Chloroplasts Modulate Elongation Responses to CanopyShade by Retrograde Pathways Involving HY5 andAbscisic Acid

Miriam Ortiz-Alcaide,a Ernesto Llamas,a Aurelio Gomez-Cadenas,b Akira Nagatani,c Jaime F. Martínez-García,a,d

and Manuel Rodríguez-Concepcióna,1

a Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, 08193 Barcelona, SpainbUniversitat Jaume I, 12071 Castelló de la Plana, Spainc Kyoto University, 606-8502 Kyoto, Japand Institució Catalana de Recerca i Estudis Avançats, 08010 Barcelona, Spain

ORCID IDs: 0000-0001-8813-4051 (M.O.-A.); 0000-0001-9262-2402 (E.L.); 0000-0002-4598-2664 (A.G.-C.); 0000-0002-4284-3140(A.N.); 0000-0003-1516-0341 (J.F.M.-G.); 0000-0002-1280-2305 (M.R.-C.)

Plants use light as energy for photosynthesis but also as a signal of competing vegetation. Using different concentrations ofnorflurazon and lincomycin, we found that the response to canopy shade in Arabidopsis (Arabidopsis thaliana) was repressedeven when inhibitors only caused a modest reduction in the level of photosynthetic pigments. High inhibitor concentrationsresulted in albino seedlings that were unable to elongate when exposed to shade, in part due to attenuated light perceptionand signaling via phytochrome B and phytochrome-interacting factors. The response to shade was further repressed bya retrograde network with two separate nodes represented by the transcription factor LONG HYPOCOTYL 5 and thecarotenoid-derived hormone abscisic acid. The unveiled connection among chloroplast status, light (shade) signaling, anddevelopmental responses should contribute to achieve optimal photosynthetic performance under light-changing conditions.

INTRODUCTION

Life on our planet heavily relies on photosynthesis, i.e. the use ofsolar energy (sunlight) to fix carbon into organic matter linked tothe production of oxygen from water. In plants, the quantity andquality of the incoming light strongly influence growth and de-velopment. For example, oxidative stress and eventual damagecan occur if the amount of light exceeds the photosynthetic ca-pacity of the chloroplast. By contrast, light supply and hencephotosynthetic activity can be compromised by the shading ofnearby plants. Under a canopy, plants might actually be exposedto moments of both excess light (e.g. sunflecks) and low light (i.e.shading) during the same day. Even in open habitats, plants areusually found in communities where competition for light mightresult in overgrowing and eventual shadowing by neighbors.

Light quality is an important signal that informs plants of po-tential competitors. Vegetation absorbs light from the visible re-gion (called “photosynthetically active radiation”orPAR, 400–700nm). Inparticular, it absorbs red light (R,600–700nm)but transmitsand reflects far-red light (FR, 700–800 nm), therefore causinga reduction in the R-to-FR ratio (R/FR). Both PAR (light quantity)and R/FR (light quality) are greatly reduced under a plant canopy,whereas the presence of nearby plants (without direct vegetation

shading) involves a more moderate reduction of R/FR withoutchanges in PAR (Casal, 2012; Martínez-García et al., 2014;Fiorucci and Fankhauser, 2017). Independently of the PAR level,a drop in R/FR acts as a signal that strongly and differentiallyaffects elongation of shade-avoiding plants such as Arabidopsis(Arabidopsis thaliana) and most crops (Martínez-García et al.,2014). Low R/FR signals also cause a decrease in the levels ofphotosynthetic pigments (chlorophylls [CHLs] and carotenoids[CRTs]) in seedlings and adult plants (Roig-Villanova et al., 2007;Patel et al., 2013; Bou-Torrent et al., 2015; Llorente et al., 2017).These and other responses triggered by a reduced R/FR arecollectively known as “shade avoidance syndrome” (SAS), andaim to overgrow neighboring plants, readjust photosyntheticmetabolism, and eventually launch reproductive development(Franklin, 2008; Casal, 2012; Gommers et al., 2013; Martínez-García et al., 2014).Low R/FR signals indicative of shade are perceived by the

phytochrome (phy) family of photoreceptors. Five genes encodethe phy family in Arabidopsis: phyA to phyE. Whereas phyB is themajor phy controlling the responses to shade, other phymemberssuch as phyD and phyE can also redundantly contribute to thecontrol of shade-modulated elongation growth or flowering time(Franklin, 2008; Martínez-García et al., 2014). In the case ofphotolabile phyA, an antagonistic negative role has been reportedfor the seedling hypocotyl elongation response to shade. Thus,the SAS is induced by phyB deactivation but gradually antago-nized by phyA in response to high FR levels characteristic of plantcanopy shade (Casal, 2012; Martínez-García et al., 2014). Thisintrafamily photosensory attenuation mechanism might act tosuppress excessive elongation under prolonged direct vegeta-tion shade. It remains unknown whether other SAS responses,

1 Address correspondence to jaume.martinez@cragenomica.es andmanuel.rodriguez@cragenomica.es.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) are Jaime F. Martinez-Garcia(jaume.martinez@cragenomica.es) and Manuel Rodríguez-Concepcion(manuel.rodriguez@cragenomica.es).www.plantcell.org/cgi/doi/10.1105/tpc.18.00617

The Plant Cell, Vol. 31: 384–398, February 2019, www.plantcell.org ã 2019 ASPB.

including photosynthetic pigment decrease, are also affected bythis antagonistic regulation by phyA and phyB.

In any case, the balance between positive and negative regu-lators of the SAS acting downstream phytochromes was found tobe instrumental to regulate not only hypocotyl elongation but alsoCRT biosynthesis (Franklin, 2008; Casal, 2013; Bou-Torrent et al.,2015). Positive regulators of the SAS include transcription factorsof the basic-helix-loop-helix family such as PHYTOCHROME-INTERACTING FACTORs (PIFs) andhomeodomain Leu zipperclass II family members, whereas the basic Leu zipper tran-scription factor LONG HYPOCOTYL 5 (HY5) and other basic-helix-loop-helix family members have negative roles. Amongthem, PIFs and HY5 have also been found to participate in ret-rograde signaling during deetiolation, i.e. in the communicationbetween chloroplasts and nucleus when underground seedlingssense the light and change from skotomorphogenic (i.e. hetero-trophic) to photomorphogenic (i.e. photosynthetic) development(Ruckle et al., 2007; Martín et al., 2016; Xu et al., 2016).

Alterations in the physiological status of the chloroplast in light-grown plants are also signaled to the nucleus by a variety ofretrograde pathways that readjust nuclear gene expression ac-cordingly (Baier and Dietz, 2005; Gläßer et al., 2014; Chan et al.,2016). Because exposure to shade causes a decrease in theaccumulation of CHLs andCRTs that can eventually compromisephotosynthesis and photoprotection (Roig-Villanova et al., 2007;Cagnola et al., 2012; Bou-Torrent et al., 2015), we reasoned thatthe derived effects on chloroplast homeostasis might not be justa consequence but influence the response to shade itself (e.g. interms of elongation) through retrograde signaling. The work re-ported here aimed to test this possibility.

RESULTS AND DISCUSSION

Functional Chloroplasts Are Required for Full Response toSimulated Shade

To initially test whether retrograde signals modulate the elonga-tion response to plant proximity, we used two kinds of inhibitors ofchloroplast function associated with retrograde signaling: nor-flurazon (NF), an inhibitor of CRT biosynthesis (Chamovitz et al.,1991) and lincomycin (LIN), an inhibitor of chloroplast proteinsynthesis (Mulo et al., 2003). Both inhibitors were present in themediumused for seedgermination andseedlinggrowth (Figure 1).This medium also contained Suc to sustain growth even in theabsence of photosynthesis. As expected, a concentration-dependent bleaching was observed in wild-type Arabidopsisplants grown under white light (W) with NF or LIN (Figure 1A). Theconcentration of inhibitors required to obtain albino seedlingswasadjusted to our experimental conditions. For example, an albinophenotypewas previously observed in wild-type seedlings grownwithout Suc in the presence of 5mMNFunder 100mmolm–2 s–1 orwith 50 nM NF under 5 mmol m–2 s–1 (Saini et al., 2011). We usedintermediate light intensity conditions (20–24 mmol m–2 s–1) and,most importantly, addedSuc in themedium,which togethermadeit necessary to adjust NF concentration to 200 nM to obtaincompletely albino seedlings (Figure 1A).Thepresenceof inhibitors hadnosignificant effect onhypocotyl

length underW (Figure 1B). However, exposure to FR-enrichedW(W+FR) to simulate canopy shade progressively impaired elon-gation as levels of photosynthetic pigments (CHLs and CRTs)decreased. Importantly, inhibition of shade-triggered elongation

Interaction of Light and Retrograde Pathways 385

Figure 1. Hypocotyl Elongation in Response to Shade Requires Functional Chloroplasts.

(A)Wild-type (Col) plantswere germinated and grown underW for 7 d onmediawith or without the indicated concentrations of NF or LIN. Graphs representthemean6 SEvaluesof totalCHLandCRTcontentsofat leastn=8 independentsamples (poolsofseedlings) fromtwodifferentexperiments.Pigmentswerequantified by spectrophotometric methods and represented relative to the levels found in the absence of inhibitors. Pictures show the phenotype ofrepresentative seedlings.(B) Hypocotyl elongation in 7-d-old seedlings germinated and grown on media supplemented with the indicated concentrations of NF or LIN under W orexposed toW+FR during the last 5 d. Graphs in the left represent the length of the hypocotyls (mean6 SE of n$ 100 seedlings grown in different plates in atleast two independent experiments) relative to the value in samples grown underW in the absence of inhibitors. Graphs in the right represent the elongationresponse to shade of the same samples. They show the ratio of hypocotyl length under W+FR relative to that under W. A value of “1” means no growthdifferencesbetweenWandW+FR; values > 1 indicate higher growth underW+FR; and values < 1 indicate lower growth underW+FR. Asterisksmark valuesstatistically > 1 (t test, P # 0.05), i.e. responsive to shade by increasing hypocotyl elongation.

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growth was observed at concentrations of NF or LIN that onlyslightly reduced the levels of photosynthetic pigments and had novisual impact on seedling pigmentation (e.g. 25 nM NF or 5 mMLIN), suggesting that even moderate alterations in chloroplastfunction might influence the response to shade. Hypocotyl elon-gation in response to shade was completely blocked at concen-trations of NF causing >80% loss of CHLs, whereas an even lowerreduction (50%) was required for a lack of response in LIN-treatedseedlings (Figure1B). Inbothcases,completelybleachedseedlingsdid not elongate at all when exposed to W+FR compared with Wcontrols (Figure 1B), suggesting that functional chloroplasts arerequired for the elongation response to canopy shade.

We next aimed to confirm that the disrupted elongation re-sponse to W+FR observed in bleached seedlings was not due toenergetic constraints. If nonphotosynthetic seedlings lackingCHLsmaintain an intrinsic capacity to grow, it would be expectedthat their hypocotyls would elongate when treated with growth-promoting hormones such as brassinosteroids, auxins, or gib-berellins. In agreement, seedlings grown in the presence ofNF concentrations that completely blocked photosynthetic de-velopment (2 mM) were able to elongate very similarly to controlgreen seedlings when treated with any of these hormones(Supplemental Figure 1). The same hormone treatments causeda similar growth response in the case of mutant hdr-3 seedlings,which are unable to produce the precursors for CHL and CRTbiosynthesis in chloroplasts and hence display an albino phe-notype (Pokhilko et al., 2015). We therefore conclude that func-tional chloroplasts are not required for hormone-mediatedhypocotyl elongation (at least in Suc-supplemented media), butare necessary for growth in response to shade signals.

Defective Chloroplast Function Impairs Phy-MediatedShade Signaling

Phytochromes are the main photoreceptors involved in shadeperception and signal transduction, with phyB having a pre-dominant role in Arabidopsis (Casal, 2012; Martínez-García et al.,2014; Fiorucci and Fankhauser, 2017). To address whethertreatment with NF or LIN had an impact on phy signaling, we usedtransgenic 35S:PHYB-GFPplants expressing abiologically activeversion of the phyB protein fused to the green fluorescent protein(GFP; Yamaguchi et al., 1999). Under W, the phyB-GFP fusionprotein shows a characteristic distribution in nuclear speckles,presumably the site where active photoreceptor proteins interactwith other nuclear factors to mediate light signaling (Yamaguchiet al., 1999). We observed that only minutes after exposing 35S:PHYB-GFP seedlings to an end-of-day FR treatment to simulateshade, the green fluorescence associated to the phyB-GFP re-porter becamemore disperse in the nuclei of epidermal hypocotylcells (Figure 2), likely reflecting phyB inactivation. Strikingly, thisshade-mediated inactivation process was clearly delayed in al-bino seedlingsgerminated andgrown in thepresenceofNF (5mM)or LIN (1 mM). While mock (i.e. green) seedlings displayed evenlydistributed nuclear phyB-GFP fluorescence in all analyzed cells90 min after the light treatment, inhibitor-grown (i.e. albino)seedlings still showed nuclear speckles in some cells after210 min. These results suggest that functional chloroplasts arerequired forproperphyB inactivation in response toshadesignals.

Consistent with this conclusion, the shade-triggered and phy-dependent stabilization of photolabile PIF3was attenuated in NF-bleached seedlings (Supplemental Figure 2).To further test whether phy function was altered in albino seed-

lings, we next analyzed the expression of rapidly shade-induced

Figure 2. Retrograde Signals Prevent phyB Deactivation in Response toShade.

Transgenic 35S:PHYB-GFP plants were germinated and grown under Wfor 7 d on media with or without 5 mM NF or 1 mM LIN. After a 5-min ir-radiation with FR to simulate shade, the distribution of the phyB-GFPreporter protein in nuclei from epidermal hypocotyl cells was analyzed byconfocal laser scanning microscopy. Ten seedlings from three in-dependent experiments were observed for each treatment and time point.Graph showsmean6 SE values of the percentage of cells showing nuclearspeckles ina total of90nucleiper treatmentand timepoint.Bottom images:Representative imagesof nuclei fromseedlinghypocotyl cells 0 and90minafter the light treatment.

Interaction of Light and Retrograde Pathways 387

phy primary target genes in wild-type plants either treatedor not with NF (Figure 3). In particular, we chose genes PIL1,ATHB2, HFR1, YUCCA8, and PAR1 (Roig-Villanova et al., 2006).Wild-type plants grownunderW for 7 dwere exposed toW+FR for1 h and then samples were collected and used for RNA extractionand reverse transcription-quantitative PCR (RT-qPCR) analysis.As expected, comparison of W-grown controls and shade-exposed (1 h W+FR) samples showed that all genes analyzedwere induced by shade in green seedlings, ranging from twofold(PAR1) to 80-fold (PIL1). In NF-grown seedlings, however, theinduction was much reduced (Figure 3). HFR1, YUCCA8, andPAR1 gene expression hardly changed after W+FR treatment inalbino seedlings, whereas PIL1 induction was only 10% com-pared with that detected in green seedlings and ATHB2 upre-gulationwas less than half. From these results, we conclude thatthe absence of functional chloroplasts somehow prevents nor-mal light (i.e. shade) perception and signal transduction byphytochromes.

Functional phy holoproteins require the covalent attachment ofa phytochromobilin (PFB) chromophore to each phy apoproteinmonomer (Rockwell et al., 2006). The synthesis of PFB occurs inthe plastid and the early steps are shared with those required to

synthesize heme and CHLs (Figure 4). To test whether the ob-served reduction in shade-triggered phy inactivation (and hencehypocotyl elongation) in albino seedlings could result from im-paired accumulation of PFB, we analyzed the elongation re-sponse to shade of Arabidopsis mutants defective in PFBsynthesis (Parks andQuail, 1991). In particular, we used the hy1-1allele, which was isolated from a fast-neutron mutagenizedpopulation of Landsberg erecta (Ler) and carries a short deletionthat disrupts its function (Davis et al., 1999). As shown in Figure 4,elongation in response to W+FR was still observed in the hy1-1mutant (Figure 4B). We therefore conclude that treatment withbleaching inhibitors interferes with phy-dependent signaling bymechanisms other than defective chromophore availability.Plastid retrograde signaling has been previously shown to in-

teract with components of light signaling networks to coordinatechloroplast biogenesis with both the light environment and de-velopment (LarkinandRuckle, 2008;LepistöandRintamäki, 2012;Ruckle et al., 2012; Martín et al., 2016; Xu et al., 2016). In fact,mutants defective in the PFB biosynthetic enzymes HY1/GE-NOMESUNCOUPLED 2 (GUN2) andHY2/GUN3 (Figure 4A) wereisolated in a screen for GUN mutants that retained partial ex-pression of genes encoding photosynthesis-related plastidialproteins after NF treatment (Mochizuki et al., 2001). Other GUNproteins such as GUN5 (Mochizuki et al., 2001) participate ina different branch of the tetrapyrrole pathway that leads to theproduction of CHLs (Figure 4A). Unlike otherGUNproteins, GUN1is not an enzyme but a central integrator of retrograde signalingpathways that was proposed to coordinate photomorphogenesiswith chloroplast function (Koussevitzky et al., 2007; Ruckle et al.,2007; Ruckle and Larkin, 2009). Similar to wild-type plants, mu-tants gun1-101 (Ruckle et al., 2007) and gun5-1 (Mochizuki et al.,2001) elongated in response to W+FR under normal growthconditions (i.e. when chloroplasts are functional) but not whenchloroplast development was blocked with NF (Figure 4). To-gether, the described results suggest that alteration of chloroplast

Figure 3. Shade-Triggered Induction of Phy Primary Target Genes IsAttenuated in Bleached Seedlings.

Wild-typeplantsweregerminatedandgrownunderW for 7donmediawithor without 5 mM NF. Before and after 1 h of exposure to W+FR, RNA wasisolated from seedlings and used to analyze the transcript levels of theindicated genes by RT-qPCR. Graph represents the induction response(transcript levels inW versus those after exposure toW+FR). Mean6 SD ofn = 3 pools of seedlings, from independent experiments, are shown.

Figure 4. gun Mutants Show Different Elongation Responses to Shade.

(A)Roles of GUN proteins in retrograde signaling and production of CHLs,heme, and the phy chromophore.(B)Elongation responses toshade inmutantsdefective in someof theGUNproteins represented in (A). Mutants and their respective wild-typebackgrounds (Ler for hy1/gun2 and Col for the rest) were germinatedand grown as indicated with or without 5 mM NF. Graph represents themean 6 SE values of at least two independent experiments with n $ 25seedlings each.

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function influences a retrograde signaling pathway independentof GUN proteins that modulates the phy-mediated responseto shade.

Retrograde Pathways Repressing Shade-TriggeredHypocotyl Elongation Involve HY5 but Not GUN1

To identify components of the chloroplast-modulated trans-ductionpathway involved in the response toshade,wenext testedthe possible role of SAS-related transcription factors known to beinvolved in both light and retrograde signaling: PIFs (Martín et al.,2016) and HY5 (Ruckle et al., 2007; Xu et al., 2016). A role for PIFsas positive regulators of the response to shade (including hypo-cotyl elongation) is well established (Lorrain et al., 2008; Leivaret al., 2012; Bou-Torrent et al., 2015). However, under our ex-perimental conditions thequadruplepifQmutantdefective inPIF1,PIF3, PIF4, and PIF5 activities showed a wild-type phenotype interms of shade-triggered hypocotyl elongation in both green andalbino seedlings (Figure 5).

HY5 has been proposed to have a function in the adaptation toprolonged shade and the response to sunflecks, i.e. exposure tosunlight through gaps in the canopy (Sellaro et al., 2011; Ciolfiet al., 2013). The role of this elongation-repressing transcriptionfactor in controlling the shade-promoted growth of seedling hy-pocotyls, however, remains unclear. Our previous work (Bou-Torrent et al., 2015) showed that complete loss of HY5 activityin the nullhy5-2mutant (referred to ashy5 fromnowon) hardly had

an influence on the elongation of Arabidopsis seedlings exposedto aW+FR treatment mimicking vegetation proximity (R/FR = 0.05).As shown in Figure 5, however, hy5 seedlings displayed in-creased hypocotyl elongation comparedwith thewild typewhenilluminated with light of a lower R/FR (0.02), reminiscent ofcanopy shade. These results suggest that HY5 is a repressor ofhypocotyl elongation in green seedlings exposed to low or verylow R/FR conditions. Consistent with this conclusion, shade-triggeredhypocotyl growthwas inhibited in transgenic seedlingsoveraccumulating HY5 in a hy5 background (SupplementalFigure 3).Similar to wild-type plants, the elongation response to canopy

shade of hy5 seedlings was almost completely blocked with LIN(Figure 5). However, the growth response of HY5-deficientseedlings was not abolished but just attenuated in NF-supplemented medium. Similar results were obtained in me-dium lackingSuc, but the effects of HY5gain or loss of function onthe elongation response of green or NF-treated seedlings, re-spectively, were much more obvious in the presence of Suc(Supplemental Figure 3). We therefore kept using Suc-supplemented media for the rest of the work. Double hy5 gun1-101 mutants were also found to display a partial elongation re-sponse to shade in NF but not in LIN, similar to that found for thesingle hy5mutant (Figure 5). Together, the described results showthat HY5 is a repressor of canopy-shade–triggered hypocotylelongation. When this negative regulator is lost, the elongationresponse to shade can still be blocked by a GUN1-independentretrograde pathway that is active in LIN-treated but not in NF-treated albino seedlings.We next analyzed the levels ofHY5 transcripts before and after

exposure to our shade conditions (Figure 6). In green wild-typeplants (grownwithout inhibitors), the levelsofHY5 transcriptsweresimilar underWandupto8hofourW+FRtreatment (Figure6A).Bycontrast, immunoblot analysis of a HY5-GFP reporter in com-plemented hy5 35S:HY5-GFP plants showed increased proteinlevels after the simulated shade treatment (Figure 6B). Chromatinimmunoprecipitation (ChIP) experiments also detected increasedlevels of HY5-GFP bound to target promotors in shade-exposedgreen seedlings (Figure 6C). Although the endogenous HY5protein might not behave exactly as the overexpressed GFP-tagged version of the protein, our results are in agreement withpreviousstudiesusingadifferent reporter (HY5-myc) thatconcludedthat the lowR/FR treatment stabilizesHY5 (Pacín et al., 2016). Post-transcriptional HY5 accumulation when R/FR is low or very low innatural environments (such as in deep or canopy shade) might helpto prevent seedlings from exhibiting excessive elongation.Both HY5-encoding transcripts (Figure 6D) and HY5-GFP

protein (Figure 6B) were higher in albino seedlings grownwith LINor NF independent of the light treatment, suggesting that theseinhibitors promote HY5 function by increasing gene expression(or/and transcript stability) and decreasing protein turnover. Theobservation that hypocotyl length is not reduced in W-grownseedlings in the presence of inhibitors (Figure 1B) despite accu-mulating higher HY5 levels (Figure 6B) suggests that hypocotylelongation is suppressed to a saturating level by multiple path-ways under W and hence it would not be further repressedby increasing HY5 function. In response to W+FR, however,enhanced HY5 activity together with reduced light signaling in

Figure 5. HY5 Represses Shade-Triggered Hypocotyl Elongation ina GUN1-Independent Manner.

Wild-type (Col, WT) as well as single (hy5), double (hy5 gun1), and qua-druple (pifQ) mutant lines were germinated and grown as indicated with orwithout 5mMNF or 1mMLIN. Graph represents themean6 SE elongationvaluesof at least two independent experimentswithn$25seedlings each.Asterisksmark statistically significant responses to shade (t test,P#0.05).

Interaction of Light and Retrograde Pathways 389

bleached wild-type seedlings would result in no hypocotyl elon-gation. Only when the repressor activity of HY5 is removed (i.e.in HY5-defective mutants), a second pathway that inhibits theelongation response of albino seedlings becomes apparent in thepresence of LIN but not in the presence of NF (Figure 5).

CRT-Derived Products Repress Shade-TriggeredHypocotyl Elongation

The distinct mode of action of LIN and NF and particularly theirdifferential effect on CRT levels is illustrated by theirconcentration-dependent impact on photosynthetic pigmentaccumulation (Figure 1A). High-performance liquid chromatog-raphyanalysisofCRTcontents (Supplemental Figure4) confirmedthat albino LIN-treated seedlings accumulated low but detectablelevels of lutein andviolaxanthin aswell as tracesofb-caroteneandneoxanthin. By contrast, NF blocks the desaturation of phytoene,the first committed intermediate of theCRTpathway (Figure 7). Asexpected, NF-treated seedlings accumulated phytoene (which iscolorlessandhencenotdetected in thespectrophotometric assayused in Figure 1A) but were virtually devoid of downstream CRTs(Supplemental Figure 4). Similar to that observed with LIN, otherbleaching inhibitors that prevent chloroplast development andcause albinism without specifically blocking the production ofCRTs, such as the plastid protein synthesis inhibitor chloram-phenicol (CAP) or the nitrogen assimilation inhibitor phosphino-tricin (PPT), were found to prevent shade-triggered elongationgrowth in wild type and HY5-defective mutants (Figure 7B). Bycontrast, inhibition of the CRT pathway downstream of lycopeneby blocking the activity of lycopene cyclases with 2-(4-chlorophenylthio)-triethylamine chloride (CPTA) resulted in albino seed-lings that were able to respond to shade and elongate when HY5function was lost (Figure 7).To test whether the ability to respond to shade of hy5 seedlings

grown in the presence of NF or CPTA was specifically due to theblockageof theCRTpathway,wenext usedArabidopsismutants.The enzymePHYTOENESYNTHASE (PSY) produces phytoene inthe first committed step of the CRT pathway (Figure 7A). BecausePSY is encoded by a single gene in Arabidopsis (Ruiz-Sola andRodríguez-Concepción, 2012), the knock-out mutant psy-1(Pokhilko et al., 2015) does not produce phytoene and hencecannot feed the pathway for the biosynthesis of downstreamCRTs (Supplemental Figure 4). As a consequence, the mutantdisplays an albino phenotype undistinguishable from that ob-served in wild-type seedlings treated with NF or CPTA (Pokhilkoet al., 2015). Similar to that described for wild-type seedlingsgrown in the presence of CRT biosynthesis inhibitors, psy-1seedlings were unable to elongate when exposed to W+FR(Figure 7B). However, the elongation responsewas rescuedwhenboth HY5 and CRTs were missing in double hy5 psy-1 mutantseedlings (Figure 7B).Pharmacological or genetic blockage of the CRT pathway

prevents the biosynthesis of CRTs and derived products, but itmight also cause an accumulation of upstream metabolites.Among them, methylerythritol cyclodiphosphate, an intermediateof the pathway that supplies the metabolic precursors of CRTs(Figure 7A), has been shown to act as a retrograde signal in re-sponse to stress (Xiao et al., 2012). Blockage of methylerythritol

Figure 6. HY5 Levels are Regulated by Shade and Retrograde Signals.

(A) Levels of HY5-encoding transcripts in wild-type and hy5 plants ger-minated and grown under W for 7 d and then exposed to W+FR for theindicated times. Transcript levels were quantified by RT-qPCR and rep-resented relative to those in W-grown wild-type plants (mean6 SE of n = 3samples corresponding to pools of whole seedlings grown in differentexperiments).(B) Levels of HY5-GFPprotein in hy535S:HY5-GFPplants germinated andgrown under W for 7 d with or without 5 mM NF or 1 mM LIN and thenexposed to W+FR for the indicated times. Protein levels were quantifiedfrom immunoblot analysis with a commercial anti-GFP serum. Mean6 SE

values (n $ 3 samples from pools of whole seedlings grown in differentexperiments) are represented relative to those in plants grown withoutinhibitors before exposure to shade. Asterisksmark statistically significantdifferences relative to the 0 h time point (t test, P # 0.05).(C) ChIP analysis of HY5-GFP binding to the promoters of the indicatedgenes. After germinating and growing plants of the hy5 35S:HY5-GFP lineon media without inhibitors as indicated, ChIP experiments were doneusing commercial anti-GFP serum. Chromatin from these samples andfrom no-antibody controls was then used for RT-qPCR amplification ofHY5 binding sites in the promoter of the genes. Enrichmentwas calculatedas the ratio of anti-GFP versus no-antibody values after normalization withinput samples (i.e. before ChIP). Graph shows mean 6 SE values of n = 2samples from seedlings grown in different experiments. Asterisks markstatistically significant differences in shade-treated samples (t test, P #

0.05).(D) Levels of HY5-encoding transcripts in wild-type plants germinated onmedium with or without 5 mM NF or 1 mM LIN and grown under W for 2 dfollowed by five additional days under W or under W+FR. Transcript levelswere quantified by RT-qPCR and represented relative to those in plantsgrown under W without inhibitors (mean 6 SE of n = 3 samples corre-sponding to pools of whole seedlings grown in different experiments).

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cyclodiphosphate production with the inhibitor fosmidomycin(FSM; Figure 7A), however, did not prevent the elongation re-sponse to shade of NF-treated hy5 seedlings (Figure 7B). Wetherefore conclude that what allows hy5 seedlings to respond toshade is not the accumulation of a metabolite upstream PSY butthe depletion of a CRT-derived product synthesized after the stepblocked by CPTA, i.e. downstream of lycopene (Figure 7A).

As represented in Figure 7A, lycopene cyclization leads to theproduction of CRTs with two b-rings (b,b-CRTs such asb-carotene and derived xanthophylls) with one b- and one e-ring(b,e-CRTs such as lutein). The production of b,e-CRTs in Arabi-dopsis is completely blocked in the green lut2 mutant(Supplemental Figure 4; Emiliani et al., 2018), which is defective inthe only gene encoding LYCOPENE e-CYCLASE in this plantspecies (Figure 7A; Ruiz-Sola and Rodríguez-Concepción, 2012).Loss of b,e-CRTs did not change the elongation response toshade of single lut2 (versus wild type) or double hy5 lut2 (versushy5) seedlings (Supplemental Figure 5). We therefore concludedthat the effect observed with CPTA (Figure 7B) is not due to theabsence of b,e-CRTs but most likely to defects in the b-carotenebranch of theCRTpathway (Figure 7A). Considering all these datatogether, we speculated that unidentified products derived fromb,b-CRTs can repress shade-induced elongation growth inseedlings bleachedwith LIN and other inhibitors that do not targetthe CRT biosynthesis pathway. The absence of these products inseedlings treated with NF or CPTA, or in the psy-1mutant, allowshypocotyl elongation in response to shade but only when thegrowth-inhibitory effect of HY5 is released.

Abscisic Acid Represses the Elongation Response to Shade

Among the biologically activemetabolites derived fromb,b-CRTs(Hou et al., 2016), we decided to evaluate the role of abscisic acid

(ABA) as this plant hormone was found to participate in thetransduction of chloroplast-derived reactive oxygen species/re-dox signals (Baier and Dietz, 2005; Gläßer et al., 2014; Chan et al.,2016), to modulate hypocotyl growth (Lau and Deng, 2010;Humplík et al., 2017) and to act togetherwith HY5 in the regulationof several plant cell responses (Chen et al., 2008; Xu et al., 2014).Furthermore, treatmentwith lowR/FRwas reported to induceABAproduction and signaling in tomato (Solanum lycopersicum) andArabidopsis (Cagnola et al., 2012; González-Grandío et al., 2013;Holalu and Finlayson, 2017). Indeed, a tendency to increase ABAcontents was observed in green seedlings after only 1 h of ex-posure to our simulated canopy shade conditions (SupplementalFigure 6).As expected, ABA was absent in NF-treated seedlings but it

could be detected in LIN-grown seedlings (Supplemental Fig-ure 6). If the presence of ABA in LIN-treated hy5 seedlings con-tributed to inhibit their response to shade, it would be expectedthat preventing the formation of this hormone would be sufficientto rescue their response to shade. In agreement, a geneticblockage of the last step of ABA biosynthesis, catalyzed by theABA2 protein (Figure 7A), allowed LIN-treated double hy5 aba2seedlings to elongate in response to shade (Figure 8). Single hy5and double hy5 aba2 seedlings had a very similar response toshade in NF-supplemented medium. By contrast, in greenseedlings grown in the absence of inhibitors (i.e. with functionalchloroplasts), the double mutant elongated more than single hy5seedlings when exposed to shade (Figure 8A). We thereforeconcluded that HY5 and ABA likely repress shade-induced hy-pocotyl elongation by independent pathways.This conclusion was further supported by the results of treating

NF-grown wild type and hy5 seedlings with increasing concen-trationsofABA (Figure8B). Althoughnoeffectwasobserved in thewild type, the ability of hy5 seedlings to elongate in response to

Figure 7. Blockage of the CRT Pathway Derepresses Shade-Triggered Elongation of Bleached HY5-Defective Seedlings.

(A)Pathways for thebiosynthesisofCRTsandderivedhormones. Thesteps targetedbyNFandother inhibitors and the reactionscatalyzedbyenzymes thatdetermine metabolic flux to CRTs (PSY) and ABA (ABA2) are shown. Xanthophylls are boxed in yellow.(B) Elongation responses to shade in wild-type and mutant plants defective in HY5, PSY, or both. Wild-type (WT) and single hy5 mutant plants weregerminated and grown as indicated on media either supplemented or not with concentrations of NF, CPTA, FSM, LIN, CAP, or PPT producing albinoseedlings. Single psy-1 and double hy5 psy-1mutants were only grown without inhibitors. Graph represents the mean6 SE values of n$ 30 seedlings ina representative experiment.

Interaction of Light and Retrograde Pathways 391

W+FR exposure was progressively repressed as ABA concen-tration increased. At concentrations of the hormone of 200 nM orhigher, which are within the physiological range (Waadt et al.,2014), NF-treated hy5 seedlings did not respond to shade(Figure 8B), similar to that observed with the LIN treatment.

Exogenous ABA treatment was also able to repress shade-promoted hypocotyl elongation in green wild-type seedlingsgrown without inhibitors (Figure 9). We used this phenotype toidentify ABA-related transcription factors involved in this re-sponse.Mutantsdefective inABA-INSENSITIVE3 (ABI3) andABI4elongated slightlymore thanwild-typeseedlingswhen illuminatedwith W+FR and this response was not repressed by ABA. Bycontrast, ABI5-defective seedlings showed a wild-type pheno-type in terms of sensitivity of shade-triggered elongation to ABAtreatment (Figure 9A). These results suggest that ABI3 and ABI4but not ABI5 are required for ABA to inhibit hypocotyl elongation.

If these transcription factorsalso transduce theABAsignal in themolecular pathway that blocks elongation in shade-exposed al-bino seedlings, it would be expected that double mutants lackingbothHY5andABI3orABI4andgrown in thepresenceofLINwould

be able to elongatewhen exposed toW+FR. Indeed, these doublemutants elongatedmore than their parental lineswhen bothmock(green) and LIN-treated (albino) seedlings were grown undersimulated shade (Figure 9B). Shade-triggered elongation of LIN-treated hy5 abi3 and hy5 abi4 seedlings, however, was reducedcompared with that of ABA-defective hy5 aba2 seedlings(Figure 9B). These results suggest that ABI3 and ABI4 might notparticipate in the same ABA signaling pathway eventually re-pressing hypocotyl elongation, but have partially redundant rolesin this process (Figure 10).

A Mechanistic Model for the Modulation of ShadeElongation Responses by Plastid-Dependent Signals

A model generated based on the described results is shown inFigure 10. In high-plant–density environments, like those found inforests, prairies, or orchard communities, a set ofR/FR-dependentadaptive responses are unleashed in shade-avoiding plants.Compared with plant proximity (without direct vegetative shading),canopy shade in nature involves lower R/FR values associated

Figure 8. ABA Represses Shade-Triggered Hypocotyl Elongation Independent of HY5.

(A)Elongation responses to shade inwild-type (WT) andmutant plants defective inHY5,ABA2, or both. Plantsweregerminatedandgrownas indicatedwithor without 5mMNFor 1mMLIN.Graph represents themean6 SE values of a total of n$ 25 seedlings from at least two independent experiments. Asterisksmark statistically significant responses to shade (t test, P # 0.05).(B)Effect of ABA on the elongation of NF-treated seedling hypocotyls in response to shade.Wild-type (WT) and hy5 plants germinatedwith or without 5mMNF plus the indicated concentrations of ABA were grown under W for 2 d followed by five additional days under W or under W+FR. Graphs representhypocotyl length (mean 6 SE of n $ 25 seedlings in a representative experiment).

392 The Plant Cell

with a reduction in the amount of PAR. Although phyB is themajorphy controlling these responses, the photolabile phyA has anantagonistic negative role in the shade-mediated regulation ofhypocotyl elongation (Ciolfi et al., 2013; Martínez-García et al.,2014; Wang et al., 2018; Zhang et al., 2018). Independently of thePAR level, phyB is deactivated by shade of intermediate, low, andvery low R/FR, whereas phyA signaling is activated by shade oflow and very low R/FR. As a result, hypocotyl elongation isderepressed under conditions mimicking vegetation proximity (aresponse aimed at overgrowing neighbors for optimal light ex-posure). Under R/FR values typical of canopy shade, however,phyA activation prevents seedlings from exhibiting excessiveelongation (Figure 10). Our results reported here and elsewhere(Bou-Torrent et al., 2015) suggest that HY5 represses the hypo-cotyl elongation response more strongly under canopy shade. Aspreviously proposed, HY5 might be principally involved in thephyA-dependent pathway (Ciolfi et al., 2013; Wang et al., 2018;Zhang et al., 2018) whereas other transcription factors, includinggrowth-promoting PIFs, would bemostly associated to the phyB-dependent pathway (Figure 10). These antagonistic phyB/PIF andphyA/HY5 pathways likely provide young seedlings with the ca-pacity to rapidly elongate when impending competition is nearbybut also to attenuate excessive growth when growing undera canopy.

During seedling deetiolation, the phyB/PIF pathway convergeswith a GUN1-dependent retrograde pathway to antagonisticallyregulate the transcriptional photomorphogenic network (Martínet al., 2016). TheGUN1-mediated retrogradesignal involved in thisparticular process was proposed to attenuate photomorpho-genesis when chloroplast function is challenged, and to be in-dependent of ABI4 and HY5 (Martín et al., 2016). Our resultsreported here suggest that in shade-exposed seedlings,

a completely different retrograde network that is independent ofGUN1 but does depend on HY5, ABI3, and ABI4 modulates theantagonistic action of phyA/HY5 and phyB/PIFs signaling path-ways (Figure 10).Prolonged exposure to shade causes a decrease in the accu-

mulation of CHLs and CRTs that can eventually compromisephotosynthesis and photoprotection (Roig-Villanova et al., 2007;Cagnola et al., 2012;Bou-Torrent et al., 2015).Our results suggestthat such a challenge to the chloroplast functional status might inturn feed-back to regulate the response to shade (Figure 10).Treatmentwith low concentrations of NF or LIN (i.e. those causingweak to moderate reduction in the level of photosynthetic pig-ments) was sufficient to repress the hypocotyl elongation re-sponse to low R/FR (Figure 1), likely due to delayed phyBdeactivation after a reduction in R/FR (Figure 2). Decreased phyBdeactivation correlated with impaired PIF accumulation(Supplemental Figure 2) and attenuatedgeneexpression changes(Figure 3). NF or LIN treatments also caused an enhanced ac-cumulation of HY5 transcripts and increased the stability of theHY5-GFP reporter protein (Figure 6). Together, our findingssuggest that retrograde signals inhibit the SAS by repressing the(positive) phyB/PIFs pathway and by promoting the (negative)phyA/HY5 pathway (Figure 10).Our work further unveiled ABA as a component of the feedback

mechanism. This CRT-derived hormone was found to repressshade-triggered hypocotyl elongation (Figure 8), likely through theaction of the transcription factors ABI3 and ABI4 (Figure 9). ABI4has been proposed to participate in GUN1-dependent retrogradesignaling (Koussevitzky et al., 2007; Sun et al., 2011; Guo et al.,2016; Xu et al., 2016). However, the results supporting this claimhave been repeatedly challenged (Kacprzak et al., 2018). Our datasuggest thatABI4 (andABI3)mayact redundantly to transduce the

Figure 9. ABI3 and ABI4 But Not ABI5 Participate in the ABA-Mediated Repression of Shade-Induced Hypocotyl Elongation.

(A)EffectofABAon theelongation responses toshadeofwild-type (WT)seedlingsandmutantsdefective inABI3,ABI4,orABI5.Plantsweregerminatedandgrown as indicated on media with or without 0.2 mM ABA. Graph represents the mean 6 SE values of a total of n $ 25 seedlings from two independentexperiments.(B) Elongation responses to shade of double mutants defective in HY5 and either ABI3 or ABI4. Plants of the indicated genotypes were germinated andgrown as illustrated with or without 1 mM LIN. Graph represents the mean 6 SE values of a total of n$ 25 seedlings from two independent experiments.Asterisks mark statistically significant responses to shade (t test, P # 0.05). WT = wild type.

Interaction of Light and Retrograde Pathways 393

ABA-dependent signal that represses shade-triggered hypocotylelongation in response to chloroplast dysfunction (Figure 9).WhileHY5 was previously shown to directly bind and activate thepromoter of ABI5 to promote light-induced hypocotyl inhibitionduring deetiolation (Chen et al., 2008; Xu et al., 2014), our resultssuggest that thismechanism does not participate in the control ofshade-dependent hypocotyl growth. First, HY5 and ABA appearto repress hypocotyl growth by independent pathways (Figure 8).Second, ABI5 is not required to transduce the ABA signal even-tually repressing the response to shade (Figure 9).

Arabidopsis mutants defective in phyB were found to accu-mulate greater amounts of ABA under well-watered conditionsand to be less sensitive to exogenous ABA treatments (Gonzálezet al., 2012). Further supporting a negative role of light in ABAsynthesis,dark treatmentofpreviously light-grownplants resultedin increased ABA contents (Weatherwax et al., 1996). A shade-triggered increase in ABA production was reported here(Supplemental Figure 6) and elsewhere (Cagnola et al., 2012;

González-Grandío et al., 2013; Holalu and Finlayson, 2017). It ispossible that W+FR treatment might promote ABA production torepress the elongation response to shade as part of the mecha-nism that prevents a too-intense commitment (Figure 10). Theseresults together support ABA as a central signal connecting thefunctional status of the chloroplast with light responses.Interestingly, the plastid-synthesized metabolite 39-

phosphoadenosine 59-phosphate (PAP), which functions asa retrograde signal during oxidative stress caused by high lightexposure and drought, was recently shown to act in concert withABA signaling in guard cells to mediate stomatal closure and inseeds tomediate dormancy and germination (Pornsiriwong et al.,2017). PAP accumulates when the SAL1 phosphatase that nor-mally degrades this metabolite is inactivated during oxidativestress (Estavilloetal., 2011).SAL1-defectivemutantsshowashorthypocotyl phenotype in the light, indicating that accumulation ofPAP can repress hypocotyl elongation (Kim and von Arnim, 2009;Chen and Xiong, 2011). This phenotype is rescued (at least par-tially) in double sal1 phyB and sal1 hy5 mutants (Kim and vonArnim, 2009; Chen and Xiong, 2011), suggesting that functionalphyB and HY5 are required for the PAP-promoted and light-dependent repression of hypocotyl growth. Further experi-ments should explore whether PAP is the retrograde signal de-duced fromourdata toattenuate the response toshade in termsofhypocotyl elongation by independently inhibiting phyB de-activation, increasing HY5 accumulation, and promoting ABAsignaling (Figure 10).Besides ABA, it is possible that other CRT-derived products

might also contribute to the repression of shade-triggered hy-pocotyl elongation detected in hy5 seedlings bleached with LIN,CAP, or PPT but not with NF or CPTA (Figure 7). In particular,strigolactones are hormones derived from b-carotene (Figure 7A)that inhibit hypocotyl elongation in the light by a mechanism re-quiring phytochromes and involving upregulation of HY5 ex-pression and protein (Tsuchiya et al., 2010; Jia et al., 2014). Othermetabolites produced after cleavage of CRTs includeb-cyclocitral, and unknown compounds that modulate de-velopmental and stress responses (Hou et al., 2016). Whileb-cyclocitral is a relatively well-established retrograde signalassociated with oxidative stress (Ramel et al., 2012), its contri-bution tohypocotyl elongation isunknown.Similarly, nohypocotylgrowth alterations have been reported in mutants lacking CRT-derivedsignals thatdohavean influenceon leafdevelopment (VanNorman and Sieburth, 2007; Avendaño-Vázquez et al., 2014).Whether any of these CRT-related metabolites participate in theelongation response to shade remains to be investigated.Collectively, our data support the notion that chloroplasts are

plant cell compartments with fundamental roles not only forphotosynthesis and metabolism but also for environmental (light)sensing and signaling. Here we show that HY5 and ABA (via ABI3and ABI4) are nodes of a plastid-modulated network that at-tenuates the response to shade in terms of hypocotyl elongation.In green plants with functional chloroplasts, light signals asso-ciated with canopy shade rapidly promote hypocotyl elongationvia the phyB/PIF pathway. Exposure to low R/FR also triggersnegative (growth-repressing) circuits involving the phyA/HY5pathway and the CRT-derived hormone ABA, likely to prevent anexcessive response and facilitate the return to non-shade

Figure 10. Model for the Modulation of Shade Elongation Responses byRetrograde Signals.

In green plants with functional chloroplasts, low R/FR (i.e. canopy shade)signals promote accumulation of growth-promoting PIFs (via phyB de-activation) and also of growth-repressing HY5 (via phyA), likely to preventan excessive elongation response. Persistent shading or other environ-mental factors challenging chloroplast function (including exogenoustreatment with LIN or NF) can repress phyB inactivation, enhance HY5expression, and likely promote HY5 stability, eventually resulting in de-creasedelongationgrowth.An independentpathway involvesABA,aCRT-derived hormone that represses shade-triggered hypocotyl elongation viaABI3andABI4.NF (but not LIN) prevents theproductionofABA.Asa result,loss of both HY5 and ABA in NF-treated hy5 seedlings allows them toelongate when exposed to low R/FR, whereas this hypocotyl response isblocked by low but detectable levels of ABA in LIN-treated mutants.

394 The Plant Cell

conditions if the lowR/FR signal disappears (e.g. if a commitmentto the shade-avoidance lifestyle isunnecessary).Whenmaintained,shade further causes a decrease of CHL and CRT contents, whichmight eventually disrupt chloroplast homeostasis. Such situationwould be then signaled to feedback-regulate the response to thelight signal by independently inhibiting phyB deactivation, in-creasing HY5 accumulation, and promoting ABA signaling. Thismechanism connecting the metabolic status of the chloroplastwith light (shade) signaling and developmental responses likelycontributes to achieve optimal photosynthetic performance.

METHODS

Plant Material

All mutants used in this work are listed in Supplemental Table 1. Arabi-dopsis (Arabidopsis thaliana) lines used here were in the Columbia (Col)background with the only exception of hy1-1, a Landsberg erecta (Ler)mutant (Rodríguez-Concepción et al., 2004). Some of those lines werealready available in our lab and previously used in published works, in-cludinghdr-3 (Pokhilko et al., 2015),gun1-101 (Llamaset al., 2017),gun5-1(Llamas et al., 2017), pifQ (Toledo-Ortiz et al., 2010), hy5-2 (Bou-Torrentet al., 2015), psy-1 (Pokhilko et al., 2015), lut2 (Emiliani et al., 2018), aba2(Ruiz-Sola et al., 2014), and hy5 35S:HA-HY5 (Toledo-Ortiz et al., 2014).Lines abi3-8 (Nambara et al., 2002), abi4-1 (Finkelstein et al., 1998), abi5-7(Tamura et al., 2006), and 35S:GUS-PIF3 (Monte et al., 2004) were re-quested. For generation of double mutants, single homozygous plantswere crossed and the F2 progeny was first screened for the characteristiclong hypocotyl phenotype associated to the hy5mutation in homozygosis.Long individuals were then PCR-genotyped to identify homozygousmutants for the second gene and confirm that theywere also homozygousfor hy5. For the generation of the 35S:HY5-GFP construct, the full codingregion of the Arabidopsis HY5 complementary DNA (cDNA) was PCR-amplified using primers HY5-attB1-F and HY5-attB2-R (SupplementalTable 2) and cloned into Gateway pDONR-207. Cloning into GatewaypGWB405 eventually generated the construct for the 35S promoter-drivenexpression of a C-terminal fusion of the sGFP reporter protein to HY5. Thisconstructwasused to transformthehy5-2mutantbyfloraldipping. Thehy535S:HY5-GFP line used for the experiments reported here was selectedbased on complete complementation of the long hypocotyl phenotypeassociated with the hy5 mutation and high levels of nuclear GFP fluo-rescence. Line 35S:PHYB-GFP was generated by transforming Col-0 plants with the same construct previously found to work in an Arabi-dopsis phyBmutant in the Ler background (Yamaguchi et al., 1999). Fromthe resulting transformants, we selected for further experiments one of thelines showing a clearer accumulation of the phyB-GFP protein in nuclearbodies under W.

Growth Conditions and Treatments

Seeds were surface-sterilized and germinated on solid Murashige andSkoogmediumsupplementedwith10mg/mLofSuc toprovidecarbonandenergy for albino seedlings to grow. When indicated, the medium wasfurther supplemented with different concentrations or NF (Zorial), LIN(Sigma-Aldrich), or ABA (Sigma-Aldrich). Other chemicals added to themedium included epibrassinolide (1mM), gibberellic acid (10mM), picloram(5mM),CPTA (25mM), FSM (500mM),CAP (50mM), or PPT (100mM).Whencomparing different lines (e.g. wild type versus mutant), they were growntogether on the sameplate insteadof growing each line on adifferent plate.After stratification for at least 3 d at 4°C in the dark, plates were incubatedin growth chambers at 22°C under W of 20 to 24 mmol m–2 s–1 PAR(R/FR = 1.6). When indicated, W was supplemented with FR provided

byGreenPowerLEDmoduleHF far-red (Philips)QB1310CS-670-735 light-emitting diode hybrid lamps (Quantum Devices) to simulate canopy shade(20–24mmolm–2s–1PAR,R/FR=0.02). Fluence ratesweremeasuredusinga Spectrosense 2 meter associated with a 4-channel sensor (Skye In-struments) as described in Martínez-García et al. (2014). Grown seedlingswere laid out flat on the growth media and digital images were taken toquantify hypocotyl length using the software ImageJ (NIH).

Microscopy

Whole35S:PHYB-GFPseedlingsgerminatedandgrownunderW for 7donmedia with or without 5mMNFor 1mMLINwere exposed to a 5-min pulseof FR (735 nm, 60 mmol/m2/s) and then kept in the dark. At different timepoints, treated seedlings were placed on glass slides under a safety greenlight and kept in the dark until observation with a BX60 FLUOVIEW FV300microscope (Olympus). Confocal laser scan images of the hypocotyl areacloser to the cotyledons were obtained at different time points in the darkwith a combination of 488-nm laser excitation and 515-nm long-pass filter(LP515; Carl Zeiss). For each time point, three sequential images fromdifferent focus planes were recorded automatically.

ChIP

Approximately 800mL of seeds from hy5 35S:HY5-GFP plantswere platedon 8-square (10 3 10 cm) plates of Suc-supplemented medium. Aftergrowth for 2 d under W, four plates were left under W and four weretransferred to W+FR for five additional days. For ChIP (Moon et al., 2008),eachsamplewasdivided in threealiquotsafter crosslinkingandsonication:one input, one to be incubated with a 1:1000 dilution of anti-GFP antibody(A-11122; Invitrogen), and the last one tobeprocessedsimilarlybutwithoutantibody. After DNA isolation, the three samples were used for RT-qPCRanalysis of promoter sequence abundance with the primers shown inSupplemental Table 2. After normalization with the input, enrichment wascalculated as the ratio of the signal with, versus without, antibody.

Gene Expression and Immunoblot Analyses

Total RNA was extracted from whole seedlings and used for RT-qPCRanalysis as described in Llamas et al. (2017). Briefly, RNA was extractedwith the Maxwell 16 LEV Plant RNA Kit (Promega) and quantified witha NanoDrop (Thermo Fisher Scientific). After producing cDNA with thecDNA Synthesis Kit (Roche), RT-qPCR was performed using LightCycler480 SYBR Green I Master (Roche) with the gene-specific primers listed inSupplemental Table 2. Expression of target genes was normalized usingUBC21as theendogenous referencegene.Proteinextraction, immunoblotanalysis, and quantification of protein abundance were performed asdescribed in Llamas et al. (2017) using a 1:1,000 dilution of anti-GFP serum(A-11122; Invitrogen).

Quantification of Metabolite Levels

Whole seedlings were frozen in liquid nitrogen, lyophilized, and ground ina mortar for extraction and quantification of photosynthetic pigments andABA.CHLandCRT levelsweremeasuredeither by spectrometricmethodsor by high-performance liquid chromatography (Bou-Torrent et al., 2015).ABA content was quantified by liquid chromatography-electrosprayionization-tandem mass spectrometry as described in Ruiz-Sola et al.(2014).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis GenomeInitiative or GenBank/European Molecular Biology Laboratory databases

Interaction of Light and Retrograde Pathways 395

under the following accession numbers: ABA2 (AT1G52340), ABI3(AT3G24650), ABI4 (AT2G40220),ABI5 (AT2G36270),GUN1 (AT2G31400),GUN5 (AT5G13630), HDR (AT4G34350), HY1 (AT2G26670), HY5(AT5G11260), LUT2 (AT5G57030), PIF1 (AT2G20180), PIF3 (AT1G09530),PIF4 (AT2G43010), PIF5 (AT3G59060), PSY (AT5G17230).

Supplemental Data

Supplemental Figure 1. Albino seedlings are able to elongate whentreated with growth regulators.

Supplemental Figure 2. NF treatment prevents shade-dependentaccumulation of the GUS-PIF3 protein.

Supplemental Figure 3. HY5 represses hypocotyl elongation inresponse to shade.

Supplemental Figure 4. CRT levels in inhibitor-treated wild-typeplants and CRT-defective mutants.

Supplemental Figure 5. Shade-induced elongation responses in theabsence of lutein.

Supplemental Figure 6. ABA levels in shade-exposed and inhibitor-treated wild-type plants.

Supplemental Table 1. Mutants used in this work.

Supplemental Table 2. Primers used in this work.

ACKNOWLEDGMENTS

This work was supported by the Spanish Ministry of Science, Innovation,and Universities (MICINN grants BIO2014-59895-P, BIO2014-59092-P,BIO2015-71703-REDT, BIO2017-90877-REDT, BIO2017-85316-R, andBIO2017-84041-P), the Generalitat de Catalunya (grants 2017SGR-1211to J.F.M.G. and 2017SGR-710 to M.R.C.), the Generalitat de CatalunyaCERCA Programme (to the Centre for Research in Agricultural Genomics),the Japan Society for the Promotion of Science (KAKENHI grant JP-15H04389 to A.N.), the Spanish Ministry of Economy and Finance SeveroOchoa Programme for Centres of Excellence in R&D 2016–2019 (grantSEV-2015-0533),SpanishMinistryofEconomyandFinance (MINECOPhDfellowship BES-2012-052597 to M.O.A.), and Mexican Consejo Nacionalde Ciencia y Tecnología (MINECO PhD fellowship 421688 and SEP “BecaComplemento” to E.L.). We thank Elena Monte (Centre for Research inAgricultural Genomics) for comments on the manuscript. Technical sup-port from M. Rosa Rodríguez-Goberna and members of the Centre forResearch in Agricultural Genomics core facilities is greatly appreciated.

AUTHOR CONTRIBUTIONS

M.O.A., J.F.M.G., andM.R.C.designed the research;M.O.A., E.L., andA.G.C.performed research; A.N. contributed analytic tools; M.O.A., E.L., A.N.,J.F.M.G., andM.R.C. analyzed data; J.F.M.G. andM.R.C. wrote the paper.

Received August 17, 2018; revised January 2, 2019; accepted January 30,2019; published February 6, 2019.

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398 The Plant Cell

DOI 10.1105/tpc.18.00617; originally published online January 31, 2019; 2019;31;384-398Plant Cell

Martínez-García and Manuel Rodríguez-ConcepciónMiriam Ortiz-Alcaide, Ernesto Llamas, Aurelio Gomez-Cadenas, Akira Nagatani, Jaime F.

HY5 and Abscisic AcidChloroplasts Modulate Elongation Responses to Canopy Shade by Retrograde Pathways Involving

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