Tempo al and spa ial amewo ks suppo ing plan
esponses o ege a ion p oximi y
Ped o Pas o -And eu,
1
Jo di Mo eno-Rome o,
1,2,3
Mikel U din-B a o,
2
Julia Palau-Rod iguez,
2
Sandi Paulisic,
1
Elizabe h Kas anaki,
4
Vicen e Vi es-Pe is,
5
Au elio Gomez-Cadenas,
5
Anna Es e e-Codina,
6
Bea iz Ma ín-Mu ,
6
An ía Rod íguez-Villalón,
4
Jaume F. Ma ínez-Ga cía
1,2,
*
1
Cen e o Resea ch in Ag icul u al Genomics (CRAG), CSIC-IRTA-UAB-UB, Ba celona 08193, Spain
2
Ins i u e o Plan Molecula and Cell Biology (IBMCP), CSIC-UPV, València 46022, Spain
3
Depa amen de Bioquimica I Biologia Molecula , Uni e si a Au ònoma de Ba celona, Ba celona 08193, Spain
4
G oup o Plan Vascula De elopmen , Swiss Fede al Ins i u e o Technology (ETH) Zu ich, Zu ich CH-8092, Swi ze land
5
Depa amen de Biologia, Bioquimica I Ciències Na u als, Uni e si a Jaume I, Cas elló de la Plana 12071, Spain
6
Func ional Genomics Team, Cen o Nacional de Análisis Genómico (CNAG), Uni e si a de Ba celona, Ba celona 08028, Spain
*Au ho o co espondence: [email p o ec ed]
The au ho esponsible o dis ibu ion o ma e ials in eg al o he indings p esen ed in his a icle in acco dance wi h he policy desc ibed in he Ins uc ions o
Au ho s (h ps://academic.oup.com/plphys/pages/Gene al-Ins uc ions) is: Jaume F. Ma ínez-Ga cía ([email p o ec ed]).
Abs ac
A e he pe cep ion o ege a ion p oximi y by phy och ome pho o ecep o s, shade-a oide plan s ini ia e a se o esponses known as
he shade a oidance synd ome (SAS). Shade pe cep ion by he phy och ome B (phyB) pho o ecep o unleashes he PHYTOCHROME
INTERACTING FACTORs and ini ia es SAS esponses. In A abidopsis (A abidopsis haliana) seedlings, shade pe cep ion in ol es apid
and massi e changes in gene exp ession, inc eases auxin p oduc ion, and p omo es hypoco yl elonga ion. O he componen s, such as
phyA and ELONGATED HYPOCOTYL 5, also pa icipa e in he shade egula ion o he hypoco yl elonga ion esponse by ep essing i .
Howe e , why and how so many egula o s wi h ei he posi i e o nega i e ac i i ies modula e he same esponse emains unclea .
Ou physiological, gene ic, cellula , and ansc ip omic analyses showed ha (i) hese componen s a e o ganized in o 2 main
b anches o modules and (ii) he connec ion be ween hem is dynamic and changes wi h he ime o shade exposu e. We p opose a
model o he egula ion o shade-induced hypoco yl elonga ion in which he empo al and spa ial unc ional impo ance o he
a ious SAS egula o s analyzed he e helps o explain he coexis ence o di e en ia ed egula o y b anches wi h o e lapping ac i i ies.
Recei ed Oc obe 17, 2023. Accep ed July 7, 2024.
© The Au ho (s) 2024. Published by Ox o d Uni e si y P ess on behal o Ame ican Socie y o Plan Biologis s.
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion-NonComme cial-NoDe i s licence (h ps://c ea i ecommons.o g/
licenses/by-nc-nd/4.0/), which pe mi s non-comme cial ep oduc ion and dis ibu ion o he wo k, in any medium, p o ided he o iginal wo k is no al e ed o ans-
o med in any way, and ha he wo k is p ope ly ci ed. Fo comme cial e-use, please con ac [email p o ec ed] o ep in s and ansla ion igh s o ep in s. All
o he pe missions can be ob ained h ough ou Righ sLink se ice ia he Pe missions link on he a icle page on ou si e— o u he in o ma ion please con ac
[email p o ec ed].
In oduc ion
When plan s g ow in high densi y, he close p oximi y o ege a ion
migh obs uc sunligh and pose a h ea o plan su i al. Plan s
ha e adop ed con as ing a oidance o ole ance s a egies o
deal wi h ege a ion p oximi y o shade (Ma inez-Ga cia and
Rod iguez-Concepcion 2023). Speci ically, when shade-a oide
(sun-lo ing) plan s ace his scena io, hey display a se o esponses
known as he shade a oidance synd ome (SAS). Some o he SAS e-
sponses acclima e pho osyn hesis o e en ual ligh sho age caused
by he p esence o neighbo ing plan s; o he s ocus on edi ec ing
g ow h o escape om shade by p omo ing ei he s em elonga ion
and/o apical dominance ( educed b anching) o lowe ing o p o-
duce seeds (Casal 2012; Roig-Villano a and Ma ínez-Ga cia 2016;
Mo elli e al. 2021). A he seedling s age, hypoco yl elonga ion is
likely he bes cha ac e ized and mos conspicuous SAS esponse
in he shade-a oide plan A abidopsis (A abidopsis haliana) (Casal
2012; Ma ínez-Ga cia e al. 2014) and he ocus o his wo k.
Plan s de ec neighbo ege a ion as changes in he ed (R) o
a - ed ligh (FR) a io (R:FR). Plan s abso b R and e lec mainly
FR om sunligh . Unde low plan ing densi y, he in ensi y o in-
coming sunligh du ing he day changes bu he R:FR (>1.2)
emains ela i ely cons an (Smi h 1982). By con as , when neigh-
bo ing plan s a e close enough, hey can sense plan p oximi y by
de ec ing he e lec ed FR om o he plan s ha combines wi h
sunligh and esul s in a mode a e dec ease in he R:FR (R:FR 0.5
o 0.3) wi hou educing ligh in ensi y. When neighbo ing ege a-
ion is dense o ming a plan canopy, pho osyn he ic pigmen s o
he uppe lea es ac as selec i e il e s ha p e e en ially abso b
and deple e blue and R om sunligh bu ansmi pa o g een
and mos FR (Casal 2012). Plan canopy shade p esen s a d as ic e-
duc ion o R compa ed o he FR ha esul s in a e y low R:FR a io
(R:FR<0.06) and a low ligh in ensi y in he pho osyn he ic ac i e
adia ion egion (Ma ínez-Ga cia e al. 2014; Pie ik and de Wi
2014; Fio ucci and Fankhause 2017). The educed R:FR occu ing
unde bo h p oximi y and canopy shade ac s as a eliable signal in-
dica i e o he nea by p esence o ege a ion ha is pe cei ed by
he phy och ome pho o ecep o s (Fig. 1).
As molecula swi ches, phy och omes exis in 2 pho ocon e i-
ble iso o ms (an inac i e R-abso bing P o m and an ac i e
FR-abso bing P o m) ha a e p esen in an equilib ium ha de-
pends on he p e ailing R:FR. Unde high R:FR (low ege a ion
densi y), mos phy och omes a e in he ac i e P o ms and SAS
Plan Physiology, 2024, 196, 2048–2063
h ps://doi.o g/10.1093/plphys/kiae417
Ad ance access publica ion 14 Augus 2024
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is supp essed, whe eas unde low R:FR (high ege a ion densi y),
he pho oequilib ium mo es owa d he inac i e P o m and
SAS is induced. F om he 5 phy och omes cha ac e ized in A. hali-
ana (phy och ome A [phyA] o phyE), phyA and phyB ha e he
main oles in con olling SAS esponses. Gene ic and physiological
analyses indica e ha pho os able phyB is he majo phy o-
ch ome con olling he SAS (Casal 2012; Ma ínez-Ga cia e al.
2014). Addi ional gene ic analyses also showed ha phyA, he
only pho olabile phy och ome, has an an agonis ic ole o e phyB
in he SAS con ol, pa icula ly unde e y low R:FR mimicking
plan canopy shade. Unde low R:FR (p oximi y shade), wild- ype
and phyA mu an seedlings p esen a simila hypoco yl elonga ion
whe eas phyB mu an s display longe hypoco yls. In con as ,
unde e y low R:FR (canopy shade), wild- ype and phyB seedlings
elonga e less han when g own unde low R:FR and phyA seedlings
p esen an exagge a ed hypoco yl leng h. This indica es ha phyB
is deac i a ed by bo h p oximi y (low R:FR) and canopy ( e y low R:
FR) shade whe eas phyA ac i i y is induced only by e y low
R:FR (Fig. 1) (Yano sky e al. 1995; Ma ínez-Ga cia e al. 2014;
Molina-Con e as e al. 2019). I has been shown ha unde
e y low R:FR condi ions, phyA p o ein ends o accumula e
(Ma ínez-Ga cia e al. 2014; Yang e al. 2018; Molina-Con e as
e al. 2019). Fo cla i y, we will use he e m simula ed shade o e-
e o any ea men , including p oximi y, canopy, o o he simila
condi ions, ha lowe s he R:FR bu has no been speci ically de-
ined as such (Roig-Villano a and Ma inez-Ga cia 2022).
SAS implemen a ion is egula ed by, a leas , he in e ac ion o
ac i e phyB wi h PHYTOCHROME INTERACTING FACTORS (PIFs),
a amily o basic-helix-loop-helix (bHLH) ansc ip ion ac o s.
When in e ac ing wi h ac i e phyB, PIFs a e phospho yla ed.
Phospho yla ion igge s he deg ada ion o PIF1, PIF3, PIF4, and
PIF5 (known as he PIF qua e [PIFQ]) ia he 26S p o easome. By
con as , PIF7 phospho yla ion has li le e ec on i s s abili y bu
i inhibi s i s DNA-binding ac i i y (Li e al. 2012) and p omo es cy-
oplasm e en ion, educing i s nuclea impo (Huang e al. 2018).
Ei he case, unde high R:FR, PIF ansc ip ional ac i i y is inhibi ed
by he ac i e o m o phyB, whe eas deac i a ion o phyB unde low
R:FR esul s in PIF accumula ion in he nucleus and/o p omo ion o
hei DNA-binding ac i i y. This ini ia es a ansc ip ional cascade
ha leads o he exp ession o dozens o PHYTOCHOME RAPIDLY
REGULATED (PAR) genes, se e al o which encode ansc ip ion
ac o s om a ious amilies (e.g. bHLH, HD-Zip, and BBX) ha ing
posi i e, nega i e, o e en complex oles in implemen ing he hypo-
co yl elonga ion esponse (Sessa e al. 2005; Roig-Villano a e al.
2006, 2007; So in e al. 2009; Ci uen es-Esqui el e al. 2013; Ciol i
e al. 2013; Gangappa e al. 2013; Kohnen e al. 2016; Gallemi e al.
2017; Gomme s e al. 2017; Bu i e al. 2020).
PIF7, oge he wi h a mino con ibu ion o PIF4 and PIF5, has a
majo and posi i e ole in p omo ing he shade-induced hypoco yl
elonga ion. pi 7 and he pi 4 pi 5 pi 7 ( om now on pi 457) showed
an a enua ed and almos null hypoco yl elonga ion in esponse
o simula ed shade (Li e al. 2012; de Wi e al. 2015). F om he a -
ious PAR genes, induc ion o YUCCAs (YUCs) con ibu es o auxin
p oduc ion oge he wi h SHADE AVOIDANCE 3 (SAV3, also known
as TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1/WEAK
ETHYLENE INSENSITIVE 8 [TAA1/WEI8]) in he 2-s ep indole-3-ace ic
acid (IAA) pa hway om yp ophan (T p). Indeed, he T p amino-
ans e ase encoded by SAV3 ca alyzes he con e sion om T p
o indole-3-py u ic acid (IPA), and he la in monoxigenase encoded
by YUC genes ca alyzes he IPA oxida i e deca boxyla ion o IAA
(Fig. 1) (B umos e al. 2014; Zheng e al. 2016). The single sa 3 and
mul iple mu an s in YUC genes (yuc2 yuc5 yuc8 yuc9 and yuc3 yuc5
yuc7 yuc8 yuc9) had a enua ed shade-induced hypoco yl elonga ion
(Li e al. 2012; Kohnen e al. 2016). Toge he , hese esul s highligh
he impo ance o SAV3- and YUC-media ed p oduc ion o IAA in
his SAS esponse.
Ano he PAR gene wi h a well-known nega i e ole in he
shade-induced hypoco yl elonga ion is LONG HYPOCOTYL IN
FAR-RED 1 (HFR1) ha encodes a ansc ip ional co ac o o he
bHLH amily s uc u ally ela ed o PIFs bu lacks he phyB- and
DNA-binding abili y (Gals yan e al. 2011; Ho ni schek e al. 2012).
HFR1 he e odime izes and inhibi s he ac i i y o all 4 PIFQ membe s
(PIF1, PIF3, PIF4, and PIF5) (Fai child e al. 2000; Ho ni schek e al.
2009; Shi e al. 2013) and PIF7 (Zhang e al. 2019; Bu i e al. 2020;
Paulisic e al. 2021). h 1 hypoco yls display an opposed pheno ype
o ha o pi 7 o pi 457 seedlings; i.e. hey a e longe han wild- ype
ones unde simula ed shade (Roig-Villano a e al. 2007; Ciol i e al.
2013; de Wi e al. 2016). Ano he PIF an agonis is ELONGATED
HYPOCOTYL 5 (HY5), known o encode a ansc ip ion ac o o he
basic domain-leucine zippe (bZIP) amily. HY5 exp ession is
phyA-dependen , and i is no apidly o s ongly induced in esponse
o ce ain shade condi ion (Ciol i e al. 2013). Hypoco yls o he hy5
mu an seedlings elonga e mo e han he wild- ype ones unde
low R:FR (Sella o e al. 2011; Bou-To en e al. 2015; an Gelde en
e al. 2018; O iz-Alcaide e al. 2019). The e o e, HY5 ac s as a nega-
i e SAS egula o . CONSTITUTIVELY PHOTOMORPHOGENIC 1
(COP1) is an E3 ubiqui in ligase ha in e ac s wi h and modula es
he abundance o se e al SAS egula o y componen s, including
HY5 and HFR1. COP1 accumula es in he nucleus unde shade,
coun e ac ing he nega i e impac o HY5 and HFR1 accumula ion
and modula ing he e o e his esponse (Pacín e al. 2013, 2016).
Nuclea -po e complex componen s, chlo oplas -de i ed signals,
and epigene ic componen s also p e en an excessi e esponse o
shade, p o iding addi ional le els o egula ion o his esponse
(Gallemi e al. 2016; O iz-Alcaide e al. 2019; Ma ínez-Ga cia and
Mo eno-Rome o 2020).
Figu e 1. Simpli ied model ha depic s he gene ic componen s
analyzed in his wo k in ol ed in plan neighbo de ec ion. Colo
indica es he posi i e (blue) o nega i e (pink) con ibu ion o he
shade-induced hypoco yl elonga ion. Aspec o ep esen a i e seedlings
jus be o e (W-g own 2-d-old, bo om le ) and a e he shade
ea men (shade-g own 7-d-old, bo om igh ) is shown. A ows
indica e posi i e and ba s ep esen nega i e egula o y ela ionships.
Ques ion ma k indica es an unknown egula o y ela ionship be ween
he connec ed componen s.
Shade-induced hypoco yl elonga ion in A abidopsis | 2049
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The mechanisms ha connec SAS componen s ha e been es-
ablished in a ew cases: (i) HFR1 inhibi s PIF ac i i y; (ii) HY5 ap-
pea s o be mainly associa ed wi h phyA ac ion (Ciol i e al.
2013; Zhang e al. 2018), al hough i has no been explo ed; and
(iii) o he ansc ip ion ac o s, including he g ow h-p omo ing
PIFs, would be mos ly linked o he phyB-dependen pa hway
(Casal 2012; Roig-Villano a and Ma ínez-Ga cia 2016) (Fig. 1).
The an agonis ic phyA/HY5/HFR1 and phyB/PIF/SAV3 ac i i ies
likely p o ide young seedlings wi h he capaci y o apidly elon-
ga e when impeding compe i ion is nea by and also o a enua e
excessi e g ow h when g owing unde a canopy. Howe e , se e al
key aspec s o he gene ic a chi ec u e o he SAS egula o y ne -
wo k emain unclea . These include ea u es ega ding whe he
hese pa hways o componen s ope a e concu en ly on he
same cell ype and, i hey do, how hey a e connec ed (Fig. 1).
To add ess hese issues, we ha e ca ied ou (i) gene ic analyses,
o es ablish i di e en SAS componen s wo k in he same o di -
e en egula o y b anches o modules o he ne wo k; (ii) empo-
al analyses, o lea n when he di e en componen s analyzed ac
in con olling he shade-induced hypoco yl elonga ion; and (iii)
spa ial analyses, o iden i y he cells a ge ed along he hypoco yl
axis epide mis by each SAS egula o . Besides, we explo ed molec-
ula connec ions be ween HY5 and PIFs, 2 an agonis ic SAS
componen s. Ou indings indica ed ha hese componen s a e
g ouped in, a leas , 2 main modules o b anches ha ac a di e -
en imes and impac he elonga ion o dis inc cells along he hy-
poco yl axis. We also show ha HY5 ac s as a node, al hough
i s unc ional ela ionship wi h PIF457 changes wi h he ime o
shade exposu e.
Resul s
The SAS egula o y ne wo k is o ganized in a
leas 2 gene ically di e en ia ed modules o
b anches
We i s p epa ed a se ies o gene ic c osses ocusing on a ew mu-
an s in nega i e (phyA, phyB, HY5, and HFR1) o posi i e (SAV3,
PIF4, PIF5, and PIF7) SAS componen s (Fig. 1). These mu an s e-
sul in s ong shade- ela ed hypoco yl pheno ypes. F om hese
componen s, only PIF4, PIF5, and PIF7 (PIF457) show some edun-
dancy in con olling he shade-induced hypoco yl elonga ion
(Li e al. 2012; He sch e al. 2014).
The hypoco yl leng h o he single and double phyA and phyB
mu an seedlings in esponse o simula ed shade was i s ana-
lyzed (Fig. 2). In con inuous whi e ligh (W) ( ha simula es sun-
ligh o high R:FR), he leng h o Col-0 and phyA hypoco yls was
simila , whe eas ha o phyB hypoco yls was longe and hose o
phyA phyB double mu an seedlings we e he longes , as expec ed.
In con inuous W+ FR ( e y low R:FR), phyA hypoco yls we e longe
and phyB hypoco yls sho e , espec i ely, han he wild ype
(Fig. 2, A and B). The an agonis ic ac i i y o phyA and phyB unde
simula ed shade indica es ha he W +FR condi ions employed in
hese expe imen s mimic canopy shade, in con as wi h hose
p oximi y shade condi ions in which phyA ac ion is negligible
(Ma ínez-Ga cia e al. 2014). Impo an ly, he phyA phyB hypoco-
yl leng h in W+FR was e en longe han in W (Fig. 2B), in ag ee-
men wi h he conclusion ha o he phy och omes egula e he
shade-induced hypoco yl elonga ion (De lin e al. 2003). To be e
isualize he e ec o simula ed shade in con olling hypoco yl
elonga ion, he di e ence in hypoco yl leng h in W+FR and
W (HYP
W + FR
−HYP
W
) was calcula ed (Fig. 2C). This ep esen a ion
showed ha phyA phyB double mu an hypoco yls had an
in e media e shade-induced elonga ion esponse compa ed o
hose o phyA and phyB single mu an s (Fig. 2C), sugges ing ha
he e ec o he 2 phy och omes is addi i e. This is in e p e ed
as indica i e ha hese 2 phy och omes ac likely independen ly
o one ano he in con olling he shade-induced hypoco yl leng h.
In he ollowing se o expe imen s, he HYP
W + FR
−HYP
W
is shown
when compa ing he di e en mu an s ( aw da a a e included as
Supplemen a y da a).
We nex p oduced double mu an s de icien in o he nega i e
egula o s (phyA h 1, hy5 h 1, and phyA hy5) and analyzed
hei shade-induced hypoco yl elonga ion esponse (Fig. 3;
Supplemen a y Fig. S1) (no phyB mu an was included in hese
c osses as i s pheno ype was obse ed mo e clea ly in W).
Seedlings o phyA h 1 and hy5 h 1 double mu an s elonga ed
mo e han he single mu an s (Fig. 3, A and B), sugges ing ha hey
wo ked addi i ely, in ag eemen wi h p e ious in o ma ion (Kim e
al. 2002; Ciol i e al. 2013). By con as , phyA hy5 seedlings elonga ed
as much as he phyA single mu an (Fig. 3C), indica ing ha phyA
was epis a ic o e HY5. Du ing dee iola ion unde monoch oma ic
FR, hund eds o phyA-associa ed genes ha a e phyA egula ed
ha e been iden i ied as pu a i e di ec a ge s o phyA. These di ec
a ge s a e likely o be co a ge ed by phyA in associa ion wi h many
known ligh - ela ed ansc ip ion ac o s, such as HY5 (Chen e al.
2014). I is he e o e expec ed ha se e al o he DNA-binding o
ligh - ela ed ansc ip ion ac o s ac downs eam phyA. Following
his model, epis asis e lec s he ups eam ac i i y o phyA o e
HY5 unde ou e y low R:FR condi ions, and i also in ol es he addi-
ional ac ion o o he ac o s (e.g. HYH). Toge he , ou gene ic anal-
yses sugges ed ha (i) phyA and HY5 ac in he same b anch o he
SAS egula o y ne wo k and ha (ii) HFR1 ac s independen ly o
phyA and HY5 in con olling his esponse.
We also gene a ed mul iple mu an s o posi i e and nega i e
SAS egula o s (Fig. 4; Supplemen a y Fig. S2). As be o e, phyB mu-
an s we e excluded om hese c osses. Pheno ypic analyses
Figu e 2. Gene ic in e ac ion o phyA and phyB in he shade-induced
hypoco yl elonga ion. A) Ca oon showing he design o he expe imen .
Seedlings we e ge mina ed and g own in W (R:FR>1.5) o 2 d, and hen
hey ei he main ained in W o ans e ed o simula ed shade (W+FR,
R:FR, 0.02) o 5 mo e days. On Day 7, pic u es we e aken and hypoco yl
(HYP) leng h was measu ed. B) HYP leng h o Col-0, phyA-211, phyB-9,
and phyA-211 phyB-9 double mu an seedlings a e g owing in W
(HYP
W
) o W+ FR (HYP
W + FR
). Values a e means and e o ba s a e SE o 3
independen eplica es. C) Elonga ion esponse (HYP
W + FR
−HYP
W
) o
lines shown in B). Values o HYP
W
and HYP
W + FR
(shown in B) we e used
o calcula e HYP
W + FR
−HYP
W
. SE was p opaga ed acco dingly. In B),
di e en le e s deno e signi ican di e ences (2-way ANOVA wi h he
Tukey es , P<0.05) among means. In C), as e isks indica e signi ican
di e ences (based on he 2-way ANOVA) be ween he mu an and
wild- ype geno ypes in esponse o simula ed shade (**P<0.01).
2050 | Plan Physiology, 2024, Vol. 196, No. 3
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showed ha h 1 pi 7 leng h was in e media e be ween he
single mu an s (Fig. 4A; Supplemen a y Fig. S2A) consis en wi h
HFR1 in e ac ing wi h and inhibi ing PIF7 ac i i y (Fio ucci and
Fankhause 2017; Bu i e al. 2020; Paulisic e al. 2021). The
shade-induced elonga ion o hypoco yls o an h 1 pi 457 quad u-
ple mu an line was educed compa ed o h 1 pi 7. This is
Figu e 3. Gene ic in e ac ion o SAS-nega i e egula o s in he shade-induced hypoco yl elonga ion. Di e ence o hypoco yl leng h (HYP) in simula ed
shade, W + FR (HYP
W + FR
) and W (HYP
W
) o Col-0, A) phyA-211, h 1-5, phyA-211 h 1-101, B) hy5-2, h 1-5, hy5-2 h 1-1, C) phyA-501, hy5-2, and phyA-501
hy5-2. Seedlings we e g own as in Fig. 2A. Means and SE o 3 independen eplica es we e used o calcula e he shown alues o HYP
W + FR
−HYP
W
and o
p opaga e he SE. E o ba s a e he p opaga ed SE. Black as e isks indica e signi ican di e ences (based on he 2-way ANOVA) be ween he mu an and
wild- ype geno ypes in esponse o simula ed shade (**P<0.01). In A) and C), ed as e isks indica e signi ican di e ences (based on he 2-way ANOVA)
be ween he double mu an s and phyA single geno ypes in esponse o simula ed shade (**P<0.01; ns, no signi ican ).
Figu e 4. Gene ic in e ac ion o pai s o SAS-nega i e and -posi i e egula o s in he shade-induced hypoco yl elonga ion. Di e ence o hypoco yl
leng h (HYP) in simula ed shade, W+ FR (HYP
W + FR
) and W (HYP
W
) o Col-0 (A) h 1-5, pi 7-1, h 1-5 pi 7-1, B) h 1-5, pi 457, h 1-5 pi 457, C) h 1-5, sa 3-5,
h 1-5 sa 3-5, D) phyA-501, pi 7-1, phyA-501 pi 7-1, E) phyA-501, pi 457, phyA-501 pi 457, F) phyA-501, sa 3-5, phyA-501 sa 3-5, G) hy5-2, pi 7-1, hy5-2 pi 7-1,
H) hy5-2, pi 457, hy5-2 pi 457, and I) hy5-2, sa 3-5, and hy5-2 sa 3-5. Seedlings we e g own as in Fig. 2A. Means and SE o 3 independen eplica es we e
used o calcula e he shown alues o HYP
W + FR
−HYP
W
and o p opaga e he SE. E o ba s a e he p opaga ed SE. As e isks indica e signi ican
di e ences (based on he 2-way ANOVA) be ween he mu an and wild- ype geno ypes in esponse o simula ed shade (**P<0.01; ns, no signi ican ).
Shade-induced hypoco yl elonga ion in A abidopsis | 2051
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consis en wi h he epo ed ole o HFR1 as a ansc ip ional
co ac o by he e odime izing and inhibi ing he ansc ip ional
ac i i y o PIF4 and PIF5 (Ho ni schek e al. 2009; Gals yan
e al. 2011). Howe e , he elonga ion esponse o he h 1 pi 457
mu an was s ill highe han ha o pi 457 seedlings (Fig. 4B;
Supplemen a y Fig. S2B), e lec ing he mino con ibu ion o ad-
di ional ac o s. Mu an h 1 plan s we e also c ossed wi h sa 3,
known o ha e a ole in he shade-induced auxin biosyn hesis
(Tao e al. 2008). Pheno ypic analyses (Fig. 4C; Supplemen a y
Fig. S2C) showed ha he pheno ype o h 1 sa 3 mu an s was sig-
ni ican ly longe han in sa 3 seedlings and i ually as sho as
h 1 pi 457, in ag eemen wi h he desc ibed ole o PIF457 in p o-
mo ing he SAV3-dependen IAA biosyn hesis (Ho ni schek e al.
2012; Li e al. 2012). These esul s no only poin ed o a e y mino
con ibu ion o o he PIFs bu also o a SAV3-independen auxin
mechanism in hese shade condi ions.
In con as wi h he p e ious c osses, phyA pi 7, phyA pi 457, and
phyA sa 3 shade-induced hypoco yl elonga ion was simila be ween
hem and close in leng h o ha o phyA han o pi 7, pi 457, o sa 3
(Fig. 4, D o F; Supplemen a y Fig. S2, D o F). Toge he , hese esul s
sugges ed ha he elonga ion ep ession imposed by phyA unde
simula ed shade is mos ly independen on PIF457 o he apid
shade-induced and SAV3-dependen auxin biosyn hesis. Finally, hy-
poco yls o hy5 pi 7, hy5 pi 457, and hy5 sa 3 seedlings showed an in-
e media e elonga ion compa ed o he pa en al pi 7, pi 457, sa 3,
and hy5 lines (Fig. 4, G o I; Supplemen a y Fig. S2, G o I). Toge he ,
hese esul s sugges an addi i e ac i i y o hese egula o s in he
con ol o his shade-induced elonga ion esponse.
The a ia ions in SAV3 equi emen s be ween he 2 p oposed
b anches led us o explo e he con ibu ion o auxin syn hesis
and anspo in hese modules. The co yledons o A. haliana and
B assica apa seedlings pe cei e shade and igge local IAA syn he-
sis in he co yledons hemsel es (Fig. 1). Then, IAA is anspo ed
om co yledons o he hypoco yl, whe e cellula elonga ion occu s
(P ocko e al. 2014). T ea men s o wild- ype seedlings wi h he
auxin biosyn hesis inhibi o L-kynu enine (L-kyn) ha e ec i ely
and speci ically a ge s T p amino ans e ases such as SAV3 (He
e al. 2011) o wi h he auxin anspo inhibi o naph hylph hala-
mic acid (NPA) abolish he shade-induced hypoco yl elonga ion e-
sponse in wild- ype seedlings (Fig. 5) (So in e al. 2009; Keuskamp
e al. 2010; He sch e al. 2014; Zheng e al. 2016). In ou W +FR con-
di ions, he ex a elonga ion o h 1 compa ed o wild- ype hypoco-
yls was comple ely abolished by he highes doses o L-kyn applied
(Fig. 5A). Auxin quan i ica ion indica ed ha IAA le els in Col-0 and
h 1 seedlings inc eased o simila alues a e 1 h o W+FR ea -
men (Fig. 5B), in con as wi h published in o ma ion (He sch e al.
2014). None heless, his esul sugges s ha HFR1 does no ha e a
s ong and measu able impac on he IAA le els in ou g ow h/
shade condi ions, a leas a he ime o shade ea men analyzed.
The ex a hypoco yl elonga ion o phyA and hy5 seedlings, which
was less a ec ed by L-kyn han he wild ype (Fig. 5A), and he a -
enua ed IAA le els a e 1 h o W +FR in bo h hy5 and phyA (Fig. 5B)
sugges ed ha shade-induced elonga ion in hese 2 mu an s was
no ully dependen on auxin le els. As IAA le els seem o be unde
nega i e eedback con ol, as in se e al auxin-signaling mu an s
(Suzuki e al. 2015; Taka o e al. 2017), hese esul s a e consis en
wi h phyA and hy5 ha ing an al e ed auxin esponsi eness (Cluis e
al. 2004; Yang e al. 2018).
The ex a elonga ion o h 1 mu an seedlings is almos abolished
by he applica ion o he auxin anspo inhibi o NPA (Fig. 5C),
which indica es ha he ac ion o PIF-HFR1 in modula ing he
shade-induced hypoco yl elonga ion is mos ly dependen on auxin
p oduced somewhe e else and anspo ed o he hypoco yl, as
p oposed (He sch e al. 2014). By con as , he esis ance o he in-
hibi o y e ec o NPA o phyA and hy5 (Fig. 5C) sugges ed ha hese
2 ac o s sha e simila mechanisms o ep ess shade-induced elon-
ga ion and, in con as wi h HFR1, a e less dependen on auxin
anspo o p omo e elonga ion.
Figu e 5. Con ibu ion o auxin syn hesis and pola anspo in he
shade-induced hypoco yl esponse o phyA, hy5, and h 1 seedlings. A)
E ec o L-kyn on he shade-induced hypoco yl (HYP) leng h o Col-0,
h 1-5, hy5-2, and phyA-501 seedlings. B) IAA con en in Col-0, h 1-5,
hy5-2, and phyA-501 seedlings g own in W o 7 d and hen ea ed o
1 h wi h W + FR (R:FR =0.02). Whole seedlings we e collec ed o measu e
IAA le els. Da a a e means and e o ba s a e SE o 4 biological eplica es.
FW, esh weigh . C) E ec o NPA on he shade-induced HYP leng h o
Col-0, h 1-5, hy5-2, and phyA-501 seedlings. In A) and C), inhibi o s we e
applied in he media, seedlings we e g own in W o 2 d and hen
ans e ed o W+FR o 5 d, and alues a e means and e o ba s a e SE
o 3 independen eplica es. As e isks indica e signi ican di e ences
(S uden ’s - es ) ela i e o he wild ype g owing unde he same ligh
ea men (ns, no signi ican , *P<0.05, **P<0.01).
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Toge he , hese esul s a e consis en wi h a ne wo k a chi ec-
u e in which hese componen s a e likely o ganized in a leas 2
sepa a e b anches o modules, one in ol ing he ac i i y o
PIF457, HFR1, and SAV3 o p omo e he shade-induced hypoco yl
elonga ion and a second one equi ing he ac i i y o PHYA and
HY5 o ep ess i .
SAS egula o y componen s ac in di e en
momen s du ing he shade-induced hypoco yl
elonga ion
We nex s udied when he di e en componen s ac upon exposu e
o young seedlings o W+FR. G ow h a es we e i s de e mined in
wild- ype (Col-0) seedlings g own unde W and W+FR. To do so, hy-
poco yl leng h was measu ed daily om Days 2 o 7 in di e en
g oups o seedlings, and he a ia ions in he daily g ow h a e
we e es ima ed o each geno ype and ligh ea men (Fig. 6A).
Unde W, Col-0 g ow h a e emained low bu cons an along he
pe iod analyzed (Fig. 6, B and D), whe eas unde W +FR, i wen
up om Day 5 onwa ds (Fig. 6, C and E). As an addi ional con ol,
he g ow h a e o he phyB mu an hypoco yls was also es ima ed.
Impo an ly, unde W, phyB g ow h a e inc eased wi h he age o
he seedlings (Fig. 6B). Unde W +FR, phyB g ow h a e mimicked
ha o Col-0 bu he peak a Day 5 was a enua ed in he mu an
(Fig. 6C), consis en wi h i s educed elonga ion compa ed o Col-0
(Fig. 2) (Ma ínez-Ga cia e al. 2014; Molina-Con e as e al. 2019).
Unde W, he g ow h a e o phyA, hy5, and h 1 hypoco yls was con-
s an along ime and simila o ha o Col-0 (Fig. 6, B and D). Unde
W+ FR, phyA and, o a lowe ex en , hy5 g ow h a e was much high-
e han ha o Col-0 hypoco yls on Days 2 o 4 bu p og essi ely
d opped o alues close o hose o Col-0 (Fig. 6, C and E). By con-
as , h 1 ollowed a pa e n o g ow h a e simila o Col-0 and, i
any hing, i elonga ed sligh ly as e han Col-0 in he second hal
o he pe iod o ime analyzed (Fig. 6E). To isualize he ep esso ac-
i i y o he di e en egula o s, he g ow h a e o he wild ype was
sub ac ed o ha o each mu an g own in hose condi ions whe e
he pheno ype is mo e ob ious: W o phyB and W+FR o h 1, hy5,
and phyA. This ep esen a ion con i med ou p e ious conclusions
(Supplemen a y Fig. S3). In summa y, al hough he empo al ac i -
i y o he egula o s o e lapped, phyA ep esses hypoco yl elonga-
ion mo e s ongly a he beginning o seedling de elopmen
( om Days 2 o 4), HY5 a he beginning bu shows a peak in he
middle o his pe iod (Day 4) and HFR1 (and phyB) appea ed as
mo e ac i e a he second hal o he pe iod analyzed ( om Days
5 o 7). These esul s p o ided a amewo k ha sepa a es he em-
po al ac ion o he pa icipa ing componen s.
SAS egula o y componen s a ge o e lapping
bu di e en egions along he hypoco yl axis
In A. haliana, hypoco yl elonga ion is a esul o cell elonga ion (no
cell di ision). Among he di e en issues o his o gan, he epide mis
is o pa icula impo ance in media ing auxin-induced g ow h in he
hypoco yl (P ocko e al. 2016). In W-g own hypoco yls, he pa e n o
epide mal cell leng h akes place in all cells o e he en i e g ow h
pe iod ( om 1 o 9 d a e ge mina ion), al hough he a ea o as es
g ow h mo es ac ope ally, om he base (Cells 2 o 4) on Days 1 o 2
o he middle (Cells 10 o 12) o he hypoco yl on Days 7 o 9
(Gend eau e al. 1997). In da k-g own seedlings, g ow h also ini ia es
in he hypoco yl basal cells bu , in his case, cells ha elonga ed as -
es mo e up much mo e apidly and only a ew cells upwa ds: om
Cell 1 a 36 o 48 h o Cells 3 o 4 a 72 h om ge mina ion (Gend eau
e al. 1997). As he e is no much in o ma ion abou how A. haliana
hypoco yls elonga e in esponse o simula ed shade a he cell le el,
we i s es ablished he pa e n o epide mal cell leng h in wild- ype
(Col-0) hypoco yls g own unde W and W +FR. Using con ocal mic o-
scopy, he leng h o se e al iles o epide mal cells along he hypoco-
yl longi udinal axis pe ea men was measu ed (Supplemen a y
Fig. S4). Cell leng h in W-g own hypoco yls was simila along he
hypoco yl (Supplemen a y Fig. S5). By con as , W+ FR ea men en-
hanced he elonga ion o all cells compa ed o W ea men ,
al hough he pa e n o epide mal cell leng h was no uni o mly
dis ibu ed, wi h cells loca ed in he lowe hal o he hypoco yl elon-
ga ing he mos (Supplemen a y Fig. S5). A simila conclusion was
eached when ep esen ing he di e ence in leng h be ween cells
g own in W +FR and W in each posi ion, wi h Cells 7 o 8 being he
ones ha g ew he mos , becoming abou 170 o 250 µm longe
han cells in he same posi ion o W-g own hypoco yls (Fig. 7, Col-0
panels). These esul s indica e ha he shade-induced hypoco yl
elonga ion o wild- ype seedlings esul ed om a bell-shaped
non-symme ical (skewed) elonga ion pa e n o epide mal cells
peaking a ound Cells 7 o 8 om he base. A skewed dis ibu ion o
cell elonga ion in he hypoco yl has also been obse ed in da k-,
W-, o low blue-g own hypoco yls o A. haliana (Gend eau e al.
1997; Keuskamp e al. 2010), shade-exposed B. apa hypoco yls
(P ocko e al. 2014), and end-o -day-FR- ea ed cowpea epico yls
(Ma inez-Ga cia and Ga cia-Ma inez 1992).
Independen ly o he p ima y si e o ac ion o he s udied SAS eg-
ula o s (e.g. co yledons o hypoco yls), hei ac i i ies con e ge on
he elonga ion o hypoco yls. To es ablish whe he he con e gence
a ec ed he same o di e en hypoco yl cells, we nex analyzed he
shade-induced cell leng h in hypoco yls o seedlings de icien in spe-
ci ic SAS egula o s (Supplemen a y Fig. S5; Fig. 7). The hypo espon-
si e sa 3 hypoco yls showed a simila pa e n o cell elonga ion as
wild ype bu s ongly a enua ed and sligh ly shi ed o lowe cells
(elonga ion peak in Cells 5 o 7 ha elonga ed ∼40 µm mo e han
Figu e 6. E ec o phyA, phyB, HFR1, and HY5 mu a ions on he
hypoco yl g ow h a e unde W and simula ed shade. A) Ca oon o he
expe imen design. Seeds we e ge mina ed and g own o 2 d unde W
and hen ei he kep unde W o ans e ed o simula ed shade (W + FR,
R:FR=0.02) o 5 mo e days. Ci cles indica e he days on which
hypoco yl leng hs we e measu ed o es ima e he daily g ow h a e.
G ow h a e o Col-0, phyA-501, and phyB-9 hypoco yls g own in W B) o
W+FR C). G ow h a e o Col-0, phyA-501, h 1-5, and hy5-2 hypoco yls
g own in W D) o W+ FR E). In B) o E), g ow h a e alues we e es ima ed
as he di e ence o a e age hypoco yl leng h a e 2 consecu i e days.
Values a e means and e o ba s a e SE o 3 independen biological
eplica es.
Shade-induced hypoco yl elonga ion in A abidopsis | 2053
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he same cells in W-g own hypoco yls) (Fig. 7A). In he shade-
hype esponsi e h 1 seedlings, he peak o cell leng h was widened,
wi h Cell 9 showing he maximum o elonga ion (∼280 µm longe
han Cell 9 in W-g own hypoco yls) (Fig. 7B). In he case o hy5 and
phyA, also hype esponsi e o shade, cell leng h was s ongly en-
hanced and he elonga ion peak mo ed o he uppe hal o he hypo-
co yl (Cell 12 in hy5 ha elonga ed ∼640 µm mo e han he same cell
in W-g own hypoco yls; Cell 15 in phyA ha elonga ed ∼830 µm mo e
han he same cell in W-g own hypoco yls) (Fig. 7, A and C).
As he peak cell numbe was associa ed wi h he di e ence in hy-
poco yl leng h in W+FR and W (HYP
W + FR
−HYP
W
) (Supplemen a y
Fig. S6), we wonde ed i he edis ibu ion o cell g ow h was a
consequence o he enhanced hypoco yl shade-induced elonga ion
shown by hese geno ypes. To check his possibili y, we analyzed
he cell leng h in phyA sa 3 hypoco yls, whose shade-induced hypo-
co yl elonga ion was simila o ha o h 1 and lowe han hy5 hypo-
co yls (Fig. 6). The peak o cell elonga ion in phyA sa 3 seedlings (Cell
13) was close o ha o hy5 (Cell 12) and phyA (Cell 15). In addi ion,
he mos esponsi e cell in phyA sa 3 seedlings elonga ed mo e (Cell
13, ∼340 µm) han in h 1 (Cell 9, ∼280 µm). These esul s ein o ced
he conclusion ha phyA ac ed by ep essing he elonga ion o a
g oup o cells loca ed in he uppe hal o he hypoco yl (Fig. 7A).
In his case, peak cell numbe did no associa e wi h he HYP
W + FR
−HYP
W
(Supplemen a y Fig. S6).
Figu e 7. Dis ibu ion o he epide mal cell leng h om base o op induced by simula ed shade in hypoco yls o wild- ype o SAS mu an seedlings.
Schema ic ep esen a ion o he 20 cells composing a cell ow o he epide mis along he longi udinal axis o an A. haliana hypoco yl (le ). Di e ence in
leng h in simula ed shade (W + FR) and W o each o he 20 epide mal cells in hypoco yls o Col-0, A) sa 3-5, phyA-501, phyA-501 sa 3-5, B) h 1-5, and C)
hy5-2. Seedlings we e g own as in Fig. 2A. A ows poin o cell wi h a highes di e ence in leng h. Values ha e been es ima ed a e mean leng hs o a
leas 15 cells o 2 cell ows pe hypoco yl om 7 di e en hypoco yls pe geno ype and g ow h condi ion. E o ba s ep esen SE.
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Al oge he , hese analyses indica e ha (i) he hypoco yl cells
mo e esponsi e o simula ed shade a e loca ed in he lowe
hal o he wild- ype hypoco yls (cen e ed in Cells 7 o 8), (ii) de i-
ciency in SAS-nega i e egula o s keeps he pa e n o epide mal
cell leng h bu a ec s he peak cell numbe , and (iii) al hough he
a ge cells o he a ious SAS-nega i e egula o s o e lap, he
peak cell numbe due o loss o HY5 and PHYA unc ion is s ongly
shi ed owa d he uppe hal o he hypoco yl. These esul s a e
consis en wi h phyA and HY5 ac i i ies ep essing he cells o
he uppe pa o he hypoco yl whe eas HFR1 mo e clea ly e-
p essed he elonga ion o cells loca ed in he lowe hal o he hy-
poco yl, p o iding a spa ial amewo k ha sepa a es he ac ion
o he pa icipa ing componen s.
PIF457 and HY5 modula e he exp ession o
sha ed shade- egula ed genes
Despi e he empo al di e ences obse ed be ween phyA, HY5, and
PIFs/HFR1/SAV3, hei ac i i ies o e lap and e en ually con e ge in
con olling hypoco yl elonga ion. Hence, we aimed o u he in es-
iga e possible con e gence poin s be ween hese 2 g oups o egula-
o s. E idence in o he pho omo phogenic o empe a u e- egula ed
esponses showing ha HY5 di ec ly in e ac s wi h PIF1/PIF3 p o-
eins (Chen e al. 2013) and HY5 and PIF ac i i ies con e ge a a
sha ed cis- egula o y elemen (Toledo-O iz e al. 2014; Gangappa
and Kuma 2017; Zhang e al. 2017) led us o explo e shade-induced
changes o PIF457 and HY5 in he exp ession o sha ed a ge s
genes. We ocused on 1-AMINO-CYCLOPROPANE-1-CARBOXYLATE
SYNTHASE 8 (ACS8) and PAR1, iden i ied as bo h po en ially pu a i e
HY5 binding a ge s (Lee e al. 2007) and di ec PIFs a ge s (Khanna
e al. 2007; Gallego-Ba olome e al. 2011; Yang e al. 2018). The ex-
p ession o bo h genes was signi ican ly p omo ed in Col-0 a e 1
o 8 h o W+FR ea men (compa ed o he beginning o he ea -
men ) and dec eased a e 24 h o he shade ea men (Fig. 8). In
hy5, ACS8, and PAR1, exp ession was also induced a e 1 o 8 h. By
con as , in pi 457 and hy5 pi 457, he exp ession o ACS8 and PAR1
emained i ually una ec ed by he W+FR ea men (Fig. 8B).
These esul s sugges ha PIF457 ac i a es whe eas HY5 ep esses
ACS8 and PAR1 exp ession. Impo an ly, HY5 ac i i y depends on
PIF457 ansc ip ional ac i a ion. These exp ession analyses we e
ca ied ou in 7-d-old seedlings. These olde seedlings elonga ed
mildly o W+ FR ea men s (Supplemen a y Fig. S7), al hough he
p o ile o esponse was consis en o wha was obse ed in younge
seedlings (Fig. 4H) sugges ing ha he same gene ic componen s and
molecula mechanisms a e s ill unc ional.
To expand ou unde s anding o he ole and in e ac ion o HY5
and PIF457 ac i i ies, we ca ied ou RNA sequencing (RNA-seq) o
he ime poin s 0, 1, and 8 h a e shade exposu e o he 4 geno-
ypes (Col-0, hy5, pi 457, and hy5 pi 457) (Fig. 9A). We iden i ied di -
e en ially exp essed genes (DEGs) up egula ed ( old change [FC] ≥
1.5, P<0.05) and down egula ed (FC≤0.667, P<0.05) a e 1 and
8 h o shade ea men compa ed o 0 h o each geno ype ana-
lyzed (Supplemen a y Tables S1 o S4). A e 1 h o W +FR, 386
and 791 DEGs we e induced and 177 and 351 we e ep essed in
wild- ype and hy5 seedlings, espec i ely. Impo an ly, only 1
and 3 DEGs we e induced and 31 and 17 we e ep essed in pi 457
and hy5 pi 457 seedlings, espec i ely (Fig. 9, B o E). F om hese
ea ly shade-modula ed DEGs, 294 up egula ed genes we e sha ed
be ween hy5 (ou o 791 genes, 37.2%) and Col-0 (ou o 386 genes,
76.2%) (Fig. 9D) and 100 down egula ed genes we e sha ed be-
ween hy5 (ou o 351, 28.5%) and Col-0 (ou o 674, 14.8%)
(Fig. 9E). As 748 DEGs (497 up egula ed and 251 down egula ed)
appea ed only in hy5 bu no in Col-0, we concluded ha HY5
has a dual ole as bo h ac i a ing and ep essing apid shade-
modula ed gene exp ession. The as majo i y o hese DEGs did
no change in pi 457 and hy5 pi 457 (Fig. 9, D and E), indica ing
ha PIF457 is basically equi ed o all he changes in gene exp es-
sion ha ake place a e 1 h o simula ed shade exposu e. An im-
po an bu weake impac o PIF457 on gene exp ession was
de ec ed a e 3 h o shade exposu e (Ince e al. 2022).
A e 8 h o W +FR, 826 and 542 DEGs we e induced and 654 and
568 we e ep essed in Col-0 and hy5 seedlings, espec i ely. A e
his ime o W+ FR exposu e, a subs an ial numbe o DEGs we e
de ec ed in pi 457 (323 up egula ed and 435 down egula ed) and
hy5 pi 457 (279 up egula ed and 690 down egula ed) seedlings
(Fig. 9, B o G). Venn diag ams indica ed ha , om he o al num-
be o DEGs iden i ied in all geno ypes (1,347 up egula ed and
1,865 down egula ed), a la ge ac ion appea ed as up egula ed
(65.9%: 416 in Col-0, 144 in hy5, 159 in pi 457, and 168 in hy5
pi 457) o down egula ed (78.4%: 340 in Col-0, 319 in hy5, 282 in
pi 457, and 521 in hy5 pi 457) only in 1 geno ype, whe eas he
es appea ed in a leas 2 geno ypes (Fig. 9, F and G). Based on
he highes signi icance and he en ichmen old o o e lapping
genes, we concluded ha he se o DEGs o hy5 pi 457 (up egula ed
Figu e 8. E ec o hy5 and pi 457 mu a ions on he shade egula ion o
he exp ession o ACS8 and PAR1. A) Ca oon o he expe imen design.
Seeds we e g own o 7 d unde W and hen ans e ed o simula ed
shade (W+FR, R:FR=0.02) o he indica ed ime be o e ha es ing
samples (ci cles). B) Rela i e exp ession o ACS8 ( op) and PAR1 (bo om)
in Col-0, hy5-2, pi 457, and hy5-2 pi 457 a he indica ed imes o
simula ed shade ea men . Values a e means and e o ba s a e SE o 3
independen biological eplica es. Exp ession is p esen ed ela i e o
he Col-0 geno ype a 0 h. As e isks a ound he symbols indica e
signi ican di e ences (S uden ’s - es ) ela i e o he same geno ype a
0 h. As e isk a he igh indica e signi ican di e ences be ween he
di e en mu an s and he wild ype in esponse o simula ed shade
(2-way ANOVA); ns, no signi ican , *P<0.05, **P<0.01.
Shade-induced hypoco yl elonga ion in A abidopsis | 2055
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and down egula ed) is close o his in hy5 han pi 457. The num-
be o mis egula ed DEGs in hy5 pi 457 is lowes when compa ed o
hy5 and highes when compa ed o pi 457 (Supplemen a y Fig. S8).
These esul s indica e ha , a e 8 h o simula ed shade, (i) he ex-
p ession o a subs an ial amoun o DEGs does no equi e PIF457
ac i i y and (ii) he DEG iden i y is close o hy5 han pi 457, in con-
as o wha happens a 1 h.
Rega ding he unc ional p edic ion, he DEGs belonged o
simila GO e m ca ego ies in all geno ypes (excep in pi 457 and
hy5 pi 457 a e 1 h o W +FR, in which no GO e m en ichmen
was ound because o he massi e d op in DEG numbe )
(Supplemen a y Table S5). Impo an ly, no ob ious and speci ic
p ocesses we e di e en ially a ec ed by HY5 (a 1 h) o HY5 and
PIF457 a la e imes ha could easily explain he di e ences in
g ow h de ec ed among he geno ypes (Fig. 5H).
Toge he , we concluded ha (i) PIF457 and HY5 ha e a s ong
impac in he ea ly shade- egula ed changes in gene exp ession,
al hough (ii) he leading ole o PIF457 a his ea ly ime o shade
exposu e dissipa es a e longe pe iods (8 h) o ea men .
Discussion
In he A. haliana shade-induced hypoco yl elonga ion, he unc ion
o phyB and i s e ec on he PIF457-HFR1 and auxin biosyn hesis
ia SAV3/YUCs in he co yledons a e well es ablished (Tao e al.
2008; Li e al. 2012; Ciol i e al. 2013; Kohnen e al. 2016; Fio ucci
and Fankhause 2017; Paulisic e al. 2021) (Fig. 1). The obse ed ge-
ne ic in e ac ions be ween sa 3/pi 7/pi 457 and h 1 (Fig. 4, A o C)
and he pha macological applica ions o L-kyn and NPA on h 1
seedlings (Fig. 5A) a e consis en wi h his scena io. The gene ic
Figu e 9. E ec o hy5 and pi 457 mu a ions on he shade- egula ed ansc ip ome. A) RNA-seq was pe o med wi h RNA ex ac ed om Col-0, hy5-2,
pi 457, and hy5-2 pi 457 seedlings a he indica ed imes (ci cles) o simula ed shade ea men . Seedlings we e g own as in Fig. 8A. Th ee independen
biological eplica es we e used o each geno ype and ea men . E olu ion o he numbe o up egula ed B) and down egula ed C) DEGs in esponse o 1
and 8 h o W + FR in Col-0, hy5-2, pi 457, and hy5-2 pi 457 seedlings g own as indica ed in A). Venn diag ams showing he o e lap o up egula ed D, F) and
down egula ed E, G) DEGs a e 1 D, E) and 8 h F, G) o W+FR ea men be ween Col-0, hy5-2, pi 457, and hy5-2 pi 457 seedlings.
2056 | Plan Physiology, 2024, Vol. 196, No. 3
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