT and n c seeds
FIGURE 3 Gene On ology e m en ichmen analysis o DEGs in he n c mu an a seedling and adul s ages o plan de elopmen . En iched
map plo showing he 50 mos en iched gene on ology e ms o di e en se s o genes in n c mu an e sus WT compa isons a adul (A, B) and
seedling (C, D) s ages. Biological p ocesses en iched in he n c mu an among he up egula ed genes a adul (A) and seedling (C) s ages and
among down egula ed genes a adul (B) and seedling (D) s ages. Node size ep esen s he numbe o genes associa ed wi h each gene on ology
en iched e m, wi h edges connec ing o e lapping gene se s. Colou g adien om blue o ed ep esen s low o high signi icance le els. GO
e ms clus e s we e manually anno a ed.
6o 14 RODRÍGUEZ-MARÍN ET AL.
Physiologia Plan a um
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we e ge mina ed and g own o 4 days unde con inuous ligh in a
syn he ic medium supplemen ed wi h 50 mM Fe-EDTA (Fe-con ol,
su iciency condi ions), 100 mM e ozine and no added Fe-EDTA
(Fe-de iciency condi ions) and 500 mM Fe-EDTA (Fe-excess condi-
ions) (Figu e 6A). To analyze he e ec a adul s age, WT and n c
plan s g own o 21 days unde sho -day pho ope iod and Fe-
su icien condi ions (Fe-con ol) we e ans e ed o Fe-con ol, Fe-
de iciency o Fe-excess syn he ic media and g own o 9 days. The
pho osyn he ic pa ame e s F /Fm, which e lec s PSII s abili y, and
quan um yield o PSII pho ochemis y (Y(II)) we e de e mined as ead-
ou s o he ea men s (Figu e 6A-C).
The Fe de iciency ea men caused no signi ican di e ences in
he le els o F /Fm ei he a adul o seedling s ages o he WT and
n c mu an (Figu e 6A, B). Howe e , he quan um yield o PSII (Y(II))
e ealed a sligh e ec o his ea men a he adul s age in he n c
mu an , which was mo e se e e a he seedling s age (Figu e 6A, C),
hence indica ing he sensi i i y o he n c mu an o Fe de iciency,
which is highe a ea ly s ages o de elopmen . The Fe excess ea -
men , which did no signi ican ly a ec he WT a he adul s age,
caused sligh ly highe le els o F /Fm (Figu e 6A, B), bu no signi ican
a ia ion o Y(II) (Figu e 6A, C) in adul n c plan s. In con as , his
ea men had a se e e e ec on WT seedlings, as e ealed by he
lowe le els o F /Fm (Figu e 6A, B) and Y(II) (Figu e 6A, C). Rema k-
ably, he lack o NTRC igge ed a signi ican ole ance o Fe excess a
he seedling s age, as shown by he highe le els o F /Fm (Figu e 6A, B)
and Y(II) (Figu e 6A, C) in seedlings o he n c mu an .
I is known ha Fe excess may be po en ially oxic o plan s by
igge ing ROS o ma ion by he Fen on eac ion (Halliwell and Gu -
e idge 1992). Thus, he be e pe o mance o he n c mu an seed-
lings unde Fe excess migh be due o al e ed ROS homeos asis
caused by he lack o NTRC. To es his possibili y, seedlings o he
WT and he n c mu an subjec ed o Fe excess and de iciency
FIGURE 4 GO e m
en ichmen analysis o common
DEGs in seedling and adul
samples o he n c mu an . Venn
diag am o GO e ms en iched
among up egula ed (A) and
down egula ed (B) genes in he
n c mu an a adul and seedling
s ages as compa ed wi h he
WT. (C) Do plo ep esen ing
common GO e ms a adul and
seedling s ages. Do size
ep esen s he a io o genes
associa ed wi h he GO e m
among he o al numbe o DEGs.
Colou g adien om blue o ed
ep esen s low o high
signi icance le els.
RODRÍGUEZ-MARÍN ET AL.7o 14
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ea men s we e s ained wi h NBT o de ec he con en s o supe ox-
ide anion (Figu e 7A). No signi ican di e ences in NBT s aining
be ween he WT and n c mu an seedlings we e obse ed unde
con ol condi ions o in esponse o Fe de iciency; in con as , he
ea men wi h Fe excess caused highe le els o NBT s aining, hence
o supe oxide anion, in WT, bu no in he n c mu an seedlings
FIGURE 5 Summa y o he old-changes in he exp ession o selec ed DEGs in ol ed in Fe homeos asis in adul and seedling s ages and
alida ion o he RNA-Seq da a. (A) A colou g adien om blue o ed h ough whi e is used o ep esen changes o exp ession, ep esen ed as
log
2
( old-change), o genes encoding p o eins ela ed o Fe homeos asis selec ed om he compa a i e ansc ip omic analyses be ween he WT and
n c mu an a seedling (squa es) and adul (ci cles) s ages o de elopmen . P o eins a e placed in hei p edic ed cell compa men s (chlo oplas ,
acuole and nucleus) and issues ( oo s and ascula ). (B, C) RT-qPCR analysis o selec ed genes up egula ed o down egula ed in he n c mu an .
T ansc ip le els o selec ed genes speci ically up egula ed (FERRITIN1 and NEET) o down egula ed (bHLH38, bHLH39, FEP1 and OPT3)in hen c
mu an , acco ding o RNA-Seq da a, we e de e mined by RT-qPCR using RNA isola ed om young lea es o plan s g own unde sho -day condi ions
o 8 weeks (B) o om seedlings g own unde con inuous ligh o 6 days (C). Fo each gene, he le els o ansc ip s we e no malized agains h ee
e e ence genes (as indica ed in Ma e ials and me hods) and e e ed o hei le els o exp ession in he WT, which was a bi a ily conside ed as
100%. Values ep esen he mean ± SD o h ee echnical eplica es. Gene-speci ic oligonucleo ides a e lis ed in Table S1.
8o 14 RODRÍGUEZ-MARÍN ET AL.
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FIGURE 6 Legend on nex page.
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