arabidopsis cop1 and spa genes are essential for plant ... · to bind to dna (hornitschek et al.,...

Arabidopsis COP1 and SPA Genes Are Essential for PlantElongation But Not for Acceleration of Flowering Time inResponse to a Low Red Light to Far-Red Light Ratio1[W]

Sebastian Rolauffs2, Petra Fackendahl, Jan Sahm, Gabriele Fiene, and Ute Hoecker*

Center of Excellence on Plant Sciences, Botanical Institute, Biocenter, University of Cologne, 50674 Cologne,Germany

Plants sense vegetative shade as a reduction in the ratio of red light to far-red light (R:FR). Arabidopsis (Arabidopsis thaliana)responds to a reduced R:FR with increased elongation of the hypocotyl and the leaf petioles as well as with an acceleration offlowering time. The repressor of light signaling, CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), has been shown previouslyto be essential for the shade-avoidance response in seedlings. Here, we have investigated the roles of COP1 and the COP1-interactingSUPPRESSOR OF PHYA-105 (SPA) proteins in seedling and adult facets of the shade-avoidance response. We show that COP1 andthe four SPA genes are essential for hypocotyl and leaf petiole elongation in response to low R:FR, in a fashion that involves theCOP1/SPA ubiquitination target LONG HYPOCOTYL IN FR LIGHT1 but not ELONGATED HYPOCOTYL5. In contrast, theacceleration of flowering in response to a low R:FR was normal in cop1 and spa mutants, thus demonstrating that the COP1/SPA complex is only required for elongation responses to vegetative shade and not for shade-induced early flowering. We furthershow that spa mutant seedlings fail to exhibit an increase in the transcript levels of the auxin biosynthesis genes YUCCA2 (YUC2),YUC8, and YUC9 in response to low R:FR, suggesting that an increase in auxin biosynthesis in vegetative shade requires SPAfunction. Consistent with this finding, expression of the auxin-response marker gene DR5::GUS did not increase in spa mutantseedlings exposed to low R:FR. We propose that COP1/SPA activity, via LONGHYPOCOTYL IN FR LIGHT1, is required for shade-induced modulation of the auxin biosynthesis pathway and thereby enhances cell elongation in low R:FR.

Plants have evolved the capacity to perceive theambient light environment and to adapt their growthand development accordingly. In particular, plants likeArabidopsis (Arabidopsis thaliana) respond to compet-ing vegetation with an increased elongation growth,resulting in a longer hypocotyl, extended internodes,and elongated petioles. Furthermore, shaded plantsexhibit an increased leaf angle and an acceleration offlowering when compared with plants grown in opensunlight. These adaptive responses provide an attemptto outgrow the shading neighbors and are, therefore,collectively referred to as the shade-avoidance syn-drome (SAS; Ballaré, 1999; Casal, 2012; Ruberti et al.,2012). Plants sense the proximity of other plants basedon the associated reduction in the ratio of red light tofar-red light (R:FR). In full sunlight, the R:FR is high(greater than 1), while under shading conditions, theR:FR of the incoming light is low (less than 1) because

neighboring vegetation absorbs red light (R) but re-flects and transmits far-red light (FR). The reflected FRcan thus initiate a SAS before actual shading occurs(Ballaré et al., 1990; Casal, 2012).

The change in R:FR is perceived by the phytochromefamily of photoreceptors, which are activated by ab-sorption of R and inactivated by absorption of FR. Thus,the proportion of active phytochrome depends on the R:FR of the incoming light. In Arabidopsis, there are fivephytochromes (phyA–phyE), among which, phyB hasthe predominant role in the SAS. Hence, phyB-deficientmutants exhibit a SAS even in full sunlight and displayonly weak responses to a reduction in R:FR (Franklin,2008; Franklin and Quail, 2010). In contrast to phyB tophyE, which are primarily active at a high R:FR, thephotoreceptor phyA shows a maximum of activity incontinuous far-red light (FRc) and, therefore, counter-acts the phyB-mediated induction of the SAS in low R:FR conditions (Smith et al., 1997).

phyB transduces the perceived light signal via interac-tion with a family of basic helix-loop-helix (bHLH) tran-scription factors, the PHYTOCHROME-INTERACTINGFACTORS (PIFs). The PIF proteins are primarily active indarkness and in low R:FR conditions and are importantfor elongation responses that seedlings exhibit underthese conditions. In light with a high R:FR, phyB isconverted into the active conformation and transportedinto the nucleus, where it interacts with the PIF proteins.As a consequence, the PIF proteins are phosphorylatedand ubiquitinated, followed by their degradation in the

1 This work was supported by the Deutsche Forschungsgemein-schaft (grant nos. SFB572 and SFB635 to U.H.).

2 Present address: Brunel GmbH, Franz-Rennefeld-Weg 4, 40472Duesseldorf, Germany.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Ute Hoecker ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.112.207233

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26S proteasome (Franklin, 2008; Leivar and Quail,2011). Four members of the PIF family, PIF3, PIF4, PIF5,and PIF7, were shown to be involved in the elongationresponse of hypocotyls in response to low R:FR condi-tions. Hence, mutants with defects in these genes showa reduced or absent SAS (Lorrain et al., 2008; Leivaret al., 2012; Li et al., 2012).

The PIF-mediated elongation responses are coun-teracted by atypical bHLH transcription factors thatlack the basic domain responsible for DNA binding.Hence, mutants with a defect in these transcription fac-tors (long hypocotyl in fr light1 [hfr1], phytochrome rapidlyregulated1 [par1], par2) show an exaggerated SAS (Sessaet al., 2005; Roig-Villanova et al., 2007; Hornitscheket al., 2009). The HFR1 protein was shown to physicallyinteract with PIF4 and PIF5 via the helix-loop-helixdomain and thereby inhibit the ability of PIF proteinsto bind to DNA (Hornitschek et al., 2009). The transcriptlevels of HFR1, PAR1, and PAR2 rise shortly after theonset of low R:FR conditions, thus providing a negativefeedback loop (Sessa et al., 2005; Roig-Villanova et al.,2006).

Genetic analyses have identified additional tran-scription factors involved in the SAS. Overexpressionof the homeodomain Leu zipper protein ArabidopsisHOMEOBOX PROTEIN2 (ATHB2) leads to longer hy-pocotyls, while reduced levels of ATHB2 cause shorterhypocotyls compared with the wild type (Schena et al.,1993; Steindler et al., 1999). Consistent with this finding,ATHB2 transcript levels are strongly and rapidly up-regulated in response to low R:FR conditions (Sorinet al., 2009). Two other shade-induced members of thisfamily are also positive regulators of the SAS (Sorinet al., 2009). The B-box-containing protein B-BOX21 wasidentified as a negative regulator of the SAS (Croccoet al., 2010). The positive regulator of light signaling,ELONGATED HYPOCOTYL5 (HY5), is involved in therepression of shade avoidance by brief exposure to fullsunlight (Sellaro et al., 2011).

Microarray analyses have identified many genesthat are regulated by R:FR in seedlings. This includes anumber of auxin-related genes (Devlin et al., 2003; Sessaet al., 2005; Tao et al., 2008). Indeed, the SAS requiresfunctional auxin biosynthesis, auxin transport, andauxin signaling (Casal, 2012). Expression of the auxin-responsive DR5::GUS reporter increases in hypocotyland cotyledons of seedlings exposed to low R:FR(Salisbury et al., 2007; Tao et al., 2008). This reflects atleast in part a low R:FR-induced increase in auxin bio-synthesis via the TRYPTOPHAN AMINOTRANSFER-ASE OF ARABIDOPSIS1 (TAA1) pathway (Tao et al.,2008). While TAA1 transcript levels are not regulatedby R:FR (Tao et al., 2008), the mRNA levels of severalYUCCA (YUC) enzymes that act downstream of TAA1in the auxin biosynthesis pathway (Stepanova et al.,2011; Won et al., 2011) were shown to increase in lowR:FR. Consistent with this finding, mutants with defectsin multiple yuc genes show a reduced SAS in seedlings(Won et al., 2011; Li et al., 2012). The shade-induced in-crease in YUC expression and in auxin levels requires the

transcription factors PIF4, PIF5, and PIF7. Since thesePIF proteins directly bind the promoters of these YUCgenes, they are directly involved in up-regulating auxinbiosynthesis (Hornitschek et al., 2012; Li et al., 2012).

Besides auxin biosynthesis, the SAS also requirespolar auxin transport. Treatment of seedlings withNPA, an inhibitor of polar auxin transport, abolishesthe SAS and the low-R:FR-induced increase in DR5::GUS expression in the hypocotyl (Steindler et al., 1999;Tao et al., 2008). Recently, the auxin efflux carrier PIN3was shown to be required for the SAS. Low R:FR expo-sure increases the expression of PIN-FORMED3 (PIN3)and changes the localization of the PIN3 protein from thebasal to the lateral side of endodermal cells in thehypocotyl. This suggests that low R:FR changes theflux of auxin from inner to outer tissues in the hypo-cotyl, thus leading to cell and hypocotyl elongation(Keuskamp et al., 2010).

The analysis of mutants has also identified CON-STITUTIVELY PHOTOMORPHOGENIC1 (COP1) as acentral regulator of the SAS, since cop1 mutants lackthe low-R:FR-induced elongation response in seedlings(McNellis et al., 1994). COP1 acts in concert with SUP-PRESSOR OF PHYA-105 (SPA) proteins to suppressphotomorphogenesis in darkness. Hence, cop1 and spamutants undergo constitutive photomorphogenesis,exhibiting features of light-grown seedlings in com-plete darkness. The COP1/SPA complex is a Cullin4-based E3 ubiquitin ligase that is primarily active indarkness and catalyzes the ubiquitination of positiveregulators of the light response, such as the tran-scription factors HY5, HFR1, and B-BOX21 (Lau andDeng, 2012). At the adult stage, the COP1/SPA com-plex also regulates photoperiodic flowering by ubiq-uitinating the activator of flowering CONSTANS (CO)under dark conditions. In the light, the COP1/SPAcomplex is inactivated by photoreceptors, thus allow-ing the positive regulators of light signaling to accu-mulate and photomorphogenesis to be initiated (Lauand Deng, 2012). COP1 is a single-copy gene, whileSPA proteins are encoded by a small family of fourgenes (SPA1–SPA4). Genetic analysis has shown thatthe four SPA genes have redundant, but also partiallydistinct, functions in controlling the different facets oflight-regulated plant development: seedling skoto-morphogenesis in darkness, prevention of exaggeratedseedling photomorphogenesis in the light, petioleelongation and leaf expansion, as well as photoperi-odic flowering (Laubinger et al., 2004, 2006; Fittinghoffet al., 2006; Balcerowicz et al., 2011).

While the function of the COP1/SPA complex inphotomorphogenesis has been intensively studied, itsrole in the SAS is not understood. Moreover, little isknown about the genetic control of the SAS beyond theseedling stage. Here, we have analyzed the role ofCOP1 and the four SPA proteins in the SAS through-out Arabidopsis development. We show that COP1and SPA genes are essential for shade-induced elon-gation responses of seedling and leaf tissues but thatneither COP1 nor the SPA genes are required for the

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acceleration of flowering in response to low R:FR. Weprovide genetic evidence for an interaction of HFR1with the SPA genes in low R:FR conditions. Further-more, transcript level analyses of auxin biosynthesisgenes and a histochemical analysis of auxin responseindicate that SPA genes are involved in the elevation ofauxin biosynthesis in response to simulated shade.

RESULTS

SPA Genes Are Essential for Enhanced HypocotylElongation in Response to Low R:FR

To investigate functions of the four SPA genes inshade avoidance, we grew wild-type and spa mutantseedlings in continuous white light (Wc; high R:FR) andin Wc supplemented with FRc (Wc+FRc; low R:FR).Wild-type seedlings responded to simulated shade withincreased hypocotyl elongation (Fig. 1, A and B). Asreported previously, this phenotype was dependent on

phyB, since phyB mutants lacked the shade-avoidanceresponse and displayed even shorter hypocotyls insimulated shade than in Wc due to the FR high-irradiance response (Smith et al., 1997). All four spasingle mutants exhibited a similar phenotype to thewild type, showing increased hypocotyl elongation inWc+FRc. Since functional redundancy might mask SPAfunction, we tested multiple spa mutants. Two doublemutants, spa1 spa2 and spa3 spa4, exhibited longer hy-pocotyls in Wc+FRc than in Wc, but the elongation re-sponse to simulated shade was diminished comparedwith the wild type and the spa single mutants. Multiplespa mutants, such as the spa quadruple mutant (spa-Q)and the spa1 spa3 spa4 and spa1 spa2 spa4 triple mutants,lacked any increase in hypocotyl elongation in responseto simulated shade (Fig. 1, A and B). Similarly, cotyle-don petioles of these spa mutants failed to elongate inresponse to Wc+FRc (Fig. 1C). These results demon-strate that SPA genes have an essential, redundantfunction in the shade-avoidance response of seedlings.

Figure 1. SPA genes are essential for enhancedelongation of seedling hypocotyls and petioles inresponse to low R:FR. A, Visual seedling pheno-types of the indicated genotypes. Seedlings weregrown in Wc for 6 d or shifted to Wc+FRc after 3d in Wc. Within each pair of seedlings of onegenotype, Wc-grown seedlings are shown on theleft and seedlings grown under Wc+FRc areshown on the right. Bar = 5 mm. B, Hypocotyllength of seedlings grown as in A. Error bars rep-resent SE. C, Cotyledon petiole length of seedlingsgrown as in A. Error bars represent SE. Col-0, Wild-type accession Columbia.

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COP1/SPA in Arabidopsis Shade Avoidance Responses



We subsequently compared the phenotypes of thefour possible spa triple mutants, each retaining thefunction of only one SPA gene. We found that thesetriple mutants exhibited distinct phenotypes, with spa2spa3 spa4 and in particular spa1 spa2 spa3 mutantsretaining significant elongation responses to shade(Fig. 1). Hence, SPA1 and SPA4 are sufficient to sustaina shade-avoidance response in seedlings, while SPA2and SPA3 are not.

SPA Genes and COP1 Are Essential for the EnhancedPetiole Elongation of Adult Leaves in Response toLow R:FR

Adult plants also respond to low R:FR conditionswith increased elongation growth. In particular, thepetioles of rosette leaves strongly increase in lengthwhen plants are grown in simulated shade (Casal,2012). Since the role of COP1 or SPA genes in this as-pect of the shade-avoidance response has not beeninvestigated so far, we grew cop1-4 and spa mutantplants in Wc and under simulated shade conditions.Figure 2 shows that petioles of wild-type plants elon-gated in response to Wc+FRc. In contrast, petioles ofthe spa quadruple and cop1-4 mutants failed to showincreased elongation in response to simulated shadetreatment. Hence, SPA genes and COP1 are essentialfor shade-induced petiole elongation in adult plants.All four spa triple mutants retained a significant elon-gation response, which, however, was considerablysmaller when compared with the wild type, in partic-ular in those triple mutants retaining only SPA2, SPA3,or SPA4 function. The spa2 spa3 spa4 triple mutantretaining only SPA1 function showed an almost normalelongation response to shade. Hence, the four SPAgenes act redundantly in controlling this aspect of theshade-avoidance response, with SPA1 being the pre-dominant player among the four SPA genes.

cop1 and spa Mutants Show Normal Acceleration ofFlowering Time in Response to Low R:FR

Shade conditions cause earlier flowering, thus allow-ing plants to proceed to reproductive development at anearlier time, which likely provides a competitive ad-vantage under these suboptimal light conditions(Casal, 2012). Since COP1 and SPA proteins are re-quired for the photoperiodic regulation of floweringtime via controlling CO protein stability (Laubingeret al., 2006; Jang et al., 2008; Liu et al., 2008), we in-vestigated whether shade-dependent control of flow-ering time also depends on COP1 and SPA genes.Figure 3, A and B, shows that wild-type plants floweredat an earlier time and with fewer leaves in Wc+FRcwhen compared with Wc. All spa triple and quadruplemutants as well as the cop1-4mutant exhibited a similaracceleration of flowering time caused by low R:FR, as itwas observed in the wild type. This demonstrates that

low R:FR controls flowering time independently ofCOP1 and SPA genes.

Low R:FR increases the transcript levels of FT, whichencodes a key positive regulator of flowering time inArabidopsis (Kim et al., 2008; Wollenberg et al., 2008;Adams et al., 2009). Therefore, we tested whether FTexpression is altered in cop1 and spa mutants. Figure3C shows that simulated shade caused a similar, ap-proximately 50-fold, increase in FT transcript levels inthe wild type and in spa1 spa3 spa4 and cop1-4mutants.Hence, low-R:FR-induced FT regulation was normal incop1 and spa mutants, which is consistent with thenormal low-R:FR-induced acceleration of floweringtime observed in these mutants. Taken together, theseresults show that COP1 and SPA genes are essential forlow-R:FR-induced elongation responses in seedlingsand adult plants, while they are dispensable for earlyflowering caused by simulated shade.

Figure 2. SPA genes and COP1 are required for leaf petiole elongationin response to low R:FR. A, Photographs show representative plants ofthe wild type (Col-0), spa triple mutants, spa-Q, and cop1-4 grown inWc or Wc+FRc. Bars = 10 mm. B, Petiole length of the longest leaf of theindicated genotypes, represented as means6 SE. Plants were grown inWcfor 4 d and then shifted to Wc+FRc or kept in Wc for an additional 7 d.


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Expression of Early Shade Marker Genes in spaMutant Seedlings

We further analyzed SPA function in the shade-avoidance response of seedlings. Since SPA genes areessential for the morphological response of seedlingsto low R:FR, we subsequently asked whether this phe-notype reflects defects in the expression of early shademarker genes. Therefore, we analyzed transcript levelsof PHYTOCHROME-INTERACTING FACTOR3-LIKE1(PIL1), HFR1, and ATHB2 in Wc-grown seedlings ex-posed to low R:FR for 3 h. Figure 4A shows that thelevels of these three transcripts increased 10- to 30-fold

Figure 3. Shade avoidance-driven acceleration of flowering time doesnot require SPA and COP1 gene functions. A and B, Number of days(A) and number of rosette leaves (B) at the time of bolting in plantsgrown in Wc or Wc+FRc. Error bars represent SE. C, Relative transcriptlevels of the floral integrator FT in plants grown for 4 d in Wc and thenshifted to Wc+FRc or kept in Wc for an additional 7 d. UBQ10 wasused as an endogenous control. All data were calibrated to the Col-0Wc samples and represent means of three biological replicates 6 SE.

Figure 4. Expression of early shade marker genes in the wild type andin spa multiple mutants. A, Transcript levels of PIL1, ATHB2, HFR1,and XTR7 were determined by quantitative reverse transcription-PCR.Seedlings were grown in Wc for 4 d (Wc) and subsequently shifted toWc+FRc for 3 h (3 h Wc+FRc) or kept in Wc for 3 h (3 h Wc). UBQ10was used as an endogenous control. Data were calibrated to Col-0 Wcand represent means of three biological replicates 6 SE. B, Transcriptlevels of PIL1 were analyzed by quantitative reverse transcription-PCR.Seedlings were grown for 4 d in Wc and subsequently shifted to Wc+FRc or kept in Wc for the indicated times. UBQ10 was used as anendogenous control. All data were calibrated to the 0-h Wc (Col-0)sample and represent means of three biological replicates 6 SE.





in shade-exposed wild-type seedlings. spa triple mu-tants and the spa quadruple mutant showed a similarincrease in transcript levels in response to low R:FR.Hence, expression of these early shade marker genesappeared normal in the absence of SPA gene function.Also, over longer periods of low R:FR treatment, up to48 h, the spa1 spa3 spa4 mutant and also the cop1-4mutant retained a robust shade-induced increase inPIL1mRNA levels that was not different from the wildtype (Fig. 4B). This suggests that the induction of theseearly shade marker genes is not sufficient to elicit amorphological elongation response to shade.

We subsequently tested transcript levels of XYLO-GLUCAN ENDOTRANSGLYCOSYLASE7 (XTR7), a low-R:FR-responsive gene coding for a cell wall-modifyingenzyme that is thought to be directly involved in cellelongation (Hornitschek et al., 2009; Sasidharan et al.,2010). We found that XTR7 transcript levels increasedmore than 50-fold in wild-type seedlings exposed tosimulated shade (Fig. 4A). In spa triple and spa quadruple

mutants, in contrast, low R:FR led only to a 3- to 10-fold increase in XTR7 mRNA levels. These resultssuggest that an up-regulation of XTR7 by low R:FRcontributes to the increased hypocotyl elongation ob-served in simulated shade.

SPA Genes Are Required for Low-R:FR-Induced Increasein DR5::GUS and YUC Expression

The shade-avoidance response correlates with anincrease in the expression of DR5::GUS, an indirectartificial reporter of auxin-regulated gene expression(Salisbury et al., 2007; Tao et al., 2008). To investigatewhether DR5::GUS expression is altered in the spamutants lacking a shade-avoidance response, wecrossed the DR5::GUS transgene into the spa1 spa3 spa4and spa1 spa2 spa4 triple mutant backgrounds. Wild-type seedlings grown in Wc exhibited very low levelsof DR5::GUS that were visible only at the tips of the

Figure 5. spa triple mutants lack the low-R:FR-induced increase in the transcript levels of DR5::GUS and YUC auxin biosynthesis genes. A,Seedlings carrying the DR5::GUS transgene weregrown in Wc for 4 d and then shifted to Wc+FRcor kept in Wc for 7 h. Seedlings were then stainedfor GUS activity. B and C, Transcript levels ofTAA1 (B) and five YUC genes (C) in seedlingsgrown in Wc for 4 d and subsequently shifted toWc+FRc for 3 h or kept in Wc for 3 h.


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cotyledons and at the root tip (Fig. 5A). After exposureto low R:FR, wild-type seedlings responded with astrong increase in DR5::GUS levels, in particular in thecotyledons and in the vasculature of the hypocotyl.The two spa triple mutants, in contrast, did not exhibitany increase in DR5::GUS levels in response to Wc+FRc irradiation. These seedlings exhibited similarlylow DR5::GUS levels in Wc+FRc as the wild type didin Wc. Hence, the lack of a shade-avoidance responsein these spa triple mutants correlates with a failure toincrease DR5::GUS expression.The shade-induced increase in DR5::GUS levels may

be due to an increase in auxin levels and/or auxinsignaling. It was shown previously that an increase inauxin biosynthesis is required for hypocotyl elongationin response to low R:FR (Tao et al., 2008). Therefore,we investigated whether the transcript levels of genesin the auxin biosynthesis pathway are misregulated inspamutants. The levels of the TAA1 transcript were notresponsive to low R:FR (Fig. 5B), which is in agreementwith previous reports (Tao et al., 2008). TAA1 levelswere also not altered in spa triple or quadruple mu-tants under any of the conditions tested. Among thefive YUC transcripts that we tested, YUC2, YUC8, andYUC9 transcript levels increased 4- to 20-fold in wild-type seedlings exposed to Wc+FRc for 3 h (Fig. 5C).This is in agreement with the findings reported by Liet al. (2012). The four spa triple mutants showed a re-duced shade induction of these three YUC transcripts,while the spa quadruple mutant fully lacked any sig-nificant increase in the levels of these YUC transcriptsin response to low R:FR (Fig. 5C). Hence, SPA genes arerequired for the regulation of YUC2, YUC8, and YUC9transcripts by simulated shade. YUC1 and YUC4 tran-script levels, in contrast, were not regulated by lowR:FR in the wild type, and spa mutants did not show achange in expression of these YUC genes when com-pared with the wild type (Fig. 5C).

SPA Genes Genetically Interact with HFR1 in LowR:FR Conditions

The COP1/SPA complex acts as a negative regulatorof transcription factors that trigger light responses.These include HFR1 and HY5, whose levels are stronglyincreased in spa and cop1 mutants (Osterlund et al.,2000; Yang et al., 2005a, 2005b; Zhu et al., 2008; Nixdorfand Hoecker, 2010). HFR1 also serves a prominentfunction as a negative regulator of shade-avoidanceresponses (Sessa et al., 2005; Hornitschek et al., 2009;Galstyan et al., 2011). Therefore, we addressed thequestion of whether SPA genes might function inshade avoidance via controlling HFR1 protein levels.To this end, we analyzed the genetic interaction be-tween spa and hfr1 mutations in the shade-avoidanceresponse. As reported previously (Sessa et al., 2005),hfr1-101 mutant seedlings exhibited a longer hypocotylonly in simulated shade when compared with the wildtype (Fig. 6A). When the hfr1-101 allele was introducedinto the spa1 spa3 spa4 triple mutant, which lacks a

shade-avoidance response, the elongation response tosimulated shade was partially restored in this triplemutant: spa1 spa3 spa4 hfr1 mutant seedlings exhibiteda longer hypocotyl in Wc+FRc than in Wc (Fig. 6A).This suggests that SPA genes genetically interact withHFR1 in the response of the hypocotyl to low R:FR andsuggests that SPA proteins act positively on shadeavoidance by repressing HFR1 function.

Another target of the COP1/SPA complex, HY5,controls the light-induced reduction in cell elongation(Oyama et al., 1997; Osterlund et al., 2000). Therefore,we investigated whether HY5 is also involved in theCOP1/SPA-mediated control of shade avoidance.Figure 6B shows that spa1 spa3 spa4 hy5 mutant seed-lings lacked a shade-avoidance response just like thespa1 spa3 spa4 triple mutant did. Thus, introducing thehy5 mutation into a spa1 spa3 spa4 background did notrestore a shade-avoidance response. This indicates thatthe COP1/SPA-mediated control of HY5 protein levelsdoes not play a major role in shade avoidance.

Low R:FR Does Not Alter SPA Transcript Levels

We had observed that the four SPA genes providedifferential contributions to the seedling response to

Figure 6. The hfr1 mutation, but not the hy5 mutation, partially rescueshypocotyl elongation in response to low R:FR in the spa1 spa3 spa4mutant.Hypocotyl lengths are shown for 8-d-old seedlings grown inWc for 4 d andthen shifted to Wc+FRc or kept in Wc for 4 d. Error bars represent SE.





simulated shade, with SPA1 and SPA4 being the majorplayers and SPA2 and SPA3 being of minor importance(Fig. 1). Since the expression of SPA genes is differentiallycontrolled by light (Fittinghoff et al., 2006), we askedwhether the partially distinct functions of the four SPAgenes may be caused by differential expression in re-sponse to low R:FR. Figure 7 shows that exposure toWc+FRc did not significantly change the transcript levelsof any of the four SPA genes. This indicates that SPAgenes are not regulated by low R:FR. Thus, the partiallydistinct functions of the four SPA genes are not causedby differential regulation of SPA transcript levels in re-sponse to shade.

The Distinct Functions of SPA1 and SPA2 in the SeedlingShade-Avoidance Response Is Due to Differences in theRegulatory and Protein-Coding Sequences

We had previously generated transgenic lines expressingchimeric SPA1/SPA2 constructs that carry swaps between

the regulatory sequences (promoter; 39 untranslatedregion) and the protein-coding sequences (Fig. 8A;Balcerowicz et al., 2011). We used these transgeniclines to ask whether the difference between SPA1 andSPA2 function in the shade-avoidance response mapsto the respective regulatory and/or coding sequences.These chimeric SPA1/SPA2 constructs were expressed inthe spa-Q background, which lacks a shade-avoidanceresponse in seedlings. We found that expression of thenonchimeric SPA2::SPA2 construct did not restore ashade-avoidance response in the spa-Q mutant, as ex-pected, indicating that this construct correctly phe-nocopied the spa1 spa3 spa4 mutant (Fig. 8B). When theSPA1 protein was expressed from the SPA2 promoter(SPA2::SPA1), shade avoidance was observed. Hence,the sequence of the SPA1 protein carries intrinsic infor-mation for its function in shade avoidance. However, theSPA2 protein can restore shade avoidance when it isexpressed from the stronger SPA1 promoter (SPA1::SPA2; Fig. 8B). Hence, both the regulatory sequences andthe protein-coding sequences contribute to the distinctfunctions of SPA1 and SPA2 in shade avoidance.

DISCUSSION

Low R:FR conditions lead to a vast change in growthand development throughout the life cycle of a plant.Here, we have analyzed the role of key repressors ofphotomorphogenesis, COP1 and the four SPA proteins,during three developmental stages of low-R:FR-exposedplants: seedlings, adult plants during vegetative growth,and adult plants during the transition to the reproduc-tive flowering stage.

We found that cop1 and spa-Q mutant seedlings failedto elongate their hypocotyl and cotyledon petioles inresponse to simulated shade. This finding agrees withprevious studies showing that cop1mutants do not showa seedling shade-avoidance response (McNellis et al.,1994). We showed that also at the rosette leaf stage, cop1and spa-Qmutant plants did not show any elongation ofthe leaf petioles in response to simulated shade. In con-trast, cop1 and spa-Qmutants exhibited an acceleration offlowering time in response to low R:FR that was verysimilar to the wild type. Taken together, these resultsdemonstrate that the COP1/SPA ubiquitin ligase is a keyplayer in the etiolation of seedlings and leaves but not inthe acceleration of flowering time in response to low R:FR. Thus, the SAS involves COP1/SPA-dependent andCOP1/SPA-independent signaling activities.

The increased elongation of hypocotyls in response tolow R:FR is primarily caused by cell elongation and notby cell division (Gendreau et al., 1997). When analyzingthe transcript levels of shade-regulated genes, we foundthat SPA genes are necessary for normal up-regulationof the XTR7 transcript, which encodes a cell wall-modifying enzyme thought to function in cell elonga-tion (Sasidharan et al., 2010). Hence, the observationthat spa-Q mutants do not elongate in response to lowR:FR correlates well with the reduced up-regulation of

Figure 7. The transcript levels of the four SPA genes do not change inresponse to low R:FR conditions. Transcript levels were determined byquantitative reverse transcription-PCR. Seedlings were grown for 4 d in Wcand then shifted to Wc+FRc or kept in Wc for the indicated times. UBQ10was used as an endogenous control. Data were calibrated to Col-0 0-h Wcfor each gene and shown as means of three biological replicates 6 SE.


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XTR7 expression in shade-grown spa-Q mutants. Animportant hormone in low-R:FR-induced cell elonga-tion is auxin, which is synthesized from Trp via theenzymatic activities of TAA1 and a family of YUCproteins (Tao et al., 2008; Stepanova et al., 2011; Wonet al., 2011). It was shown previously that this pathwayis essential for hypocotyl elongation in response to lowR:FR, since taa1 mutants and yuc multiple mutantsshow no or a reduced shade-avoidance response, re-spectively, at the seedling stage. While TAA1 transcriptlevels are not regulated by shade, several YUC tran-scripts are up-regulated by low R:FR (Tao et al., 2008;Won et al., 2011; Li et al., 2012). We found that low R:FR regulation of exactly these YUC transcripts requiresSPA proteins, since the spa-Q mutant failed to showany increase in the transcript levels of YUC2, YUC8,and YUC9 in low R:FR. Other, not shade-regulatedtranscripts tested (YUC1, YUC4, TAA1), in contrast,were expressed at similar levels in wild-type and spa-Qmutant seedlings. Hence, the spa-Q mutant showed amisregulation specifically of the low-R:FR-regulatedYUC genes. In wild-type seedlings exposed to simu-lated shade, the increased expression of YUC genesleads to higher auxin levels and to higher expression ofthe auxin-responsive reporter DR5::GUS (Hornitscheket al., 2012; Li et al., 2012). In spa mutants, in contrast,exposure to low R:FR did not cause any detectableincrease in DR5::GUS expression. This is consistentwith the observed lack of YUC gene regulation by low

R:FR in spa-Q mutant seedlings. Taken together, theseresults suggest that the failure of spa-Q mutant seed-lings to elongate in low R:FR is, at least in part, causedby their failure to up-regulate the expression of YUCgenes in low R:FR.

Our epistasis analysis showed that SPA genes actupstream of the COP1/SPA ubiquitination substrateHFR1, but not of HY5, in controlling the SAS. HFR1is an atypical bHLH protein that lacks a functionalDNA-binding domain. It inhibits the SAS by formingnonproductive heterodimers with the typical bHLHproteins of the PIF family (Hornitschek et al., 2009). Itwas shown that PIF proteins promote cell elongationin the SAS by binding to the promoters of YUC8 andYUC9, thereby activating gene expression in low R:FR(Hornitschek et al., 2012; Li et al., 2012). Hence, wepropose that the COP1/SPA ubiquitin ligase is neces-sary for the SAS in low R:FR because it ubiquitinatesHFR1, thereby releasing PIF proteins from the inhibi-tory activity of HFR1 (Fig. 9). Subsequently, PIF pro-teins can activate YUC expression, which leads to anincrease in auxin biosynthesis and in the expression ofthe artificial reporter DR5::GUS. Since low R:FRstrongly induces HFR1 expression (Sessa et al., 2005;this work), COP1/SPA activity may be particularlyimportant in limiting HFR1 function in low R:FR con-ditions. According to this model, HFR1 would not bedegraded in spa-Q mutants. As a consequence, HFR1protein levels would be especially high in low-R:FR-grown spa-Q mutants and therefore counteract the low-R:FR-induced stabilization of PIF proteins, therebymaintaining the high R:FR phenotype even under lowR:FR conditions. Besides controlling PIF protein sta-bility and HFR1 expression, we speculate that the R:FRmay also affect COP1/SPA ubiquitin ligase activity,since photoreceptors, including phytochromes, arethought to inactivate the COP1/SPA complex in thelight (Lau and Deng, 2012). Hence, COP1/SPA may bemore active in low R:FR than in high R:FR (Fig. 9),which would lead to increased HFR1 degradation inlow R:FR and, therefore, increased PIF protein activity.

Apart from regulating HFR1 protein stability, theCOP1/SPA complex likely has additional roles in theSAS. The levels of the PIF protein PIF3 were shown tobe much lower in dark-grown cop1 and spa mutantswhen compared with the wild type (Bauer et al., 2004;Leivar et al., 2008). If PIF4, PIF5, and/or PIF7 proteinlevels are also up-regulated by the COP1/SPA complex,

Figure 9. Model of the function of COP1/SPA in controlling hypocotylelongation in response to low R:FR. For discussion, see text.

Figure 8. Divergent functions of SPA1 and SPA2 in simulated shadederives from their regulatory and protein-coding sequences. A, Sche-matic representation of chimeric SPA1/SPA2 constructs containing theSPA1 or SPA2 promoter (pSPA1 and pSPA2), the SPA1 or SPA2 openreading frame (ORF), the hemagglutinin (HA) tag, and the 39 un-translated region of SPA1 or SPA2 (39 SPA1 and 39 SPA2). B, Hypocotyllength of seedlings expressing the constructs shown in A in the spa-Qmutant background. Seedlings were grown in Wc for 3 d and thenshifted to Wc+FRc or kept in Wc for an additional 3 d. Error bars in-dicate SE.





this regulation will also contribute to cell elongation inlow R:FR. In addition, BBX transcription factors havebeen shown to act downstream of COP1 in the SAS(Crocco et al., 2010), and ELF3, another target of theCOP1/SPA ubiquitin ligase (Yu et al., 2008), was iden-tified as a quantitative trait locus for the shade-avoidanceresponse (Jimenez-Gomez et al., 2010). It remains to bedetermined whether these transcription factors also acton the expression of YUC genes or have a differentfunction in the SAS.

In contrast to the misregulated transcripts XTR7 andYUC, the transcript levels of two negative regulators ofthe SAS, PIL1 and HFR1, as well as of ATHB2 in-creased normally in spa-Q mutants in response to lowR:FR when compared with the wild type. This is incontrast to previous results showing that cop1 mutantsexhibit an approximately 2- to 3-fold reduced responseof these early shade marker genes when comparedwith the wild type (Roig-Villanova et al., 2006). Whilethese contrasting results may be due to the experi-mental setups, our results nevertheless show that SPAproteins have a much stronger effect on the expressionof XTR7, YUC2, YUC7, and YUC8 than on PIL1,ATHB2, and HFR1. Since all these genes are regu-lated by PIF proteins, it is tempting to speculate thatthese two sets of genes respond to different PIFproteins or exhibit different binding affinities for PIFproteins.

Our results show that COP1 and SPA genes are alsorequired for elongation of the petiole in response tolow R:FR. This aspect of the shade-avoidance responseis much less understood than the hypocotyl response.Spotlight irradiation of small areas in the leaf blade orpetiole demonstrated that the leaf blade is the photo-perceptive site for FR-induced elongation of the peti-ole. In the leaf blade, YUC2, YUC8, and YUC9 wereup-regulated in response to end-of-day FR, althoughauxin levels did not increase either in the blade or inthe petiole of the leaf (Kozuka et al., 2010). The con-served regulation of these YUC genes in leaf bladesand seedlings nevertheless suggests that COP1/SPAmay act in the leaf in a similar way to that in theseedling. In petiole elongation, the four SPA genesshow fully redundant activities, because only spa-Qmutants exhibit a lack of this facet of the shade-avoidance response. With respect to seedling shadeavoidance in low R:FR, the four SPA genes show dis-tinct actions: SPA1 and SPA4 are the primary players,while the contributions of SPA2 and SPA3 are ofnegligible importance. Our functional analysis ofSPA1/SPA2 chimeric genes demonstrates that at leastpart of the distinct functions of SPA1 and SPA2 isencoded in the respective protein sequence. Thisagrees with our earlier results showing that the SPA2protein loses repressor activity as soon as dark-grownseedlings are transferred to the light (Balcerowiczet al., 2011).

One aspect of the shade-avoidance response is ear-lier flowering in response to low R:FR. We found thatcop1 and spa-Q mutants flowered earlier in low R:FR

than in high R:FR and thus responded to low R:FRsimilarly to the wild type. Consistent with this finding,FT transcript levels increased very strongly in spa-Qmutants in response to low R:FR. This result indicatesthat the COP1/SPA ubiquitin ligase is not essential forthe control of flowering time by simulated shade. Theexpression of FT is primarily regulated by the balancingactivities of two transcription factors: the repressor offlowering FLC and the activator of flowering CO. WhileCO is a target of the COP1/SPA ubiquitin ligase indarkness (Laubinger et al., 2006; Jang et al., 2008; Liuet al., 2008), CO degradation in R was found to be in-dependent of COP1 (Jang et al., 2008). This latterfinding is in agreement with our results showing thatflowering time regulation by R:FR does not requireCOP1/SPA. Moreover, SPA proteins and phyB act indistinct tissues in the control of flowering time: whilephyB acts in the mesophyll, SPA1 acts in the phloem(Endo et al., 2005; Ranjan et al., 2011). Previous resultshave shown that white light + FR increases CO transcriptlevels and also enhances CO protein stability (Kim et al.,2008). Since CO and the activator of CO transcription GIare important for the acceleration of flowering in far-red-enriched light (Kim et al., 2008; Wollenberg et al., 2008),low R:FR clearly acts on CO expression and CO stability.However, how low R:FR regulates CO remains to beinvestigated.

In summary, we have shown that facets of theshade-avoidance response are genetically separable,with the COP1/SPA complex being required for theelongation of hypocotyl and petiole tissues in responseto low R:FR, while it is dispensable for the accelerationof flowering in low R:FR. This was similarly reportedfor the transcription factor ATHB2, which is also notrequired for the control of flowering time by low R:FR(Kim et al., 2008). Moreover, we have demonstratedthat SPA proteins are required for the increase inYUC2, YUC8, and YUC9 transcript levels in responseto low R:FR, suggesting that the COP1/SPA complexregulates cell elongation in low R:FR by controllingauxin biosynthesis. Taken together, our genetic andmolecular characterization has elucidated the role ofCOP1/SPA within the signaling cascades leading todifferent features of the shade-avoidance response.

MATERIALS AND METHODS

Plant Material

All Arabidopsis (Arabidopsis thaliana) spa single, double, triple, and qua-druple mutants have been described previously (Laubinger and Hoecker,2003; Laubinger et al., 2004; Fittinghoff et al., 2006; Balcerowicz et al., 2011),except for spa1-7 spa2-1 spa4-1, which was established by crossing spa4-1 withthe spa1-7 spa2-1 double mutant. cop1-4 (McNellis et al., 1994), hy5-215 (Oyamaet al., 1997), hy5-51 (Ruckle et al., 2007, 2012), and hfr1-101 (Fankhauser andChory, 2000) have been described earlier. The spa1-7 spa3-1 spa4-1 hfr1-101 andspa1-7 spa3-1 spa4-1 hy5-51 mutants were selected from the respective F2populations based on phenotype and subsequently confirmed using poly-morphic markers (Laubinger and Hoecker, 2003; Fittinghoff et al., 2006). Thecop1-4 hy5-215 double mutant was generated by Roman Ulm (University ofGeneva). The DR5::GUS transgene (Ulmasov et al., 1997) was crossed into thespa1-7 spa2-1 spa3-1 and spa1-7 spa2-1 spa4-1 triple mutants.


Rolauffs et al.



Growth Conditions and Light Treatment

For seedling experiments and all transcript analyses, seeds were surfacesterilized and subsequently sown on Murashige and Skoog (Duchefa) plateswithout Suc containing 1% activated charcoal. We found that the addition ofactivated charcoal to the medium enhances the shade-avoidance response inseedlings. Seeds were stratified at 4°C for 2 to 3 d in the dark and subsequentlyincubated in Wc (50 mmol m22 s21), provided by Wc light-emitting diodes(LEDs), at 21°C (FloraLED; CLF Plant Climatics) for 3 d. The R:FR was 10.3.For simulated shade treatment, 3-d-old seedlings were then exposed to thesame Wc treatment but supplemented with FRc emitted from built-in FR LEDs(FloraLED) for 3 d (R:FR = 0.22). The high-R:FR-treated control seedlings werekept in Wc for the same period of time.

For adult plant growth analyses and determination of flowering time, seedswere first incubated in water for 3 d at 4°C and then germinated on soil insingle wells of 77-well trays in a randomized fashion. For high R:FR treatment,seedlings were grown in a growth chamber (AR-36L; Percival-Scientific) in Wcprovided by cool-white fluorescent light sources (50 mmol m22 s21; R:FR = 9.8)at 21°C and 60% humidity. For simulated shade treatment, seedlings weregrown under the same conditions for 4 d and were subsequently transferred tolow R:FR conditions. These were produced in the same AR-36L growthchamber that was additionally equipped with FR LED light sources (QuantumDevices). The R:FR was 0.15.

Determination of Hypocotyl Length, Petiole Length, andFlowering Time

Seedlings were flattened on the surface of the agar plates and photographedwith a Nikon D5000 digital camera. The measurements of hypocotyl andcotyledon petiole length were carried out using ImageJ 1.43u software (WayneRasband, National Institutes of Health). A minimum of 15 seedlings pergenotype and condition were analyzed, and each experiment was repeated atleast twice. Petiole length of leaves was determined in 11-d-old plants bymeasuring the petiole of the longest leaf. At least eight leaves were measuredper genotype and condition, and the experiment was repeated twice. Floweringtimewas scored on the first day the inflorescence was visible to the unaided eye.It was determined as the number of rosette leaves formed and the number ofdays since transfer of sown seeds to 21°C. At least eight plants were analyzedfor each genotype and condition, and all experiments were repeated twice.

Analysis of Transcript Levels by QuantitativeReverse Transcription-PCR

RNA extraction, DNase digestion, complementary DNA synthesis, andquantitative PCR were performed as described previously (Balcerowicz et al.,2011). The 2-ΔΔCt method was used to determine relative transcript levelsnormalized to the UBQ10 transcript levels (Livak and Schmittgen, 2001). Threebiological replicates were used, and each was analyzed in duplicate. Theprimer pairs used in this study for the amplification of the transcripts ofUBQ10 (Balcerowicz et al., 2011), ATHB2, HFR1, and PIL1 (Lorrain et al.,2008), XTR7 (Hornitschek et al., 2009), TAA1 (Franklin et al., 2011), YUC1 andYUC4 (Rizzardi et al., 2011), YUC2, YUC8, and YUC9 (Stavang et al., 2009),and FT (Wollenberg et al., 2008) were described earlier. Primers for theanalysis of SPA1, SPA2, SPA3, and SPA4 transcript levels by quantitative PCRare provided in Supplemental Table S1.

Histochemical Analysis of GUS Activity

Seedlings were grown on plates in high R:FR as described above for 4 d,except that the temperature was raised to 27°C to boost auxin production(Gray et al., 1998). This was necessary to allow DR5::GUS detection. Seedlingswere then either kept in high R:FR or shifted to low R:FR for 7 h. They wereincubated in GUS staining buffer (Ranjan et al., 2011), but without vacuuminfiltration, at 37°C for 16 h in darkness and subsequently destained in 70%ethanol.

The Arabidopsis Genome Initiative numbers for genes mentioned in thisarticle are as follows: SPA1 (At2g46340), SPA2 (At4g11110), SPA3 (At3g15354),SPA4 (At1g53090), COP1 (At2g32950), HY5 (At5g11260), HFR1 (At1g02340),YUC1 (At4g32540), YUC2 (At4g13260), YUC4 (At5g11320), YUC8 (At4g28720),YUC9 (At1g04180), and TAA1 (At1g70560).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Table S1. Primers used for quantitative PCR.

ACKNOWLEDGMENTS

We thank Johannes Stauber, Stephen Dickopf, Vicky Tilmes, and LauraRupprecht for excellent technical assistance and Klaus Menrath and thegreenhouse staff for expert care of our plants. We are grateful to SiegfriedWerth for photography. Transgenic seed expressing the DR5::GUS reporterwas kindly provided by Tom Guilfoyle. We thank Roman Ulm for providingseed of the cop1-4 hy5-215 double mutant.

Received September 12, 2012; accepted October 22, 2012; published October23, 2012.

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arabidopsis cop1 and spa genes are essential for plant ... · to bind to dna (hornitschek et al.,...

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