Temporal and spatial frameworks supporting plant responses to vegetation proximity

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 Resea ch A icle Downloaded om h ps://academic.oup.com/plphys/a icle/196/3/2048/7733512 by Consejo Supe io de In es igaciones Cien i icas (Books) use on 21 No embe 2024 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 Downloaded om h ps://academic.oup.com/plphys/a icle/196/3/2048/7733512 by Consejo Supe io de In es igaciones Cien i icas (Books) use on 21 No embe 2024 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 Downloaded om h ps://academic.oup.com/plphys/a icle/196/3/2048/7733512 by Consejo Supe io de In es igaciones Cien i icas (Books) use on 21 No embe 2024 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 Downloaded om h ps://academic.oup.com/plphys/a icle/196/3/2048/7733512 by Consejo Supe io de In es igaciones Cien i icas (Books) use on 21 No embe 2024 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). 2052 | Plan Physiology, 2024, Vol. 196, No. 3 Downloaded om h ps://academic.oup.com/plphys/a icle/196/3/2048/7733512 by Consejo Supe io de In es igaciones Cien i icas (Books) use on 21 No embe 2024 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 Downloaded om h ps://academic.oup.com/plphys/a icle/196/3/2048/7733512 by Consejo Supe io de In es igaciones Cien i icas (Books) use on 21 No embe 2024 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. 2054 | Plan Physiology, 2024, Vol. 196, No. 3 Downloaded om h ps://academic.oup.com/plphys/a icle/196/3/2048/7733512 by Consejo Supe io de In es igaciones Cien i icas (Books) use on 21 No embe 2024 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 Downloaded om h ps://academic.oup.com/plphys/a icle/196/3/2048/7733512 by Consejo Supe io de In es igaciones Cien i icas (Books) use on 21 No embe 2024 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 Downloaded om h ps://academic.oup.com/plphys/a icle/196/3/2048/7733512 by Consejo Supe io de In es igaciones Cien i icas (Books) use on 21 No embe 2024 indole acidic acid p o ein s abili y. De Cell. 2018:44(1):29–41.e4. h ps://doi.o g/10.1016/j.de cel.2017.11.017 Yano sky MJ, Casal JJ, Whi elam GC. Phy och ome A, phy och ome B and HY4 a e in o led in hypoco yl g ow h esponses o na u al adia ion in A abidopsis: weak de-e iola ion o he phyA mu an unde dense canopies. Plan Cell En i on. 1995:18(7):788–794. h ps://doi.o g/10.1111/j.1365-3040.1995. b00582.x Zhang X, Huai J, Shang F, Xu G, Tang W, Jing Y, Lin R. A PIF1/PIF3- HY5-BBX23 ansc ip ion ac o cascade a ec s pho omo pho- genesis. Plan Physiol. 2017:174(4):2487–2500. h ps://doi.o g/10. 1104/pp.17.00418 Zhang S, Li C, Zhou Y, Wang X, Li H, Feng Z, Chen H, Qin G, Jin D, Te zaghi W, e al. TANDEM ZINC-FINGER/PLUS3 is a key compo- nen o phy och ome A signaling. Plan Cell. 2018:30(4):835–852. h ps://doi.o g/10.1105/ pc.17.00677 Zhang R, Yang C, Jiang Y, Li L. A PIF7-CONSTANS-cen e ed molec- ula egula o y ne wo k unde lying shade-accele a ed lowe - ing. Mol Plan . 2019:12(12):1587–1597. h ps://doi.o g/10.1016/j. molp.2019.09.007 Zheng Z, Guo Y, No ák O, Chen W, Ljung K, Noel JP, Cho y J. Local auxin me abolism egula es en i onmen -induced hypoco yl elonga ion. Na Plan s. 2016:2(4):16025. h ps://doi.o g/10.1038/nplan s.2016.25 Shade-induced hypoco yl elonga ion in A abidopsis | 2063 Downloaded om h ps://academic.oup.com/plphys/a icle/196/3/2048/7733512 by Consejo Supe io de In es igaciones Cien i icas (Books) use on 21 No embe 2024