SMAX1-LIKE/D53 Family Members Enable DistinctMAX2-Dependent Responses to Strigolactones andKarrikins in Arabidopsis

Ishwarya Soundappan,a,1 Tom Bennett,b,1 Nicholas Morffy,a Yueyang Liang,b John P. Stanga,a Amena Abbas,a

Ottoline Leyser,b and David C. Nelsona,2

a Department of Genetics, University of Georgia, Athens, Georgia 30602b Sainsbury Laboratory, Cambridge University, Cambridge CB2 1LR, United Kingdom

ORCID IDs: 0000-0002-3302-6341 (I.S.); 0000-0003-1612-4019 (T.B.); 0000-0003-3170-2032 (N.M.); 0000-0002-7427-1028 (Y.L.);0000-0003-3629-9932 (J.P.S.); 0000-0002-9049-5232 (A.A.); 0000-0003-2161-3829 (O.L.); 0000-0001-9792-5015 (D.C.N.)

The plant hormones strigolactones and smoke-derived karrikins are butenolide signals that control distinct aspects of plantdevelopment. Perception of both molecules in Arabidopsis thaliana requires the F-box protein MORE AXILLARY GROWTH2(MAX2). Recent studies suggest that the homologous SUPPRESSOR OF MAX2 1 (SMAX1) in Arabidopsis and DWARF53 (D53)in rice (Oryza sativa) are downstream targets of MAX2. Through an extensive analysis of loss-of-function mutants, wedemonstrate that the Arabidopsis SMAX1-LIKE genes SMXL6, SMXL7, and SMXL8 are co-orthologs of rice D53 that promoteshoot branching. SMXL7 is degraded rapidly after treatment with the synthetic strigolactone mixture rac-GR24. Like D53,SMXL7 degradation is MAX2- and D14-dependent and can be prevented by deletion of a putative P-loop. Loss of SMXL6,7,8suppresses several other strigolactone-related phenotypes in max2, including increased auxin transport and PIN1accumulation, and increased lateral root density. Although only SMAX1 regulates germination and hypocotyl elongation,SMAX1 and SMXL6,7,8 have complementary roles in the control of leaf morphology. Our data indicate that SMAX1 andSMXL6,7,8 repress karrikin and strigolactone signaling, respectively, and suggest that all MAX2-dependent growth effectsare mediated by degradation of SMAX1/SMXL proteins. We propose that functional diversification within the SMXL familyenabled responses to different butenolide signals through a shared regulatory mechanism.

INTRODUCTION

Strigolactones (SLs) and karrikins (KARs) are butenolidecompounds that have recently been identified as regulators ofplant growth and development. SLs were first found in rootexudates as compounds that stimulate germination of parasiticweeds in the Orobanchaceae family (Cook et al., 1966). Al-though parasitic weeds exploit SLs as a host-detection signal,SL exudation into the rhizosphere is beneficial to plants. Par-ticularly under nutrient-poor conditions, SLs promote arbus-cular mycorrhizal symbioses that enable exchange of carbonfor nitrogen, phosphorus, and water (Akiyama and Hayashi,2006; reviewed in Xie et al., 2010). Recently, SLs have beenrecognized as plant hormones that influence multiple aspectsof development, including shoot branching (tillering), root ar-chitecture, leaf senescence, and secondary growth (Gomez-Roldan et al., 2008; Umehara et al., 2008; Agusti et al., 2011;Kapulnik et al., 2011; Ruyter-Spira et al., 2011; Rasmussenet al., 2012; Yamada et al., 2014; Ueda and Kusaba, 2015).SLs are synthesized from carotenoids by the DWARF27 classcarotenoid isomerase and the CCD7/MORE AXILLARY

GROWTH3 (MAX3) and CCD8/MAX4 classes of carotenoidcleavage dioxygenases; the sequential action of these enzymesproduces carlactone (Alder et al., 2012). Other enzymes, in-cluding MAX1, a cytochrome P450, subsequently act uponcarlactone to produce a large range of biologically active stri-golactones (Zhang et al., 2014; reviewed in Al-Babili andBouwmeester, 2015).KARsare chemical signals found in smoke that promote seed

germination of a wide range of species (Flematti et al., 2004;Nelson et al., 2009; reviewed in Nelson et al., 2012). KARapplication also enhances seedling responses to light in Arabi-dopsis thaliana and promotes seedling vigor in crop plants(Jain et al., 2006; Kulkarni et al., 2006; van Staden et al., 2006;Nelson et al., 2010). To date, there is no evidence that plantssynthesize KARs, but genetic evidence suggests that KARsmimic an as yet unknown endogenous butenolide molecule(KAI2 ligand [KL]) that is not SL (Flematti et al., 2013;Waters et al.,2014).Remarkably, SL and KAR signaling both depend upon the

activity of the F-box protein MAX2, which forms part of a Skp-Cullin-F-box (SCF) complex (Stirnberg et al., 2002; Stirnberget al., 2007; Nelson et al., 2011). SCF complexes act by ligatingubiquitin moieties to target proteins, often resulting in theirdegradation by the 26S proteasome (Somers and Fujiwara,2009). The putative receptors for SLs and KARs are the closelyrelated a/b-hydrolases DWARF14 (D14) and KARRIKININSENSITIVE2 (KAI2), respectively. These are ancient paralogsthat are present throughout angiosperms (Waters et al., 2012,

1 These authors contributed equally to this work.2 Address correspondence to dcnelson@uga.edu.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) is: David C. Nelson(dcnelson@uga.edu).www.plantcell.org/cgi/doi/10.1105/tpc.15.00562

The Plant Cell, Vol. 27: 3143–3159, November 2015, www.plantcell.org ã 2015 American Society of Plant Biologists. All rights reserved.

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2013). D14 and KAI2 require an intact catalytic triad for signaltransduction (Hamiaux et al., 2012; Waters et al., 2014). Ligandbinding or hydrolysis is thought to induce conformationalchanges in the receptors that alter their interactions withdownstream signaling partners, including MAX2 (Hamiaux et al.,2012). The differences between SL-insensitive d14 phenotypesand KAR-insensitive kai2 phenotypes show that SL and KAR/KLregulate distinct aspects of MAX2-dependent development andthat the max2 phenotype reflects a combination of d14 and kai2effects (Waters et al., 2012). Since both signaling pathways actthrough SCFMAX2, it is unclear how specific developmental re-sponses to SL and KAR/KL are mediated. Identifying the targetsof MAX2 and understanding how they mediate specificity is akey objective for elucidating the mechanisms of SL and KAR/KLsignaling.

To date, several candidates have been suggested as targetsof SCFMAX2. Based on biochemical approaches, the DELLAfamily of transcriptional activators, which are growth re-pressors targeted for degradation by gibberellins, and theBES1 family of brassinosteroid response factors have beenproposed to be MAX2 targets (Nakamura et al., 2013; Wanget al., 2013). A third class of putative targets, SMAX1-LIKE(SMXL) proteins, was identified primarily on the basis of geneticapproaches. A screen for suppressors of max2 in Arabidopsisled to the identification of smax1, which suppresses the seedgermination and seedling growth phenotypes ofmax2 but doesnot affect its shoot architecture, lateral root growth, or se-nescence (Stangaet al., 2013). D53, a homologofSMAX1 in rice(Oryza sativa), was identified from a semidominant d53 muta-tion that has a SL-insensitive, high tillering phenotype similar tothat of d14 and d3, the rice ortholog ofMAX2 (Jiang et al., 2013;Zhou et al., 2013). SL promotes physical interaction of D14with D53 and D3, and the D53 protein is rapidly degradedfollowing SL treatment in a D3- and D14-dependent manner.The d53 mutant protein, however, is resistant to SL-induceddegradation (Jiang et al., 2013; Zhou et al., 2013). Thissuggests a mechanism in which SL promotes formation ofan SCFD3-D14-D53 complex. This leads to polyubiquitinationand degradation of D53, which enables growth responsesto SL.

SMAX1 and D53 are members of a wider, uncharacterizedSMXL protein family that has weak similarity to Class 1 Hsp100/ClpB proteins (Jiang et al., 2013; Stanga et al., 2013; Zhou et al.,2013). Convergence on the same gene family through in-dependent approaches in two species strengthens the evidencethat SMXLproteins are bona fideMAX2 targets. It also furthers theparallel between SL and KAR signaling pathways seen at thereceptor level. Therefore, a promising hypothesis is that differentaspects of MAX2-dependent signaling are mediated by degra-dation of different SMXL proteins. In this study, we perform anextensive analysis of loss-of-function mutants to determine thecontributions of SMAX1, SMXL6, SMXL7, and SMXL8 to growthresponses downstream of MAX2. We demonstrate that SMXL6,SMXL7, and SMXL8 are redundant orthologs of D53 that regulateshoot branching and other SL-regulated processes.We thereforeestablish specific relationships between SMAX1 and KAI2-KAR/KL-regulated growth and between SMXL6,7,8 and D14-SL-regulated growth.

RESULTS

SMXL6, SMXL7, and SMXL8 Control Branchingin Arabidopsis

The SMXL family in Arabidopsis is composed of eight genes thatcan be divided into four clades present in all angiosperms: (1)SMAX1 and SMXL2, (2) SMXL3, (3) SMXL4 and SMXL5, and (4)SMXL6,SMXL7, andSMXL8 (Stangaetal., 2013;Zhouetal.,2013).To investigate whether SMXL genes control shoot branching, weconstitutively expressed artificial microRNAs (amiRNAs) that tar-get SMXL4 and SMXL5 (smxl45-ami), and SMXL6, SMXL7, andSMXL8 (smxl678-ami ) in the max2-1 mutant background(Supplemental Figure 1). The increased branching phenotype ofmax2 was reduced by smxl678-ami in most transgenic lines, butnot by smxl45-ami, implicating SMXL6, SMXL7, or SMXL8 inbranching control. This approach was useful to overcome geneticredundancy in the SMXL family, but we observed variability inbranching suppression among the transgenic lines (SupplementalFigure 1). To enable straightforward genetic analyses, we isolatedmultiple T-DNA insertion alleles for SMXL6, SMXL7, and SMXL8from publicly available mutant collections. All T-DNA insertionsdisrupted the production of full-length transcripts (Figure 1).Wefirst testedwhethersmxl6,smxl7,orsmxl8allelessuppressthe

max2-1 shoot branching phenotype. Branching and inflorescenceheights of smxl6 max2 and smxl8 max2 allelic combinations wereall similar to max2 (Figure 2). However, both smxl7-3 max2 andsmxl7-4 max2 had significantly reduced branch numbers and in-creased height compared with max2 (Figure 2). To eliminate func-tional redundancyamong thesegenes,wealsocreatedhigher ordersmxl mutant combinations. Rosette branching and inflorescenceheight were restored to wild type levels in smxl7,8 max2 andsmxl6,7,8 max2 (Figure 2; Supplemental Figure 2A). Although notquitephenotypicallywild type,smxl6,7max2hadreducedbranchingrelative to smxl7 max2, while smxl6,8 max2 only showed a slightreduction in branching relative to max2 (Figure 2B). We concludethat SMXL7 has the predominant role in branching control in Arabi-dopsis but that SMXL6 and SMXL8 also contribute significantly.The function of these genes with respect to shoot branching is

most apparent in the max2 background, as rosette branching ofsmxl mutants alone was not significantly different from the wildtype (Supplemental Figures 2Band2C). The smxl6,7,8mutant hasthe same number of rosette branches as the wild type but hasa slight decrease in cauline branchnumber (Supplemental Figures3A and 3C). Contrary to our prior observations (Stanga et al.,2013), weobserved amild but significant reduction in branching insmax1-2max2 comparedwithmax2 (Figure 2B). However, smax1did not enhance branching suppression in either of two smxl6,7max2 allelic combinations (Figure 2B), so if SMAX1 has a role inbranching, it is relatively limited. This is supported by the ob-servation that smxl6,7,8 mutations were sufficient to fully sup-press max2 shoot phenotypes.

SMXL6,7,8 Promote Auxin Transport and PIN1 Accumulationin the Stem

Auxin transport within the inflorescence stem is increased inSL-deficient and SL-insensitive mutants (Bennett et al., 2006).

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This is attributed to increased accumulation of the auxin effluxcarrier PIN1 at the basal plasmamembrane of xylem parenchymacells in the polar auxin transport stream (Bennett et al., 2006;Crawford et al., 2010; Shinohara et al., 2013). PIN1 levels at theplasma membrane are rapidly reduced following rac-GR24treatment in a MAX2-dependent, translation-independent man-ner, and altered dynamics of PIN1 membrane localization are atleast partially responsible for the increase in shoot branchingobserved in SLmutants (Bennett et al., 2006; Prusinkiewicz et al.,2009; Shinohara et al., 2013). PIN1has thus been suggested as aneffector of SL signaling (Shinohara et al., 2013).

We tested whether SMXL proteins regulate PIN1-mediatedauxin transport. We observed that the increased level of auxintransport in max2 was suppressed to wild-type levels in smxl7,8max2 and even further reduced in smxl6,7,8max2 (Figure 3A). Thesmxl6,7,8mutant alsohas reducedauxin transport comparedwiththewild type (Supplemental Figure 3D). Thus, there is a correlationbetween auxin transport level in the stem and axillary bud out-growth, although exceptions such as smxl6,7 max2 can be ob-served (Figure 3A). Consistent with these results, we observedreduced PIN1-GFP accumulation at the basal plasma membrane

of xylem parenchyma cells in the basal internodes of smxl6,7,8max2 (Figures 3B and 3D). In contrast, PIN1-GFP accumulation attheplasmamembrane inmax2wasnot reducedbysmax1 (Figures3C and 3E). These data indicate that accumulation of PIN1 at themembrane is regulated by SMXL6,7,8 activity, as a downstreamoutcome of MAX2-dependent signaling.

SMXL6,7,8 Repress BRC1/TCP18 Expression inAxillary Buds

The TCP domain (TEOSINTE BRANCHED1, CYCLOIDEA, andPCF) transcription factor BRC1/TCP18 has been implicated asanother downstream effector of MAX2 signaling in the regulationof shoot branching. brc1 mutants have increased, SL-resistantshoot branching,BRC1 expression is upregulated after rac-GR24treatment, and BRC1 transcription is reduced in max2, d14, andSL-deficient mutants (Aguilar-Martínez et al., 2007; Dun et al.,2012; Chevalier et al., 2014). We thus tested whetherBRC1mightalso act downstream ofSMXL6,7,8. Consistent with prior reports,BRC1 expression was reduced more than 10-fold in the axillarybuds of max2 rosette leaves. Conversely, BRC1 expression wasupregulatedmore than 60-fold in smxl6,7,8 max2 relative tomax2but was not recovered in smax1max2 (Figure 4). This observationis consistent with the suppression of branching seen in smxl6,7,8max2plants andsuggests that repressionofBRC1 transcription isone mechanism by which SMXL6,7,8 promote bud outgrowth.

SMXL6,7,8 Promote Lateral Root Growth

Themax2mutant has increased lateral root density, which can beattributed partially to SL insensitivity, as SL-deficient max1 andmax4mutantshavean intermediate lateral root density phenotypebetween the wild type and max2 (Kapulnik et al., 2011; Ruyter-Spiraetal., 2011).WeassessedwhetherSMXL6,7,8mightalsoactdownstream of MAX2 in this response. We found that smxl6,7,8had lower lateral root density than the wild type and stronglysuppressed the increased lateral root density phenotype ofmax2(Figure 5; Supplemental Figure 3H). In contrast, lateral root densityin smax1max2 seedlingswas not significantly different frommax2(Figure 5).

rac-GR24 Triggers Rapid Degradation of SMXL7

Our data support the hypothesis that SMXL6,7,8 are targets ofSCFMAX2 in Arabidopsis. To test this, we examined the post-translational regulation of SMXL7 by SL andKAR1.We introduceda35Spro:SMXL7-YFP reporter intoawild-type (Col-0)background.SMXL7-YFP, which localizes to the nucleus, was degraded by1 mM rac-GR24, but not KAR1, within 20 min (Figures 6A and 6B).At a 10 mM rac-GR24 concentration, SMXL7-YFP was noticeablyreduced after 5 min of treatment and was undetectable after 20min (Figure 6C). SMXL7-YFP was not degraded after rac-GR24treatment in themax2 and d14 backgrounds (Figures 6D and 6E).Consistent with SL-induced proteolysis via SCFMAX2, pre-treatment with the 26S proteasome inhibitor MG132 reduced rac-GR24-triggered destabilization of SMXL7-YFP (Figure 6F). Theseresults support a role for D14 and SCFMAX2 in targeting SMXL7 fordegradation after SL perception and are consistent with the

Figure 1. Mutant Alleles of SMXL6, SMXL7, and SMXL8.

(A) T-DNA insertion positions for smxl6-1 (SAIL_86_H04), smxl6-2(SAIL_1285_H05), smxl6-4 (SALK_049115), smxl7-3 (WiDsLox339_C04),smxl7-4 (SALK_082032), smxl8-1 (SALK_025338C), and smxl8-2(SALK_126406C). Boxes indicate exons, open triangles indicate the po-sitions of T-DNAs, and small dark triangles indicate left borders. Mostinsertions had two outward-facing left borders, implying T-DNA con-catamers. Left border insertion positions in the gene sequence were de-fined by Sanger sequencing and are described in Supplemental Table 2.(B) End-point RT-PCR analysis of SMXL6 (30 cycles), SMXL7 (30 cycles),andSMXL8 (40 cycles) expression in respective T-DNAmutant lines.ACT2(30 cycles) was used as an internal reference gene. RT-PCR primers spanthe T-DNA insertion sites, as indicated in (A).

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regulationofD53bySL in rice (Jianget al., 2013; Zhouet al., 2013).Notably, KAR1 had no effect on SMXL7 and the d14mutation wassufficient to stabilize SMXL7. This suggests that KAI2 does notregulate SMXL7.The semidominant d53mutation in rice is a 15-bp deletion that

results in substitution of the amino acids RGKTGI with a singlethreonine residue. This form of d53 is resistant to rac-GR24-induced degradation (Jiang et al., 2013; Zhou et al., 2013). TheRGKTcomponent is a conservedmotif in theArabidopsisSMAX1,SMXL2, and SMXL6,7,8 proteins (and their rice homologs) but isnot present in SMXL3,4,5 (Supplemental Figure 4). It is similar tothe phosphate binding P-loop (or Walker A) consensus sequenceGx4GK[T/S] and therefore may bind ATP or GTP (Saraste et al.,1990). For instance, deletion of an RGKT motif from the proteinphosphatase2CABI2,which is involved inABAsignaling, stronglyreduced itsphosphataseactivity (Sunet al., 2011). To testwhetherthis motif is required for SCFMAX2-dependent degradation ofSMXL7-YFP, we created a DP-loop variant of SMXL7 (deletion ofamino acids 718 to 725). Similar to d53, SMXL7DP-loop wasresistant to degradation after rac-GR24 treatment (Figure 6G).Based on the evidence of similarity in sequence, developmentalroles, and posttranslational regulation by SL, we conclude thatSMXL7 and D53 are orthologs.

SMXL6,7,8 Affect Cotyledon Expansion but Not Germinationor Hypocotyl Elongation

Wenext examinedwhetherSMXL6,7,8maybeSCFMAX2 targets inother developmental contexts. max2 seed are insensitive to thegermination-promoting effects of karrikins and have increasedprimary dormancy (Nelson et al., 2011). The dormancy phenotypeof max2 is suppressed by smax1 (Stanga et al., 2013). Loss ofSMXL6,7,8 did not significantly increase germination ofmax2 nordid smxl6,7 further enhance smax1max2 germination (Figure 7A).Thus, SMXL6,7,8 do not regulate Arabidopsis seed dormancy.max2 seedlings grown under continuous red light have elon-

gated hypocotyls, consistent with decreased responses to light(Stirnberg et al., 2002; Shen et al., 2007). We tested whetherSMXL6,7,8 might regulate this process but did not observeconsistent suppression of themax2 hypocotyl phenotype amongseveral smxl6,7 max2 and smxl6,7,8 max2 allelic combinations(Figure 7B; Supplemental Figure 5A). The max2 hypocotyl phe-notypewasstrongly suppressedby smax1, producingeven shorterseedlings than the wild type, but the hypocotyl length of smax1smxl6,7 max2 was not different from that of smax1 max2. Weconclude that SMXL6,7,8 do not promote hypocotyl elongation.max2mutant seedlingsalsohave reducedcotyledonexpansion

(Shen et al., 2007). Consistent with the results above, this phe-notype is suppressed by smax1, producing significantly largercotyledons than the wild type (Stanga et al., 2013). In contrast, weobserved that two smxl6,7 alleles actually enhanced the max2phenotype (Figure 7C). Thus,SMAX1andSMXL6,7haveoppositeand antagonistic effects on cotyledon expansion (Figure 7C).Neither SMAX1 or SMXL6,7 may be considered epistatic to theother, as the smax1 smxl6,7 max2 phenotype is intermediate tosmax1 max2 and smxl6,7 max2. Curiously, smxl6,7,8 max2 hadcotyledons that were larger than smxl6,7 max2 and not differentthan max2 (Figure 7C; Supplemental Figure 5B), suggesting that

Figure 2. SMXL6, SMXL7, and SMXL8 Promote Shoot BranchingDownstream of MAX2.

(A) Representative images of adult Col-0 (wild type), max2-1, smxl7-3max2-1, smxl6-4,7-3 max2-1, and smxl6-4,7-3,8-1 max2-1 plants.(B) Primary rosette branch number.(C) Height of the primary inflorescence.Plantsweregrownunder a LDphotoperiod (16h light/8 hdark). Imagesanddata were collected for each plant at 10 d postanthesis. For (B) and (C),graphs show mean 6 99% CI, n $ 17. Bar colors indicate no significantdifference toCol-0 (white), nosignificantdifference tomax2 (darkblue), andsignificant differences to both Col-0 andmax2 (gray). P < 0.001 (P = 0.02,with Bonferroni correction for 19 comparisons), Student’s t test.

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SMXL8may have an opposite effect on cotyledon expansion thanSMXL6 and SMXL7 or that loss of all three genes affects growthcapacity differently than the loss of two genes.

These results implicateSMAX1, but notSMXL6,7,8, as the likelytarget of MAX2 during germination and seedling photomorpho-genesis. It shouldbenoted that thedormancyandseedlinggrowthphenotypes of max2 are not seen in SL-deficient mutants or d14and appear to be kai2-specific defects (Nelson et al., 2011; Shenet al., 2012;Waters et al., 2012). Moreover, (1) KAR but not naturalSL (stereoisomers with a 29R configuration) treatments promoteArabidopsis germination; (2) KARpromotes cotyledon expansion,

but rac-GR24, which acts through both KAI2 and D14, inhibitscotyledon expansion; and (3) SL-deficientmaxmutants and d14,but not kai2, have increasedaxillary branching (Nelsonet al., 2010;Waters et al., 2012; Scaffidi et al., 2013, 2014). Therefore smax1suppresses max2 phenotypes associated with KAR/KL-KAI2regulated growth, and smxl6,7,8 suppresses max2 phenotypesassociated with SL-D14-regulated growth.

SMAX1 and SMXL6,7,8 Influence Different Aspects ofLeaf Morphology

To further investigate this correlation, we examined the con-tributions of SMXL genes to leaf morphology, which is regulatedby both KAI2 and D14. SL-deficient mutants and d14 havea distinct leaf phenotype under long-day (LD) photoperiods (16 hlight/8 h dark), with reduced petiole and blade lengths (Figure 8)(Stirnberg et al., 2002; Booker et al., 2004; Scaffidi et al., 2013). Inkai2 leaves, blade length and width are both increased. Themax2mutant shares the general rosette morphology of d14, includingshorterpetiole length, buthasa rounder,widerblade thand14; thismay represent a hybrid of the d14 and kai2 phenotypes (Figure 8).We observed that petiole elongation was inhibited specifically

by SMXL6,7,8. The shortened petiole phenotype ofmax2was thesame asd14, andmax2petiole elongationwas additively restoredby smxl6, smxl7, and smxl8 mutations (Figure 8B; SupplementalFigures 3F and 6A). KAI2 and SMAX1 had little or no effect onpetiole elongation. However, SMAX1 does promote leaf bladegrowth, as smax1causeda reduction in thebladewidthand lengthofmax2 and smxl6,7max2 (Figure 8C;Supplemental Figure 6B). Incontrast, smxl6,7mutations increased blade elongation ofmax2,suggesting that SMXL6 and SMXL7 repress leaf growth underthese conditions. Consistent with the idea that D14 targetsSMXL6,7,8 for degradation and KAI2 may target SMAX1 fordegradation, d14 effects on leaf growth were opposite tosmxl6,7,8and kai2effectswereopposite to smax1.Moreover,d14shared leaf morphology traits with smax1 max2, and kai2 had

Figure 4. SMXL6,7,8 Repress BRC1 Expression in Axillary Buds.

RT-qPCR analysis of BRC1/TCP18 gene expression in nonelongatedaxillary buds of Col-0, max2-1, smax1-2 max2-1, and smxl6-4,7-3,8-1max2-1collected7dafteranthesis. ExpressionofBRC1 is relative toCACSinternal reference gene. Expression values are scaled to the Col-0 level ofexpression. Expression for max2-1 and smax1-2 max2-1 is indicated inparentheses. Mean6 SE; n = 3 to 4 pooled tissue samples, four plants perpool. ANOVA with post-hoc Fisher’s LSD, P < 0.01.

Figure 3. SMXL6,7,8 Promote Auxin Transport and PIN1 Accumulation.

(A) Auxin transport in stem segments from basal internodes. Mean6 95%CI, n = 15 to 20.(B)Confocal laser scanning fluorescencemicroscopy ofPIN1-GFP in stemsegments from basal internodes of Col-0, max2, and smxl6,7,8 max2.(C)Confocal laser scanning fluorescencemicroscopyofPIN1-GFP in stemsegments from basal internodes of Col-0, max2, and smax1 max2.(D) PIN1-GFP fluorescence intensity at the basal plasma membrane instem segments, as exemplified in (B). Mean 6 99% CI, n = 40 (five basalmembranes in eight plants per genotype).(E)PIN1-GFPfluorescence intensityat thebasalplasmamembrane instemsegments, as exemplified in (C). Mean 6 99% CI, n = 40 (five basalmembranes in eight plants per genotype).Bar colors indicate no significant difference to Col-0 (white), no significantdifference tomax2 (dark blue), andsignificant differences tobothCol-0andmax2 (gray). Student’s t test, P < 0.05 for (A) and P < 0.01 for (D) and (E).PIN1-GFP is from Xu et al. (2006).

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similar morphology to smxl6,7 max2 and smxl6,7,8 max2; thus,different aspects of the hybrid d14-kai2 phenotype ofmax2 weresuppressed by removing subsets of the SMXL family. Suppres-sion of both subsets of genes restoredmax2 leaves to essentiallywild type dimensions, as shown by smax1 smxl6,7 max2 (Figure8C; Supplemental Figure 6B).

Tissue-Specific Expression Patterns of SMAX1and SMXL6,7,8

The different developmental roles of SMAX1 and SMXL6,7,8could result from distinct expression patterns. Among the SMXLfamily, SMAX1 is expressed most highly in seed, seedlings, andleaves, and SMXL7 is expressed most highly in axillary branches(Stanga et al., 2013). This suggested that further correlationmightbe found between the expression of SMXL family members andtheir functions.

We examined the tissue-specific expression of SMAX1,SMXL6, SMXL7, and SMXL8 through promoter fusions to a GUS-GFP reporter. We observed similar patterns of expression for theSMXL6, SMXL7, and SMXL8 transcriptional reporters in the pri-mary rosette buds and expanding leaves of adult rosettes, thevasculature of the hypocotyls, cotyledons, and mature roots, andin the midvein and petioles of young leaves (Figures 9A to 9D and9F). The SMAX1 transcriptional reporter had a similar pattern ofexpression in these tissues but also producedGUS staining in theyoung leaf periphery, stomata, and the root cap of primary roots(Figures 9B, 9E, and 9G). SMAX1pro:GUS-GFP expression wasalso visible in the caps of lateral roots but not during the earlystages of lateral root emergence (Supplemental Figures 7A to 7D).SMAX1pro:GUS-GFP expression in seedling hypocotyls wassomewhat variable, showing strong expression in the hypocotylvasculature in some seedlings and little or no expression in others(Figure 9C; Supplemental Figure 7E). A clear correlation betweenSMAX1 and SMXL6,7,8 expression and their mutant phenotypeswas not apparent in these data, suggesting that tissue-specifictranscriptional regulation is not sufficient to explain the differentdevelopmental roles of these genes.

SMXL7 and SMAX1 May Interact with TOPLESS Proteins

An alternative explanation for the differences between SMAX1and SMXL6,7,8 functions might be that they have different

protein-protein interactions with downstream signaling partners.The downstream signal transduction partners of the SMXL familyare unknown, but three potential ethylene-responsive elementbinding factor amphiphilic repression (EAR) motifs in D53 thatwere identified by Jiang et al. (2013) provide clues about theiridentity. EAR motifs were initially identified as transcriptional re-pression domains in ethylene signaling (Ohta et al., 2001). Sub-sequent analysis has shown that EAR domains are required forinteractions with the C-terminal to lissencephaly homology(CTLH) domain of the TOPLESS (TPL) and TOPLESS-RELATED(TPR) family of transcriptional corepressors (Szemenyei et al.,2008). EAR motifs are typically defined as LxLxL, DLNxxP, or the

Figure 5. Lateral Root Formation Is Suppressed by smxl6,7,8.

Lateral root density of 10-d-old seedlings. Mean6 SE; n = 6 experiments,$10 roots measured per genotype in each experiment. ANOVA with post-hoc Student’s paired t test, P < 0.05.

Figure 6. rac-GR24 Triggers Rapid Degradation of SMXL7.

(A) and (B) Col-0 35Spro:SMXL7-YFP roots after 0 and 20min of treatmentwith 1 mM rac-GR24 (A) or 1 mM KAR1 (B).(C) Col-0 35Spro:SMXL7-YFP after 0, 5, 10, and 20 min treatment with10 mM rac-GR24.(D)max2-1 35Spro:SMXL7-YFP after 0 and 20min of treatment with 10 mMrac-GR24.(E) d14-1 35Spro:SMXL7-YFP after 0 and 20 min of treatment with 10 mMrac-GR24.(F) Col-0 35Spro:SMXL7-YFP after 20 min of treatment with 10 mM rac-GR24, without and with a 1-h pretreatment with 50 mM MG132, a 26Sproteasome inhibitor.(G) An eight-amino acid sequence (FRGKTVVD) containing a putativeP-loopwas removed fromSMXL7-YFP.Col-0 35Spro:SMXL7DP-loop-YFPafter 0 and 20 min of treatment with 10 mM rac-GR24.

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hybrid L/FDLNL/FxP (Ohtaetal., 2001;Kagaleetal., 2010).Onlyone

of the candidate EARmotifs from D53 is evolutionarily conservedacross angiospermSMXL sequences (Bennett and Leyser, 2014),but it is often a noncanonical sequence that only retains strictconservation of the L/FDLN portion of the motif (SupplementalFigure 8A). Therefore, TPL and TPR corepressors are reasonablecandidates for proteins that interact with SMXL family members.Indeed, D53 was previously reported to interact with rice TPL-related proteins TPR1, TPR2, and TPR3 in a mammalian two-hybrid assay, and the TPR2-D53 interaction was supported by anin vitro pull-down assay (Jiang et al., 2013). SMXL6 was alsoidentified as a TPR3 interactor in a yeast two-hybrid whole-plantlibrary screen with TPL/TPR baits (Causier et al., 2012).We tested whether SMAX1 and SMXL7 can interact with

TPL/TPR proteins in a yeast two-hybrid assay. SMAX1 interactedwith TPL, TPR2, and TPR4, and to a lesser extent with TPR1.Mutationof theputativeEARmotif (mEAR)weakenedor abolishedgrowth on selective media (Figure 10A). SMXL7 had a similar, butweaker, pattern of interactions than SMAX1. SMXL7mEAR mu-tantsdidnotshowbettergrowth than thenegativecontrol testwithRalGDS. In comparison to the strong positive control RalGDS-Krev1 interaction, yeast two-hybrid interactions involving SMAX1or SMXL7 were weak. b-Galactosidase activity was also weak inall interactions and did not appear substantially different betweenmost wild-type andmEAR versions of SMAX1 and SMXL7 (Figure10A).As a complementary approach, we tested whether SMAX1

and SMXL7 can interact with TPR2 through a transient bimo-lecular fluorescence complementation (BiFC) assay in Nicotianabenthamiana leaves. We observed associations between SMAX1and SMXL7 with TPR2, which were localized to subnuclearspeckles. These interactions were abolished by mutation of theEARmotif, even though themEAR isoformsofSMAX1andSMXL7were present in equivalent amounts as the wild-type isoforms(Figure 10B; Supplemental Figure 8B).These data, interaction studies by others (Jiang et al., 2013;

Zhou et al., 2013), and the strong conservation of the EAR motifsuggest thatSMXLproteins interactwithTPL/TPR inanEAR-motifdependent manner. Therefore, it is tempting to speculate thatSMXL proteins function in a transcriptional corepressor complex,similar to Aux/IAAs in auxin signaling and NINJA in jasmonatesignaling (Tiwari et al., 2004; Pauwels et al., 2010). However,further analysis is required todeterminewhether theEARmotif is infact necessary for SMXL function, whether SMXL complexesare associated with DNA, and whether SMXL have repressiveeffects on expression of KAR- and SL-responsive genes. OtherEAR motif-interacting proteins, such as SIN3-ASSOCIATEDPOLYPEPTIDEP18andLEUNIG/LEUNIG_HOMOLOG,must alsobeconsidered as candidate interaction partners. In any case, there iscurrently no evidence for preferences among the SMXL family fordifferent TPL/TPR. Therefore, discrimination among other signal

Figure 7. Distinct Roles for SMXL6,7,8 and SMAX1 during Germinationand Seedling Growth.

(A) Germination of primary dormant seed after 66 h imbibition at 21°C in LDphotoperiodon0.8%(w/v)Bacto-agarmedia.Mean6SE;n=6to7batchesofseed,75seedtestedperbatch.ANOVAwithpost-hocFisher’sLSD,P<0.025.(B) Hypocotyl lengths of 5-d-old seedlings grown under continuousred light (30 mE) for 4 d at 21°C. Mean 6 99% CI; n = 45 seedlings

(15 seedlings from three replicate plates). ANOVAwith post-hoc Fisher’sLSD, P < 0.01.(C)Cotyledon surface area of seedlings grown as described in (B).Mean699% CI; n = 36 cotyledons from 18 seedlings. ANOVA with post-hocFisher’s LSD, P < 0.01.

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transduction partners may be necessary for differential growthcontrol by SMXL proteins.

DISCUSSION

In this article, we describe the characterization ofSMXL6,SMXL7,andSMXL8 fromArabidopsis andshow that theyareco-orthologsof rice D53. The original d53mutation is hypermorphic, and whilesuch mutations are useful because they can overcome func-tional redundancy, they can also give misleading interpreta-tions of genetic relationships or obscure the roles of individualgenes within a family. Here, we use genetic resources in Arabi-dopsis to perform an extensive loss-of-function analysis of theSMXL6,7,8/D53 clade. SMXL6,7,8 have conserved roles in theregulation of shoot branching and act upstreamof the auxin effluxcarrierPIN1and theTCP transcription factorBRC1 in thisprocess.Our analysis of rac-GR24-induced SMXL7 protein degradationsupports it as a proteolytic target ofMAX2 andD14 activity, whichcorrespondswell to thepreviouslydescribed regulationof theD53protein (Jiang et al., 2013; Zhou et al., 2013). We show thatSMXL6,7,8 regulate multiple aspects of development includingleaf morphology and root architecture. We demonstrate thatSMAX1andSMXL6,7,8havedistinct roles indevelopmentbutcanalso have overlapping, antagonistic effects within the same tis-sues (e.g., cotyledons and leaves). Finally, we provide support forin vivo interactions between SMAX1 and SMXL7 with TPR2,suggesting a potential function for SMXL proteins in transcrip-tional regulation. Several themes emerge fromour analysis, whichwe discuss below.

SMXL6,7,8 Are the Major Targets of MAX2-DependentSL Signaling

In this study, we observed that smxl6,7,8max2phenotypesmimicmost wild-type responses to SL, but smax1 max2 phenotypesmimicwild-type responses toKAR. For example, KAR, but not SL,promotes germination, and we observed reduced dormancy insmax1 max2 but not smxl6,7,8 max2. rac-GR24 suppressescotyledon expansion in seedlings, but KAR promotes expansion;correspondingly, smxl6,7,8 max2 have smaller cotyledons, butsmax1 max2 have enlarged cotyledons. Finally, SL, but not KAR,represses axillary branching, and smxl6,7,8 max2 have a clearreduction in primary rosette branch numbers.As SCFMAX2 mediates the signaling of at least two different

butenolide molecules, the max2 phenotype is a combination ofSL-insensitive phenotypes (as observed in d14) and KAR/KL-insensitive phenotypes (as observed in kai2). This is particularlyapparent in rosettes, inwhichmax2 is phenotypically intermediateto kai2 and d14 (Figures 8A and 11A). Interestingly, SMAX1 andSMXL6,7 have very different effects on leaf morphology in shortdays versus long-day conditions (Figures 8A and 11A). None-theless, under both photoperiods, it is clear that smxl6, smxl7, andsmxl8 suppressmax2 phenotypes that are shared with d14; thus,the smxl6,7 max2 rosette resembles kai2. Each SL-related phe-notype inmax2 that we examined was completely suppressed bythe loss of SMXL6, 7, and 8, which strongly suggests that theseproteins are major proteolytic targets of SCFMAX2-mediated SL

Figure 8. SMXL6,7,8 and SMAX1 Have Different Effects on Leaf Mor-phology.

(A)Rosettes of 28-d-old plants grown under LD photoperiod (16 h light/8 hdark). d14-1, kai2-1, max2-1, smax1-2, smxl6-4, smxl7-3, and smxl8-1alleles were used. kai2-1, first identified in Landsberg erecta, was back-crossed twice into the Col-0 background.(B) Petiole length of the 7th leaf of 35-d-old plants grown under LDphotoperiod. Mean6 95%CI; n = 11 to 12. ANOVAwith post-hoc Fisher’sLSD, P < 0.05.(C) Blade length (blue), not including the petiole, and width (gray) of the7th leaf of 35-d-old plants grownunder LDphotoperiod.Mean6 95%CI;n = 11 to 12. ANOVAwith post-hoc Fisher’s LSD, P < 0.05. Prime symbol(e.g., a9) differentiates statistical tests of width measurements fromlengthmeasurements.Mean length:width ratio6 95%CI is shownbelowthe x axis. Statistical groups for length:width ratios are indicated in eachcolored box.

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Figure 9. Tissue-Specific GUS Expression from SMAX1, SMXL6, SMXL7, and SMXL8 Promoters.

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signaling. Consistent with a lack of germination or seedlingphenotypes in d14 and SL biosynthesis mutants, the smxl6,7,8max2 mutants were not different from max2 in germination andhypocotyl elongation (Figure 7). In contrast, we show that smax1suppresses max2 phenotypes that are shared with kai2, and assuch the smax1 max2 mutant rosettes resemble d14 (Figures 8Aand 11A). Thus, SMAX1 is likely to be themajor target of SCFMAX2

during KAR/KL-KAI2 signaling. We did not examine phenotypessuch as leaf senescence, drought tolerance, secondary growth,adventitious rooting, or root hair initiation in this study, but wehypothesize that these phenotypes are also regulated by SMXLproteins.

The discovery that MAX2mediates signaling in response to theperception of two types of butenolide molecules raises questionsabout how specific downstream responses could occur (Nelsonet al., 2011). Our results show that SMAX1 and SMXL6,7,8 ex-pression overlaps in many tissues and furthermore that SMAX1and SMXL6,7,8 can have antagonistic effects within the sametissues. It is thus unlikely that differential expression of theseproteins is sufficient to account for the specific outcomes ofMAX2-dependent signaling. Instead, our results support a modelin which specific protein-protein interactions between KAI2-SMAX1 and D14-SMXL6/7/8 confer the specificity of MAX2 sig-naling (Figure 11B) (Bennett and Leyser, 2014). This hypothesis issupported by recent KAI2 and D14 promoter-swapping experi-ments,whichdemonstrated that thedistinct rolesofKAI2andD14in plant growth are not due to transcriptional regulation (Waterset al., 2015). Physical interactions between D14 and D53 havebeen demonstrated (Jiang et al., 2013; Zhou et al., 2013), andyeast two-hybrid assays also support interaction betweenSMXL7and D14 in the presence of SL analogs (Umehara et al., 2015).However, KAI2 and D14 affinities for different SMXL proteinpartners have not been reported. It should be noted that undersome experimental conditions, there might be some cross-interaction between these anticipated receptor-target pairs. Forinstance, exogenous SL application inhibits hypocotyl elongationthrough D14 [e.g., GR24 or (+)-5-deoxystrigol application is ef-fective in the kai2mutant, but not in kai2 d14] (Scaffidi et al., 2014;Toh et al., 2014; Umehara et al., 2015). However, smxl6,7,8 max2,which otherwise shows constitutive SL responses, has no sup-pression of hypocotyl elongation (Figure 7B). This suggests thatSMAX1, or another SMXL family member that can regulate hy-pocotyl growth, is targeted by D14 following exogenous SLtreatment. However, we do not believe that this response to ex-ogenous SL is physiologically relevant, as d14 and SL-deficientmutants have normal hypocotyl elongation (Shen et al., 2007,2012; Nelson et al., 2011; Waters et al., 2012; Umehara et al.,2015). Thus, even though D14 can mediate a response toexogenous SL, endogenous SL does not appear to control hy-pocotyl growth.

Evolutionary Diversification of ButenolideSignaling Pathways

Therefore, we hypothesize that the ligand-induced formation ofseparate receptor-SMXL complexes permits their interactionwith SCFMAX2 and subsequent degradation of the SMXL protein.One of the intriguing aspects of SL signaling is that the D14 cladearose within the vascular plants, but KAI2 proteins can be tracedback to charophyte algae (Waldie et al., 2014). However, SLsynthesis and responses to rac-GR24 have been identified inbasally diverging land plants such as the moss Physcomitrellapatens (Proust et al., 2011). One possible explanation for theseobservations is that SL and other butenolide molecules in ba-sally diverging land plants signal through a KAI2-SMXL path-way similar to that in angiosperms. Consistent with this, SMXLproteins are present in P. patens (Bennett and Leyser, 2014;Waldie et al., 2014). It is possible that wholesale duplication ofthis signaling pathway in the vascular plant lineage gave rise tothe D14 and SMXL6,7,8/D53 clades, and that subsequent co-evolutionof the receptor-target pairs createdseparate signalingpathways for SL and KAR/KL (Bennett and Leyser, 2014). In-terestingly, this signaling system has evolved more recentlyat the receptor level in parasitic plants in the Orobanchaceaefamily, in which SL perception through KAI2 is the basis of host-triggered germination (Conn et al., 2015; Toh et al., 2015;Tsuchiya et al., 2015).

SMXL Proteins Are Likely the Only Proteolytic Targetsof SCFMAX2

All the max2 phenotypes we have examined so far can be at-tributed to excess SMAX1 and/or SMXL6,7,8 activity, which isstrikingly illustratedby thebroadlywild-typephenotypeof smax1smxl6,7max2mutant rosettes (Figures 8 and11A). This suggeststhat SMXL proteins are likely to be the only direct proteolytictargets of SCFMAX2-dependent signaling. Among the four otherunstudied SMXL proteins in Arabidopsis, SMXL3, SMXL4, andSMXL5 lack the RGKT motif that is required for MAX2-mediatedprotein degradation of D53/SMXL7 (Supplemental Figure 4). Assuch, these proteins may not be proteolytic targets of MAX2.Nevertheless, we did not examine every max2 phenotype, andit is quite possible that SMXL3,4,5 function in other MAX2-regulatedprocesses.SMAX1andSMXL2are very closely relatedproteins that likely represent a recent duplication; as such,SMXL2 may have redundant functions with SMAX1 (Stangaet al., 2013).Our analysis calls into question prior assertions that BES1

and DELLA proteins are targets of SCFMAX2. The identificationof BES1 was primarily through biochemical means, and theconclusion that it is a MAX2 target hinges on the report that

Figure 9. (continued).

Expression of promoter:GUS-GFP fusions in the primary rosette buds and young leaves of 4.5-week-old plants (A), shoot apex and developing leaves (B),hypocotyl (C), cotyledonary veins (D), stomataon first true leaves (E), vasculature in thematuration zoneof primary roots (F), andprimary root cap (G). (B) to(G)are tissuesof 10-d-old light-grownseedlings. For each tissue, thedifferentSMAX1andSMXL transcriptional reporterswere stainedat the same time, forthe same duration.

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BES1-RNAi lines suppress themax2 shootbranchingphenotype(Wang et al., 2013). However, the three transgenic lines de-scribed by Wang et al. had severe growth defects, includingdwarfism, reduced inflorescence height, highly curled leaves,and reduced fertility, and appeared even more severely affectedin the max2 background than in the wild type. It is thereforeunlikely that max2 branching suppression by BES1-RNAi isa specific epistatic interaction, rather than an effect caused bya highly pleiotropic mutant phenotype. In contrast, smxl6,7,8mutations restore max2 to an approximately wild-type shootmorphology, thus showing a highly specific genetic interactionconsistent with being bona fide targets of max2. In the case ofDELLA proteins, the biological relevance of the reported in-teraction between the DELLA protein SLR1 from rice and thestrigolactone receptor D14 has not yet been demonstrated(Nakamura et al., 2013). Protein-protein interaction studies canprovide powerfulmeans to identify newcomponents of signalingpathways or modes of crosstalk, and we do not necessarilyexclude the possibility of crosstalk between strigolactone sig-naling and brassinosteroid or gibberellin signaling. However,cautious interpretation of these results is advised in the absenceof supporting genetic evidence.

SMXL Interactions with Auxin

We demonstrated that SMXL6,7,8 promote auxin transport in thestemandaccumulationofPIN1-GFPat thebasalplasmamembraneof cells in the polar auxin transport stream. Cycloheximide does notprevent the rapid removal of PIN1-GFP from theplasmamembraneafter rac-GR24 treatment, indicating that de novo protein synthesisis not required for SL effects on auxin transport capacity (Shinoharaet al., 2013). Thisobservationchallenges thecurrenthypothesis thatSMXL proteins act through transcriptional regulation and suggeststhat transcription-independent functions should also be consid-ered. For instance, it is possible that a heat shock protein-likefunction of SMXL proteins may be involved in PIN1 regulation.It also remains to be determinedwhether SMXL genes influence

other PIN proteins or affect auxin transport in other tissues. Auxindose–responsecurvesoften showabell shape, inwhich too littleortoo much auxin can cause similar, nonoptimal growth effects(Collett et al., 2000; Ruyter-Spira et al., 2011). It is tempting tospeculate that unexpected observations such as elongatedsmxl6,7andsmxl6,7,8hypocotylscomparedwith thewild type,andreduced phenotypic severity in smxl6,7,8 max2 cotyledons com-pared with smxl6,7 max2 may be caused by shifts in auxin distri-bution or availability (Figures 7B and 7C; Supplemental Figure 5).

Figure 10. EAR Motif-Dependent Interaction of SMAX1 and SMXL7 with TOPLESS Family Proteins.

(A) Yeast two-hybrid interaction tests of SMAX1 and SMXL7 with TPL and TPR proteins TPR1-TPR4. SMAX1/SMXL7 proteins were bait (N-terminal GAL4DNAbindingdomain fusions), andTPL/TPRproteinswereprey (N-terminalGAL4activationdomain fusions). RalGDSwasanegative control for interactionswithSMAX1andSMXL7.RalGDS+Krev1wasa strongpositive control. SMAX1andSMXL7 variantswith amutatedEARdomain (mEAR, sequenceFDLNQorLDLNLmodified toADANA)werealso tested.Growth isshown forequallydilutedcoloniesgrown for3dat30°Con-Leu/Trp/His (-LTH)dropoutmediawith10mM3-amino-1,2,4-triazole (3-AT). Qualitative LacZ assayswere performed by growing colonies on -Leu/Trp (-LT) double dropoutmedia with 80mg/mLX-a-Gal for 30 h.(B) BiFC tests for interaction between SMAX1, SMAX1mEAR, SMXL7, and SMXL7mEAR with TPR2. The N-terminal portion of GFP was fused to the Nterminus of TPR2, and theC-terminal portion ofGFPwas fused to theN terminus of SMAX1/SMXL7 proteins. Confocal fluorescencemicroscopy images oftransiently transformedN. benthamiana leaveswere takenwith the same exposure settings across construct comparisons. A representative field of view isshown (left). Representative nuclei (right) are shown with GFP signal (green) overlaid onto 49,6-diamidino-2-phenylindole (DAPI) signal (blue).

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Figure 11. SMAX1 and SMXL6,7,8 Have Complementary Roles in MAX2-Regulated Development.

(A) Rosettes of 37-d-old plants grown under a short-day photoperiod (8 h light/16 h dark). The kai2/htl allele is htl-3 (Toh et al., 2014).(B)Model of SL andKAR/KL signaling. SMXL6,7,8 act in processes controlled byD14, andSMAX1 acts in processes controlled byKAI2. SMAX1 repressesgermination and seedling responses to light (e.g., reduces cotyledon expansion and promotes hypocotyl elongation) and promotes medio-lateral bladeexpansion (i.e., bladewidth) andelongation in longdays.SMXL6,7,8promotebranching, auxin transport, PIN1accumulationat thebasal plasmamembrane(data not shown), and lateral root density but inhibit petiole elongation andproximo-distal blade expansion in long days (i.e., blade elongation). *LD denotescontrol ina long-dayphotoperiod; undershort-dayconditions,SMAX1andSMXL6,7,8havedifferenteffectson leafgrowthbutarestill antagonistic (A). Lossof SMXL6,7 inhibits cotyledon expansion, although this is countered somewhat by loss of SMXL8.D53/SMXL7 interactswithD14after SLperception and istargeted for degradation in aMAX2-dependentmanner (Figure 6; Jianget al., 2013; Zhouet al., 2013;Umehara et al., 2015). A similarmechanism for SMAX1degradation after KAR/KL perception by KAI2 is hypothesized.

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SMXL6,7,8 Are Epistatic to MAX2

A notable feature of our data is that the suppression of max2 bysmxl6,7,8 often “overshoots” the wild type, resulting in pheno-types that are quantitatively opposite to the effect of max2. Forinstance, lateral root density is ;50% less than the wild type insmxl6,7,8 max2 (Figure 5), while PIN1-GFP and auxin transportlevels are ;40 and ;60% less than the wild type, respectively(Figure3A). Intriguingly,we found thatseveralof thesephenotypesare replicated in a MAX2 background, such that smxl6,7,8is similar to smxl6,7,8 max2 in growth habit and branchingpattern (Supplemental Figures 3A and 3C), lateral root growth(SupplementalFigure3H),andauxin transport level (SupplementalFigure 3D). There are obvious differences in leaf shape betweenthese genotypes, but these result from the residual KAI2-relatedphenotypes in the max2 background (Supplemental Figures 3B,3E, and 3F). In a wild-type MAX2 background, without theseconflicting phenotypes, smxl6,7,8 causes narrowing of the leafblade and an increase in petiole length, emphasizing the quali-tative andquantitative differences between smxl6,7,8 and thewildtype (Supplemental Figure 3). Taken together, these data showthat SMXL6,7,8 are epistatic to MAX2-mediated SL signaling,such that theirmutation causes the samephenotypes irrespectiveof whether SL signaling is functional or not. This suggests thatSMXLs are not simply repressors of SL signaling, but are growthregulators whose activity is repressed by SL signaling. Wild-typemorphology in Arabidopsis thus represents a balance betweentoo little SMXL activity (e.g., smxl6,7,8) and unrestrained SMXLactivity (e.g., max2).

METHODS

Plant Materials

The max2-1 (Stirnberg et al., 2002), d14-1 (Waters et al., 2012), kai2-1(Waters et al., 2012), htl-3 allele (Toh et al., 2014), smax1-2 max2-1(Stanga et al., 2013), and PIN1pro:PIN1-GFP (Xu et al., 2006) lines havebeen described previously. T-DNA alleles described here are smxl6-1(SAIL_86_H04), smxl6-2 (SAIL_1285_H05), smxl6-4 (SALK_049115),smxl7-3 (WiDsLox339_C04), smxl7-4 (SALK_082032), smxl8-1(SALK_025338C), and smxl8-2 (SALK_126406C). T-DNA alleles weregenerated by Alonso et al. (2003), Sessions et al. (2002), and Woodyet al. (2007). All lines are in the Col-0 background. To validate theknockout effects of the T-DNA alleles, RNAwas extracted from 7-d-oldseedlings with the Spectrum Total Plant RNA extraction kit (Sigma-Aldrich) and reverse transcribed with Superscript III (Invitrogen) andanchored oligo(dT) primers. PCR was performed on cDNA for 30 or40 cycles, as noted. Primers for genotyping and RT-PCR analysis aredescribed in Supplemental Table 1. Except where other alleles areindicated, simplified mutant notation in figures (e.g., smax1 smxl6,7max2) refers to the max2-1, smax1-2, smxl6-4, smxl7-3, and smxl8-1alleles.

Plant Growth Conditions

Mature plants for leafmorphology, auxin transport, andPIN1-GFPanalysiswere grownonLevington’sF2 compost under a LDphotoperiod (16 h light/8 hdark) at 22°C/18°Cday/night in controlled environment roomswith lightprovided by white fluorescent tubes (;150 mmol m22 s21). For other as-says, seedswereplatedon0.53Murashige andSkoog (MS)basalmedium

with Gamborg’s vitamins (Sigma-Aldrich M0404), pH 5.7, 0.8% (w/v)Bacto agar plates, stratified 3 d in the dark at 4°C, and grown for 7 d un-der LD photoperiod (;50 to 70 mmol m22 s21) at 21°C before trans-fer to soil (Fafard 3B) and further growth under LD photoperiod (;80 to110 mmol m22 s21) at 21°C. Light intensity for plants grown in short days(8 h light/16 h dark) was ;150 to 200 mmol m22 s21.

Statistical Analysis

Confidence intervals were calculated with the t-distribution. ANOVA andpost-hoc comparisons of means with Student’s t test (Fisher’s LSD) wereperformed in JMP Pro 11.

amiRNAs

amiRNAs were designed by Web MicroRNA Designer (WMD3) at http://wmd3.weigelworld.org/ (Ossowski et al., 2008). Primers for amiRNAcloning are described in Supplemental Table 1. attB adapter sequenceswere included during amiRNA construction for Gateway cloning. amiRNAswere cloned into pDONR221 entry vector and then transferred intoa pEarlyGate100 vector (Earley et al., 2006) that drives expression froma 35S promoter. Constructs were introduced into max2-1 and max2-1PIN1-GFP by the Agrobacterium tumefaciens floral dip method (Cloughand Bent, 1998) and selected for BASTAr. Homozygous transgenic lineswere acquired for T2 lines that had a 3:1 segregation ratio of BASTAr,suggesting a single T-DNA insertion locus.

Axillary Branching and Height Assay

The position of plants within flats was randomized to account for envi-ronmental variation. Primary rosette branches at least 1 cm in length werecounted for each plant at 10 d postanthesis, or as noted. The primaryinflorescence height was measured on the same day as branching.

Auxin Transport Assays

Auxin transport assays were modified from Crawford et al. (2010). The18-mm stem segments from basal internodes were excised and the apicalend placed in 30mLATS solutionwithout sucrose, pH5.6, containing 1mM14C-IAA (AmericanRadiolabeledChemicals). After 6h incubation, thebasal5mmof thestemsegmentwasexcised,placed in200mLscintillation liquid,and shaken overnight at 400 rpm prior to scintillation counting (Perkin-Elmer MicroBeta2).

PIN1-GFP Quantitation

Hand sections of the vascular bundles of basal internodal stem segmentsof 6-week-old plants were embedded in agar plates. PIN1-GFP was im-aged by confocal laser scanning microscopy using a Zeiss LSM700 im-aging systemwith 203water immersion lenses (final magnification 2003).Excitation was performed using 488-nm (15% laser power) and 555-nm(6%) lasers. Chloroplast autofluorescence was detected above 600 nmandGFP fluorescence below555 nm. The same settings forGFPdetectionwere used within experiments for each line. Fluorescence intensity in theGFP channel was performed on nonsaturated images using Zeiss ZENsoftware.

Gene Expression Analysis

RNA was prepared with the Sigma-Aldrich Spectrum Total Plant RNAextraction kit from nonelongated axillary buds (collected 7 d afteranthesis) of plants grown in LD photoperiod. Real-time quantitativeRT-PCR was performed as previously described (Stanga et al., 2013).

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Primers for BRC1 and CACS reference genes are described inSupplemental Table 1.

SMXL7 Degradation

The SMXL7 coding sequence was cloned into a pDONR221 entry vector(Life Technologies). The SMXL7DP-loop variant, lacking amino acids 718to 725 (FRGKTVVD), was made with the Q5 Site-Directed Mutagenesis Kit(NEB). Primers are described in Supplemental Table 1. SMXL7 andSMXL7DP-loop entry clones were subcloned into a pEarlyGate101 des-tination vector (Earley et al., 2006), between the 35S promoter and aC-terminalYFP tag.The resultant35Spro:SMXL7-YFPandSMXL7DP-loop-YFP constructs were transformed into the wild type (Col-0) and max2-1genetic backgrounds using the Agrobacterium floral dip method (CloughandBent, 1998). The roots of 3- to 5-d-old T3 homozygous seedlingsweretreated as described, mounted on glass slides in 10 mM propidium iodidesolution, and imaged by confocal laser scanningmicroscopy using a ZeissLSM780 imaging systemwith 203 lenses. Excitationwas performed usinga 514-nm laser. Propidium iodide was detected above 600 nm and YFPfluorescence below 555 nm. The same settings for YFP detection wereused within experiments for each line.

Germination Assay

Plants grown under LD photoperiod at 21°C were harvested whenmost ofthe siliques were brown (;2 months of growth in soil) and dried in paperbagsat room temperature for 3d.Seedswerecleanedandstoredat280°Cto preserve primary seed dormancy. Seeds were surface sterilized witha 70% (v/v) ethanol and 0.05% (v/v) Triton X-100 solution for 5 min, rinsedwith 70% (v/v) ethanol, rinsed with 95% (v/v) ethanol, and dried on filterpaper. Seeds were plated on 0.8% (w/v) Bacto-agar (Difco) and incubatedin white light (;50 to 70 mmol m22 s21) at 21°C under LD photoperiod.Germination was indicated by the emergence of the radicle. Independentseed batches grown under similar conditions were assessed.

Seedling Photomorphogenesis Assay

Surface-sterilized seeds were plated on solid 0.53 MS medium andstratified in the dark at 4°C for 3 d. Seedswere treatedwith 3 hofwhite light(;50 to70mmolm22 s21) at 21°C, 21hofdarkat21°C, and thengrown for4d at 21°C in continuous red light (;30 mmol m22 s21; Philips GreenPowerLED Research Module HF Deep Red). Hypocotyl lengths and cotyledonareas were measured from digital images using ImageJ (NIH).

Lateral Root Assay

Seedlingswere grown using ATSmedia (Lincoln et al., 1990) with 1% (w/v)sucrose, solidified with 0.8% (w/v) plant agar, in 10-cm square plates.Plates were oriented vertically in growth chambers with a LD photoperiod(22°C/18°C), with light provided by white fluorescent tubes (intensity;150 mmol m22 s21). Roots were analyzed at 10 d postgermination (dpg).Plates were imaged using a flatbed scanner at 600 dpi. Primary root lengthwas then measured using ImageJ. Lateral roots and primordia that werevisible in the scanned images were scored. The lateral root density alongthe whole root was calculated separately for each root.

Leaf Morphology Assay

The 7th leaf of each plant was marked with indelible marker at ;4 weekspostgermination. These leaveswere provisionally measured at 35 dpg andthen again at 37 dpg to confirm that growth of these leaves was arrested.Themaximum length andwidth of the leaf bladeweremeasured, aswell asthe length of the petiole.

Promoter:GUS Reporter Assay

Approximately 3 kb of DNA 59 to the start codon of SMAX1, SMXL6,SMXL7, and SMXL8 was PCR amplified from wild-type (Col-0) genomicDNAusingPrimestarGXLhigh-fidelityDNApolymerase (Clontech), clonedby BP reaction into Gateway entry vector pDONR221 (Invitrogen), andsequenced toconfirmorientationand reading frame.Primersaredescribedin Supplemental Table 1. Reverse primers included 6 or 12 of the firstcoding nucleotides of each gene to ensure full transcriptional fusions.Promoters were subcloned by LR reaction into pHGWFS7, a vector forGUS and GFP fusions (Karimi et al., 2002). Constructs were transformedinto Col-0 plants by Agrobacterium floral dip method. T1 transformantswere selected for resistance to 25 mg/mL hygromycin. GUS analysis wasperformed in theT2generationandcomparedacrossmultiple independenttransgenic lines to ensure consistent tissue-specific expression patterns.Surface-sterilized seeds were plated on 0.53MSmedium, stratified 3 d at4°C in dark, and grown at 21°C in white light under LD photoperiod. After10 d of growth, seedlings were either stained for GUS expression or trans-ferredtosoilandgrownunderLDphotoperiod.WholeplantswereassayedforGUSexpressionafterbolting (;30to34dold).GUSstainingwasaccordingtoBomblies (2000). Seedlings were stained for 12 h and adult tissues for 16 h.Representative images are shown.

Yeast Two-Hybrid Assays

Yeast two-hybrid was performed with the Proquest system (Invitrogen).pDEST32 and pDEST22 constructs were generated by Gateway cloning(Invitrogen) from sequenced entry vectors. Primers for cloning are describedin Supplemental Table 1. mEAR site-directed mutants were generated withInFusion (Clontech). The MaV203 strain of Saccharomyces cerevisiae wastransformedwith pDEST32bait plasmidandapDEST22preyplasmidby thepolyethylene glycol-mediated transformation method (Gietz and Schiestl,2007). Transformantswere selectedon -Leu/-Trpdoubledropoutmedia andconfirmed by colony PCR. To compare growth, yeast were grown overnightin -Leu/-Trpmedia,diluted tofinal concentrationsof0.005and0.0005OD550,spotted onto -Leu/-Trp/-His plates containing 5 or 10 mM 3-amino-1,2,4-triazole, andgrown3dat 30°C. Tocompareb-galactosidaseactivity, dilutedcolonies were grown on -Leu/-Trp plates containing 80 mg/mL X-a-Gal for30 h at 30°C.

Bimolecular Fluorescence Complementation Analysis

BiFCexpressionplasmids (Gehl et al., 2009)were generatedbyGatewaycloning from sequenced entry vectors and transformed in Agro-bacterium (strain C58C1). Transient transformation of Nicotianabenthamianawas according to Voinnet et al. (2003), except the OD600 ofp19 carriers and each BiFC vector carrier strain was 0.3 and 0.4, re-spectively, for a total OD600 of 1.1 after resuspension in the inoculantbuffer (10mMMES, pH 5.6, 10mMMgCl2, and 150mMacetosyringone).In addition, cells were incubated for 2 to 4 h at room temperature in theinoculation buffer. Each interaction pair was injected into two differentplants aged 4 to 5 weeks old. Leaf clippings were collected 2 to 4 d afterinjections and viewed under a Zeiss 510 LSM confocal microscope.Images were taken at 2003 and 4003. To test whether a lack of mEARinteraction with TPR2 was due to low mEAR protein levels, tissue co-infected with SMAX1, smax1mEAR, SMXL7, or smxl7mEAR with TPR2BiFC vectors was collected 2 d after infection. Protein was extracted in400 mM sucrose, 10 mM KCl, 1 mM MgCl2, 0.01% (v/v) Triton X-100,200 mM Tris, pH7.2, and 3% (v/v) DTT. Protein concentration wasnormalized by Bradford assay (Bio-Rad). Total protein (34 mg) foreach sample was separated by 10% SDS-PAGE. Protein blots onpolyvinylidene fluoride membrane were probed with mousemonoclonalanti-HA HA-7 primary antibody (Sigma-Aldrich) at 1:1000 dilution andanti-mouse IgG horseradish peroxidase-linked secondary antibody

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(Cell Signaling) at 1:10,000dilution.GEAmershamECLWesternBlottingDetection Reagent was used for detection.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis GenomeInitiative or GenBank/EMBL databases under the following accessionnumbers: ACT2 (At3g18780), BRC1 (At3g18550), CACS (At5g46630),D14 (At3g03990), KAI2 (At4g37470), MAX2 (At2g42620), SMAX1(At5g57710), SMXL4 (At4g29920), SMXL5 (At5g57130), SMXL6(At1g07200), SMXL7 (At2g29970), SMXL8 (At2g40130), TPL (At1g15750),TPR1 (At1g80490), TPR2 (At3g16830), TPR3 (At5g27030), and TPR4(At3g15880).

Supplemental Data

Supplemental Figure 1. Targeting of SMXL6,7,8 with artificial miRNAsreduces branching in max2.

Supplemental Figure 2. Shoot phenotypes of additional smxl6, smxl7,and smxl8 mutants.

Supplemental Figure 3. Shoot, leaf, and root phenotypes of smxl6,7,8.

Supplemental Figure 4. Putative P-loop motif in the SMXL family.

Supplemental Figure 5. Seedling morphology phenotypes of addi-tional smxl6, smxl7, and smxl8 alleles.

Supplemental Figure 6. Specific contributions of SMXL6, SMXL7,and SMXL8 to leaf morphology.

Supplemental Figure 7. Additional examples of SMAX1pro:GUS-GFPexpression in 10-d-old seedlings.

Supplemental Figure 8. A conserved C-terminal EAR motif in theSMXL/D53 family in Arabidopsis and rice.

Supplemental Table 1. Primer sequences.

Supplemental Table 2. T-DNA positions of smxl alleles.

ACKNOWLEDGMENTS

We thank Shigeo Toh and Peter McCourt for the htl-3 allele of KAI2/HTL.Funding support was provided by the University of Georgia ResearchFoundationand theNationalScienceFoundation (IOS-1350561) toD.C.N.;NIGMS National Institutes of Health Award T32GM007103 to N.M.; Chi-nese Government Scholarship PhD Program (Sichuan Agriculture Univer-sity) to Y.L.; and European Research Council (294514 EnCoDe) and theGatsby Foundation (GAT3272C) to T.B. and O.L.

AUTHOR CONTRIBUTIONS

I.S., T.B., N.M., Y.L., J.P.S., and D.C.N. designed the research. I.S., T.B.,N.M., Y.L., J.P.S., A.A., andD.C.N. performed research and analyzed data.T.B., O.L., and D.C.N. wrote the article.

Received June 25, 2015; revised October 5, 2015; accepted October 18,2015; published November 6, 2015.

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