SMAX1-LIKE7 Signals from the Nucleus to RegulateShoot Development in Arabidopsis via Partially EARMotif-Independent MechanismsOPEN

Yueyang Liang,a,b Sally Ward,a Ping Li,b Tom Bennett,a,1,2 and Ottoline Leysera,2

a Sainsbury Laboratory, University of Cambridge, Cambridge CB2 1LR, United KingdombRice Research Institute, Sichuan Agricultural University, Chengdu 611130, China

ORCID IDs: 0000-0002-7427-1028 (Y.L.); 0000-0002-5520-1331 (S.W.); 0000-0003-1612-4019 (T.B.); 0000-0003-2161-3829 (O.L.)

Strigolactones (SLs) are hormonal signals that regulate multiple aspects of shoot architecture, including shoot branching.Like many plant hormonal signaling systems, SLs act by promoting ubiquitination of target proteins and their subsequentproteasome-mediated degradation. Recently, SMXL6, SMXL7, and SMXL8, members of the SMAX1-LIKE (SMXL) family ofchaperonin-like proteins, have been identified as proteolytic targets of SL signaling in Arabidopsis thaliana. However, themechanisms by which these proteins regulate downstream events remain largely unclear. Here, we show that SMXL7functions in the nucleus, as does the SL receptor, DWARF14 (D14). We show that nucleus-localized D14 can physicallyinteract with both SMXL7 and the MAX2 F-box protein in a SL-dependent manner and that disruption of specific conserveddomains in SMXL7 affects its localization, SL-induced degradation, and activity. By expressing and overexpressing theseSMXL7 protein variants, we show that shoot tissues are broadly sensitive to SMXL7 activity, but degradation normally buffersthe effect of increasing SMXL7 expression. SMXL7 contains a well-conserved EAR (ETHYLENE-RESPONSE FACTORAmphiphilic Repression) motif, which contributes to, but is not essential for, SMXL7 functionality. Intriguingly, differentdevelopmental processes show differential sensitivity to the loss of the EAR motif, raising the possibility that there may beseveral distinct mechanisms at play downstream of SMXL7.

INTRODUCTION

Shoot system architectural characteristics strongly influence theproductivity of many crop species, and architectural traits havebeen selected in both historical and contemporary breedingschemes. Understanding the mechanisms that regulate shootarchitecture, and its environmental responsiveness, is thereforean important goal for plant research. It is well established thatlong-distance hormonal signals, including auxin, cytokinin, andstrigolactone (SL), are key regulators of shoot architecture andallow communication both within the shoot system and betweenthe shoot and root (Domagalska and Leyser, 2011). For instance,cytokinin produced in the root system in response to the avail-ability of nitrate ions is systemically transported to the shoot,where it promotes branching (Kiba et al., 2011;Müller et al., 2015).Similarly, root-derived SL plays a key role in negatively regulatingbranching in response to low phosphate availability in the rhizo-sphere (Kohlen et al., 2011). However, our understanding of themolecularmechanisms thatactdownstreamof thesehormones toalter developmental processes in the shoot is currently limited.

This is particularly true of SLs. Analysis of the phenotypes of SLbiosynthesis and signaling mutants has revealed roles for SLs inthe regulation of shoot branching, branching angle, plant height,stem thickness, and leaf blade elongation (Smith and Waters,2012). The role of SLs in regulating shoot branching has beenintensively studied, resulting in two contrasting, nonexclusivemodels for their mode of action. In the first, SLs are proposed toact locally in axillary buds by upregulating the expression ofthe BRANCHED1 (BRC1) gene, which encodes a TEOSINTEBRANCHED1/CYCLOIDEA/PCNA domain transcription factor(Braun et al., 2012). BRC1 is well-established as a regulator ofbud outgrowth, and brc1 mutants have strongly increased,SL-resistantbranching (Aguilar-Martínezetal., 2007;Breweretal.,2009). In the second model, SLs are proposed to act throughoutthe shoot to trigger clathrin-mediated endocytosis of the PIN1auxin efflux carrier (Crawford et al., 2010; Shinohara et al., 2013).This hinders the establishment of positive feedback-driven,canalized auxin export from buds into the stem, which has beenproposed to be required for the outgrowth of buds (Li andBangerth, 1999; Prusinkiewicz et al., 2009; Crawford et al., 2010;Shinohara et al., 2013). For the other aspects of shoot architectureaffected in SLmutants, very little is known about the downstreammechanisms involved. It is therefore an open question as towhether there is a single downstream target or class of targets forSLsignalingorwhether different aspectsof shoot responses toSLare mediated by distinct downstream targets.While the downstream targets of SL signaling remain poorly

defined, in recent years there has been rapid progress in ourunderstanding of proximal events in SL signaling.Multiple studieshave demonstrated that the DWARF14 (D14) a/b fold protein is

1Current address: Department of Biology, University of Leeds, LeedsLS2 9JT, UK.2 Address correspondence to ol235@cam.ac.uk or t.a.bennett@leeds.ac.uk.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: Ottoline Leyser (ol235@cam.ac.uk).OPENArticles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.16.00286

The Plant Cell, Vol. 28: 1581–1601, July 2016, www.plantcell.org ã 2016 American Society of Plant Biologists. All rights reserved.

very likely to act as aSL receptor, since it bindsandhydrolyses thesynthetic SL analog GR24 and is required for sensitivity to SL (Liuet al., 2009; Hamiaux et al., 2012; Waters et al., 2012; Kagiyamaet al., 2013; Zhao et al., 2013; Chevalier et al., 2014). D14 acts inconcert with theMAX2F-box protein, which formspart of anSCF-type ubiquitin ligase complex (Stirnberg et al., 2007; Hamiauxet al., 2012). Thus, as is the case inmany other hormonal signalingpathways inplants, the regulateddegradationof targetproteinsbythe 26S proteasome appears to be a key feature of SL signaling.The principle proteolytic targets of SL signaling appear to bea clade of proteins in the SMAX1-LIKE (SMXL) family, typified bySMXL7 inArabidopsis thaliana andD53 in rice (Oryza sativa) (Jiangetal., 2013;Zhouetal., 2013;Soundappanetal., 2015;Wangetal.,2015). SMXL proteins are weakly homologous to HEAT SHOCKPROTEIN101 ClpB chaperonins and are named for the foundingmember, SUPPRESSOR OF MAX2 1 (SMAX1) (Stanga et al.,2013). SMAX1 is also a probable target of SCFMAX2, although inthis case in response to signaling mediated by the KAI2 receptorprotein, itself a close relative of D14, for which the endogenousligand is currently unknown (Waters et al., 2012; Stanga et al.,2013; Soundappan et al., 2015). In Arabidopsis, SMXL7 is rapidlydegraded in response to treatment with rac-GR24 in a D14- andMAX2-dependent manner (Soundappan et al., 2015; Wang et al.,2015). Furthermore, loss of SMXL7 function, in combination withthat of its close relatives SMXL6 and SMXL8, is sufficient tosuppress completely theSL-related shoot and root phenotypes ofmax2, suggesting that theseproteinsare theprimaryandprobablyonly direct targets of MAX2-mediated SL signaling (Soundappanet al., 2015; Wang et al., 2015). In line with both models of theregulation of shoot branching, the smxl6 smxl7 smxl8 max2quadruple mutant has both strongly upregulated BRC1 expres-sion and strongly downregulated PIN1 levels relative to max2(Soundappan et al., 2015).

SMXL proteins contain a well-conserved ETHYLENE-RESPONSE FACTOR Amphiphilic Repression (EAR) motif (Ohtaet al., 2001), which has been widely viewed as an importantcontributor to SMXL function (Jiang et al., 2013; Zhou et al., 2013;Smith and Li, 2014; Soundappan et al., 2015; Wang et al., 2015),although this isbasedonlyoncircumstantial evidence.EARmotifsallow proteins to interact with partners containing correspond-ing C-Terminal to Lissencephaly Homology (CTLH) domains(Szemenyei et al., 2008). One of the best-characterized of theseinteractions is between the EAR motif in Aux/IAA proteins (re-pressors of auxin signaling) and the TOPLESS RELATED (TPR)family of chromatin remodeling factors (Szemenyei et al., 2008).The EAR motif in SMXL7 permits relatively weak interaction withTPR proteins, particularly TPR2 (Soundappan et al., 2015; Wanget al., 2015). On this basis, it has been suggested that SMXLproteins act as regulators of transcription (Smith and Li, 2014), butthe functional relevance of this interaction, and indeed the EARmotif ingeneral, hasnotbeenestablished. Furthermore, even if theEAR motif is functionally important, as suggested by its evolu-tionary conservation, it must be noted that there are other CTLH-domain proteins in plants, and these may be functionally relevantpartners for SMXL/D53 family members in addition to, or insteadof, TPR proteins (Bennett and Leyser, 2014).

Detailed characterization of the function of relevant SMXL/D53family proteins thus represents an opportunity to dissect the

downstream events in SL signaling. In this report, we assess thefunction of SMXL7 in SL signaling and shoot development inArabidopsis. We demonstrate that SMXL7 colocalizes with SLsignaling components D14 andMAX2 in the nucleus and that thislocalization is necessary for both SMXL7 function and degrada-tion. We demonstrate the SL-dependent physical interaction ofSMXL7with D14, but notMAX2, in planta.We show that SMXL7 isexpressed, and shows SL-induced degradation, throughout theshoot system in vascular-associated tissues and that the shootis well buffered against changes in SMXL7 transcription, but notSMXL7 stability. Deleting or modifying the EAR motif in SMXL7does not affect its localization or degradation, and although thisreducesSMXL7activity, it doesnotabolish it.Wefind thatdifferentaspects of development are differentially sensitive to the loss ofthe EAR motif. This raises the intriguing possibility that there aredistinct mechanisms downstream of SMXL7.

RESULTS

Strigolactone Signaling Components Are Localized to andFunction in the Nucleus

To understand the mechanisms by which SMXL7 activity regu-lates shoot architecture, we assessed its subcellular localizationusing a translational fusion to YFP (SMXL7-YFP) (Figure 1). Nu-clear localization of SMXL7 has previously been demonstrated(Soundappan et al., 2015), and consistent with this, transientexpression of 35Spro:SMXL7-YFP in leaf epidermal cells of Ni-cotiana benthamiana resulted in a signal highly localized to thenucleus (Figure 1A; Supplemental Figure 1) (Soundappan et al.,2015). We observed the same highly enriched nuclear localizationin root cells when we introduced the 35Spro:SMXL7-YFP con-struct into Arabidopsis (Figure 1E). However, this construct onlypartially restored the shoot branching phenotype of the smxl6-4smxl7-3 smxl8-1max2-1mutant (hereafter smxl678max2) towardthe expectedmax2-like branch number (Figure 1D). This could bebecause the protein fusion is not fully functional or because the35S promoter does not recapitulate endogenous SMXL7 pro-moter activity. We thus transformed Arabidopsis with a similarfusion driven by the native SMXL7 promoter (SMXL7pro:SMXL7-VENUS). This fusion protein completely restored the branchingphenotypeof the smxl678max2mutant tomax2 levels (Figure 1D),demonstrating that the fusion protein is functional.Despite thepartially and fully rescuedphenotypes in these lines,

it proved difficult to detect the protein in wild-type shoots, pre-sumably due to its instability, especially in the presence of SL. Inrice, the orthologous D53 protein is stabilized by deletion of fiveamino acids in the first NTPase domain (Jiang et al., 2013; Zhouet al., 2013), and it has previously been shown that a slightly largerdeletion has a similar stabilizing effect on SMXL7 in Arabidopsis(Soundappanetal., 2015).We reasoned thatastabilizedversionofSMXL7 might be more easily visualized, and we thus utilizeda version of the protein (SMXL7d53) containing an equivalentmutation to that seen in d53 (812Arg-Gly-Lys-Thr-Val-Val817 →Thr). We found that, indeed, SMXL7d53-VENUS or YFP fusionscould now be clearly detected in stems when expressed from thenative promoter (Figure 1H).

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Figure 1. SL Signaling Components Function in the Nucleus.

(A) to (C)Subcellular localizationofSMXL7-YFP (A),D14-CFP (B), andMAX2-CFP (C) inN.benthamianaepidermal cells, transiently expressed fromthe35Spromoter.(D)Primary rosette branching levels inCol-0,max2-1, and smxl6-4 smxl7-3 smxl8-1max2-1 transformedwith nothing (3rd bar), SMXL7pro:SMXL7-VENUS(4th bar), 35Spro:SMXL-YFP (two independent homozygous lines, 5th and 6th bars), and 35Spro:SMXL7DNLS-YFP, in which the nuclear localization signalhasbeendeleted (two independent homozygous lines, 7th and8th bars). Plantsweregrown in longdays for 7weeks.n=13 to 22; error bars indicate SE; barswith the same letter are not significantly different from one another (ANOVA + Tukey HSD, P < 0.05).(E) to (G) Subcellular localization of SMXL7-YFP (E), D14-CERULEAN (F), andMAX2-GFP in Arabidopsis root meristems, in homozygous transgenic linesexpressing the fusion proteins from the 35S promoter ([E] and [F]) or MAX2 promoter (G).(H) and (I) Expression of SMXL7d53-VENUS (in which the protein has been stabilized by replacing amino acids 812RGKTVV817 with T) (H) and D14-CERULEAN (I) in transverse hand sections through Arabidopsis inflorescence stems, in homozygous transgenic lines expressing the fusion proteins fromtheir native promoters.(J) Subcellular localization in an Arabidopsis root meristem of SMXL7DNLS-YFP in homozygous transgenic lines expressing the fusion protein from the 35Spromoter.(K)Rosette branching levels in Col-0 and d14-1 transformedwith nothing (2nd bar) andD14pro:D14-CERULEAN (three independent lines, 3rd to 5th bars),D14pro:D14NLS-VENUS in which a strong nuclear localization signal has been added (three independent lines, 6th to 8th bars), and D14pro:D14palm/myr-CERULEAN in which a palmitoylation/myristoylationmotif has been added (three independent lines, 9th to 11th bars). The data are taken from a larger dataset and show themost, median, and least rescued lines for each construct. The full data set is shown in Supplemental Figure 1. Plants were grown in shortdays for 4 weeks, grown in long days until the inflorescence stemwas 10 cm long, and then decapitated. Primary rosette branches were counted 10 d later(Grebet al., 2003).n=13 to20; error bars indicate SE; barswith thesame letter arenot significantlydifferent fromoneanother (ANOVA+TukeyHSD,P<0.05).(L) to (N)Subcellular localizationofD14-CERULEAN (L), D14NLS-VENUS (M), andD14palm/myr-CERULEAN (N) inN.benthamianaepidermal cells, transientlyexpressed from the 35S promoter.

Shoot Developmental Regulation by SMXL7 1583

We identified a predicted nuclear localization signal (NLS)in the SMXL7 protein (amino acid position 85 to 117;RLPSSKSTPTTTVEEDPPVSNSLMAAIKRSQAT). Consistent withits predicted function, specific deletion of this motif (SMXL7DNLS)reduced the nuclear localization of SMXL7-YFP (Figure 1J;Supplemental Figure 1). It has previously been shown thattreatment with 5 mM rac-GR24 results in the rapid degradation ofSMXL7-YFP, such that little fusion protein is detectable with-in 20 min (Soundappan et al., 2015). Similar treatments ofSMXL7DNLS-YFP had little effect, suggesting that the NLS, andpresumably therefore nuclear localization, is required for its SL-mediateddegradation (Supplemental Figure1). Thedeletionof theNLS also compromised the ability of the fusion protein expressedfrom the35Spromoter to restore increasedbranching levels to thesmxl678 max2 mutant (Figure 1K). These data suggest that thenuclear localization of SMXL7 is required for its function.

Previous reports show that the other known components of SLsignaling, D14 and MAX2, are expressed in the same vascular-associated tissues as SMXL7 and are at least partially nuclear(Chevalier et al., 2014; Stirnberg et al., 2007). To explore thecolocation of these proteins inmore detail, we used both transientexpression assays in N. benthamiana and stable expression inArabidopsis. Transient expression of a D14-CFP fusion protein inN. benthamiana leaf epidermal cells driven by the 35S promoterrevealed localization to both the nucleus and cytoplasm (Figure1B; Supplemental Figure 1), as previously observed by Chevalieret al. (2014). We observed the same subcellular localization in theroot when we transformed Arabidopsis with a 35Spro:D14-CE-RULEAN FLUORESCENT PROTEIN (CER) construct (Figure 1F),including colocalization with SMXL7 (Supplemental Figure 1). Wealso transformed Arabidopsis with aD14pro:D14-CER construct.Like SMXL7, this fusion protein was difficult to detect in the shootbut could be observed with high laser intensities (Figure 1I).

Toassesswhere in thecellD14 functions,wecreatedvariantsoffluorescently tagged D14 with either a strong NLS (D14NLS-VENUS), predicted to increase its nuclear localization, or a pal-mitoylation/myristoylation motif (D14palm/myr-CER), which shouldsequester D14 at the plasma membrane. Transient expression inN. benthamiana confirmed the expected changes in protein dis-tribution (Figures 1L to 1N). We then assessed the ability of theseproteins to restorewild-type levelsof shootbranching to thed14-1mutant when driven from the D14 promoter. We found that D14-CER and D14NLS-VENUS, but not D14palm/myr-CER, could com-pletely restore wild-type branching levels to d14-1 (Figure 1K;Supplemental Figure 1). These data suggest that D14 is alsopredominantly functional in the nucleus. We observed a similarlocalization pattern for MAX2-CFP fusions as for D14 in N.benthamiana cells (Figure 1C). We confirmed this subcellularlocalization pattern in Arabidopsis roots (Figure 1G) but wereunable to detect reliably MAX2-fluorescent protein fusions inArabidopsis stems, despite the well-documented activity of theMAX2 promoter in these tissues (Stirnberg et al., 2007) as well asMAX2-dependent SL response (Shinohara et al., 2013).

SMXL7, D14, and MAX2 Interact in the Nucleus

Several recent studies have shown that SMXL7/D53 proteinsare ubiquitinated and targeted for degradation in response to

D14-mediated SL perception in a MAX2-dependent manner(Jiang et al., 2013; Zhou et al., 2013; Umehara et al., 2015;Soundappan et al., 2015; Wang et al., 2015). Consistent with thisdegradation, SL-triggered interactions between D14 and SMXLs,and between D14 and MAX2, have been detected using variousassays in several species, with evidence of a complex involving allthree proteins (Hamiaux et al., 2012; Jiang et al., 2013; Zhou et al.,2013; Umehara et al., 2015; Wang et al., 2015). To assess theseinteractions further, we tested for protein-protein interactionsbetween thesecomponents inN.benthamiana leaf epidermalcellsusing Förster resonance energy transfer (FRET) with fluorescencelifetime imaging microscopy (FLIM).For the D14-CFP/SMXL7-YFP pair, the SMXL7 signal was

rapidly lost uponGR24 treatment, sowe again used the stabilizedSMXL7d53 version of the protein, which allowed us to assess theinteraction more easily. As expected based on previous reports(Jiang et al., 2013; Zhou et al., 2013; Wang et al., 2015), we ob-served FRET between cotransfected D14-CFP and SMXL7d53-YFP only upon rac-GR24 treatment (Figures 2A and 2B). TheSMXL7 protein is clearly localized to nuclear speckles(Soundappan et al., 2015), and it primarily in these speckles thatwe detected FRET. D14 appears to be recruited to the speckles inresponse to GR24 treatment.We also observed rac-GR24-dependent FRET betweenMAX2-

CFP and D14-YFP. This occurred across the whole nucleus(Figures 2C and 2D). We tested the interaction between MAX2-CFP and SMXL7-VENUS and observed no interaction in theabsence or presence of rac-GR24 (Figures 2E and 2F). In this caseSMXL7 signal remained stable upon GR24 treatment, so we didnot need to use SMXL7d53. These results suggest that co-transfected D14, but not MAX2, is needed for efficient SMXL7degradation. We confirmed that using SMXL7d53 in this assaydoes not affect the lack of interaction with MAX2 (SupplementalFigure 2). Taken together, these data suggest that SMXL7 inter-acts only indirectlywithMAX2,withD14acting asabridge tobringthese components together. Our in planta data thus confirm thepreviously reported interactions among the Arabidopsis SL sig-naling proteins. Interestingly, we also detected FRET betweenSMXL7-YFP and SMXL6-CFP in N. benthamiana leaf cells, sug-gesting that there is dimerization among the SMXL proteinsthemselves (Supplemental Figure 2). Consistent with these ob-servations, we observed colocalization of SMXL6-mCherry andSMXL7-VENUS fusion proteins in Arabidopsis roots and theirsimultaneousdegradation in response to treatmentwith rac-GR24(Supplemental Figure 2).

SMXL7 Variants Affect Protein Stability

To gain further insights into mechanisms of SMXL7 action, wecreated several variants of the SMXL7 protein, lacking definedprotein motifs (Figure 3; Supplemental Table 1). First, wemutatedthe first NTPase domain, deleting either the whole domain(SMXL7NTP1) or using the existing SMXL7DP-loop (Soundappanet al., 2015) or SMXL7d53 (see above) variants to specifically deletethe P-loop motif. Second, we created a protein deletion that re-moved the entire second NTPase domain (SMXL7NTP2). Third, wemodified the EAR motif (Leu-Asp-Leu-Asn-Leu-Pro) in SMXL7,either by deleting it (SMXL7DEAR) or by replacing the conserved

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Figure 2. SMXL7 Physically Interacts with D14 in a SL-Dependent Manner.

Detection of FRET in N. benthamiana epidermal cells doubly transfected with D14-CFP/SMXL7d53-YFP, MAX2-CFP/D14-CFP, and MAX2-CFP/SMXL7-YFPpairs, expressed fromthe35Spromoter.Colocalizationof theacceptoranddonormolecules isshown in thefirstandsecondpanel ineach row.TheCFPdonor was excited with using a 405-nm laser line, and emission from the YFP acceptor (indicating the occurrence of FRET) was monitored by FLIM (thirdpanel in each row). The fluorescence lifetime of YFP (green) is shorter than CFP (red).

leucine residues with alanines (Ala-Asp-Ala-Asn-Ala-Pro;SMXL7alaEAR), which has previously been shown to abolish itsinteraction with TPR2 in yeast two-hybrid assays (Soundappanet al., 2015). We also created two double deletions of both theP-loopandtheEARmotif (SMXL7DP-loop,DEARandSMXL7d53,alaEAR).We then tested how these SMXL7 variants affected nuclear lo-calization by transiently expressing them in N. benthamiana epi-dermal cells. We observed that nuclear targeting is not obviouslyaffected by any of these alterations (Figures 3B to 3G). We thenassessed the SL response of these proteins in the roots ofhomozygous transgenic Arabidopsis lines expressing thesefusionproteinsfromthe35Spromoter.Consistentwiththeresults inN. benthamiana, all of these variants showed strong nuclear lo-calization (Figures 3H to 3M). As expected, we observed that thetwo new variants affecting the first NTPase domain resulted inseverely compromised rac-GR24-induced degradation of theproteins (Figures 3I and 3O). Deletion of the entire NTP1 domainalso seemed to reduce the accumulation of this protein in root tips,but it was possible to detect the protein in the transition zone(Figures 3I and 3O). In contrast, the SMXL7NTP2 variant retained theGR24-inducible degradation of the protein, while both EAR motifvariantswereboth rapidlydegraded in response toGR24 treatment(Figures 3J to 3L and 3P to 3R).

SMXL7 Expression Levels Do Not Have Dose-DependentEffects in the Wild Type

To test the function of these SMXL7 protein variants on differentaspects of shoot architecture, we created homozygous trans-genic lines in relevant genetic backgrounds (Table 1). We firstassessed the effect of SMXL7 expression levels on shoot de-velopment using transgenic lines with altered SMXL7 copynumbers and expression levels (Table 1). For each line in eachbackground, we took representative lines to homozygosity in theT3 generation. A suite of phenotypes affected by SL signaling inthe shoot has previously been described, including leaf blade andpetiole length, degree of branching, branch angle, height, andstem thickness (Smith and Waters, 2012; Soundappan et al.,2015), and we assessed these characteristics in our lines (Figure4).We first confirmed thatour constructswereexpressed in leavesand stems and that the fusion proteins respond to rac-GR24 asexpected. In leaves, wewere able to observe robust expression ofSMXL7 in petioles (Figure 4A). As expected, the SMXL7-VENUSsignal was rapidly reduced in response to 5 mM rac-GR24treatment inawild-typebackground,butnot inmax2-1 (Figures4Ato 4D). In wild-type stems, SMXL7-VENUS was very difficult todetect (Figure 4I). However, in a max1-1 mutant background,which is defective in SL biosynthesis, we detected reliableexpression, which was strongest in the vascular cambium (Figure4K) and was greatly reduced by 5mMGR24 treatment (Figure 4L).

Again, SMXL7 was stabilized in a background containing themax2-1 mutation (Figure 4J).We quantified a range of shoot phenotypes in Col-0, smxl678

max2 (no SMXL7 activity), and max2 (overactive SMXL7) back-grounds as a baseline for assessing the function of SMXL7 var-iants (Figure 5) (Supplemental Figure 3).We found that expressionofSMXL7pro:SMXL7-VENUS from its nativepromoter is sufficientto rescue all of the assessed smxl678 max2 phenotypes to anapproximately max2 phenotype (Figures 5C, 5D, 5I, 5J, 5M, and5N;Supplemental Figure3).When this constructwasexpressed inawild-typebackground, thereby increasingSMXL7copynumber,we found only minor effects on the assessed characteristics(Figures 5A, 5B, 5G, 5H, 5M, and 5N; Supplemental Figure 3).However, we found that introducing SMXL7pro:SMXL7-VENUSinto amax2-1 SL signaling-deficient background produced clearadditional effects on leaf development, in general exaggeratingthe effects of max2-1. In leaves, petiole length and blade lengthwere reduced further compared with max2, but blade width wasreduced and an additional phenotype of upward rolling of the leafmargins was observed (Figures 5E, 5F, and 5M). Plant height wasreduced below that of max2 mutants, as was branch angle andstem diameter (Figures 5K and 5L; Supplemental Figure 3).However, we did not detect any further increase in primary shootbranching, probably because this is near-maximal in max2-1(Figures 5K, 5L, and 5N). Furthermore, the plants showed a re-duced fertility phenotype not observed in max2-1. Very similarphenotypes were observed in d14-1 SMXL7pro:SMXL7-VENUSandmax1-1 SMXL7pro:SMXL7-VENUS (Supplemental Figure 3).In summary, unlike wild-type plants, genotypes deficient inSMXL7 degradation showed a clear dose-dependent response toincreasing SMXL7 copy number in most tissues (e.g., smxl678max2 < max2 < max2 SMXL7pro:SMXL-VENUS).

Shoot Tissues Are Widely Sensitive to SMXL7 Activity

The results described above suggest that, in a wild-type context,MAX2-mediated degradation can buffer against increases inSMXL7 expression in the shoot; that is, wild-type plants are re-sistant to alterations in SMXL7 expression level. To probe therelationship between SMXL7 degradation and shoot architecturefurther, we examined the effects of introducing stabilized SMXL7driven from the native SMXL7 promoter into relevant geneticbackgrounds (Table 1). We first confirmed that these constructsresulted in SMXL7 accumulation in leaves and stems and that theprotein was resistant to rac-GR24-induced degradation in thesetissues (Figures 4E to 4H and 4M to 4P). We then quantified thephenotypic effects of expressing stabilized SMXL7 (Figure 6)(Supplemental Figure 4). In the wild type, this generally resultedin a clear phenocopy of SL-deficient or SL signaling mutants.The plants typically resembled d14-1 in leaf morphology, shoot

Figure 2. (continued).

(A) and (B) FRET-FLIM analysis of D14-CFP and SMXL7d53-YFP in mock treated cells (A) or cells treated with 5 mM rac-GR24 (B)(C) and (D) FRET-FLIM analysis of MAX2-CFP and D14-YFP in mock-treated cells (C) or cells treated with 5 mM rac-GR24 (D).(E) and (F) FRET-FLIM analysis of MAX2-CFP and SMXL7-YFP in mock-treated cells (E) or cells treated with 5 mM rac-GR24 (F).(G) Color scheme for FLIM analysis, indicating fluorescence lifetimes between 3 and 1 ns.

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morphology, and shoot branching levels (Figures 6A to 6D and 6Ito 6J; Supplemental Figure 4). Expression of stabilized SMXL7from the 35S promoter resulted in leaf morphology resemblingmax2-1 leaves expressing SMXL7pro:SMXL7-VENUS, withleaf margins that rolled upward, and exaggerated petiole andblade length phenotypes (compare Figures 6F to 6H with Figures5E and 5F).

Overexpression of stabilized versions similarly resulted inqualitatively similar phenotypes to max2-1 SMXL7pro:SMXL7-VENUS, with reduced height and decreased branching anglerelative to max2-1 (Figures 6K to 6M; Supplemental Figure 4).Furthermore, there was poor fertility in these lines, and in moreextreme cases, a failure of reproductive organs to developproperly (Figures 6K to 6M). However, shoot branching was nogreater than in max2-1 or d14-1 (Figure 6N). For all of the as-sessed phenotypes, the effect of overexpressing SMXL7d53

was often weaker than that of SMXL7Dp-loop. Furthermore,plants hemizygous for 35Spro:SMXL7Dp-loop-YFP had a lessextreme phenotype than homozygous plants (Figures 6G, 6H,6L, and 6M).

These results show that stabilization of SMXL7 in its endoge-nous expression domain results in a close phenocopy of SLmutants, similar to the d53mutant in rice (Jiang et al., 2013; Zhouet al., 2013), but higher expression levels can give additionalphenotypes. Some of these are only observed when the 35Spromoter is used, and thus they may result from ectopic ex-pression of SMXL7.

SMXL7 Activity Is Sufficient to Alter PIN1 Protein and AuxinTransport Levels

We previously demonstrated that SL signaling negatively regu-lates PIN1 protein accumulation at the basal plasma membraneof xylem parenchyma cells in the stem, which is associated withreduced stem auxin transport (Crawford et al., 2010; Shinoharaet al., 2013). The smxl678 max2 quadruple mutant has a corre-sponding reduction in PIN1 relative to max2, as assessed byfluorescence of a PIN1-GFP fusion protein at the basal plasmamembranes of xylem parenchyma cells in the stem (Soundappanet al., 2015). We therefore tested whether altering SMXL7 activity

Figure 3. SMXL7 Variants Affect Protein Stability.

(A) Structure of SMXL7 protein identifying conserved domains targeted in this study.(B) to (G)Subcellular localizationofSMXL-YFP (B),SMXLDNTP1-YFP (C),SMXLDNTP2-YFP (D),SMXLDEAR-YFP (E),SMXLalaEAR-YFP (F),andSMXLDP-loop,DEAR-YFP(G) in N. benthamiana epidermal cells, transiently expressed from the 35S promoter. Bar = 50 mm.(H) to (S) Subcellular localization of SMXL-YFP ([H] and [N]), SMXLDNTP1-YFP ([I] and [O]), SMXLDNTP2-YFP ([J] and [P]), SMXLDEAR-YFP ([K] and [Q]),SMXLalaEAR-YFP ([L] and [R]), and SMXLDP-loop,DEAR-YFP ([M] and [S]) in the root tips of homozygous transgenic Arabidopsis lines expressing the fusionproteins from the 35S promoter, treated with 5 mM rac-GR24, at 0 min ([H] to [M]) and 20 min ([N] to [S]) after treatment. Bar = 100 mm.

Shoot Developmental Regulation by SMXL7 1587

was sufficient to cause changes in PIN1 protein levels (Figure 7).We first tested whether the PIN1 and SMXL7 expression patternsshow significant overlap in the stem using GUS reporter lines andfound that there is indeedclear overlap in their expressionpatternsin vascular-associated tissues (Figures 7A to 7D), particularly inthe cambium and xylem parenchyma, which are major sites ofPIN1 expression in the stem (Bennett et al., 2006). Furthermore,when we crossed SMXL7pro:SMXL7-VENUS into a PIN1pro:PIN1-GFP reporter line, we found that the proteins appeared to beexpressed in the same cells in the xylem parenchyma, in thenucleusandat theplasmamembrane, respectively (Figure7E).Wethen tested whether PIN1 protein levels are increased if SMXL7activity is increased by crossing PIN1pro:PIN1-GFP into theSMXL7pro:SMXL7d53-VENUS background. Compared withcontrol plants, PIN1-GFP protein levels at the basal plasmamembrane of xylem parenchyma cells in the stem were indeedincreased in this line (Figures 7F to 7H). Similarly, auxin transportthrough stem segments was increased in the SMXL7pro:SMXL7d53-VENUS line relative to the wild type (Figure 7I).

The EAR Motif Contributes to but Is Not Essential forSMXL7 Function

The presence of the EARmotif in SMXL7 and related proteins hasled to speculation about a potential role for SMXL proteins inregulating transcription via interaction with TPL transcriptionalrepressor proteins (Smith and Li, 2014), but the contribution of theEARmotif to the function of SMXL proteins has been unclear. Wethus used our constructs to assess the role of the EAR motif inSMXL7-mediated regulation of shoot architecture by comparing

the ability of SMXL7pro:SMXL7alaEAR-VENUS and SMXL7pro:SMXL7DEAR-VENUS to restore the smxl678 max2 mutant phe-notype toward themax2phenotype, relative to controlSMXL7pro:SMXL7-VENUS lines (Figure 8). With respect to leaf morphology,both SMXL7alaEAR and SMXL7DEAR cause a decrease in petiolelength and increase in blade length, similar to wild-type SMXL7,resulting inmax2-like leaves (Figures8A to8G).ExpressionofbothSMXL7alaEAR and SMXL7DEAR causes a large decrease inbranching angle, though not to the same extent as intact SMXL7(Supplemental Figure 5). Expression of both these proteins is alsoassociated with a slight, though not significant, increase in shootbranching relative to the smxl678 max2 and Col-0 backgrounds(Figure 8H). The effect is much less than that caused by intactSMXL7,whichcan restorebranching tomax2-like levels.Similarly,expression of both SMXL7alaEAR and SMXL7DEAR is associatedwith only a very small reduction in plant height, which is notsignificant for SMXL7alaEAR (Figure 8J). Here again, this contrastswith the effect of intact SMXL7 expression, which can fully restorethemax2 height phenotype to smxl678max2 plants (Figure 8J). Incombination, these effects result in shoots in theEARmutant linesintermediate in appearance between max2 and smxl678 max2(Figures 8A to 8F and 8I).The smxl678 max2 mutant (and smxl678) has a sprawling

growth habit with increased branching angle and stems that oftenappear unable to bear their own weight (Figure 8I) (Soundappanet al., 2015). Since load-bearing in stems is typically associatedwith thickening of the interfascicular cambium (Ko et al., 2004), weinvestigated whether there was a defect in cambial formation insmxl678 max2. We observed that, indeed, there is very little in-terfascicular cambiumpresent in smxl678max2, a phenotype that

Table 1. Arabidopsis Transgenic Lines

Background Construct Copy No. GR24 Response Total T1 Lines T1 Lines with Representative Phenotype

Col-0 SMXL7pro:SMXL7-VENUS 4 Y 4 4s678 max2 SMXL7pro:SMXL7-VENUS 2 Y 5 5max2-1 SMXL7pro:SMXL7-VENUS 4 N 5 5max1-1 SMXL7pro:SMXL7-VENUS 4 Y CrossedCol-0 SMXL7pro:SMXLd53-VENUS 4 N 6 6s678 max2 SMXL7pro:SMXL7d53-VENUS 2 N 3 3s678 SMXL7pro:SMXL7d53-VENUS 2 N CrossedCol-0 SMXL7pro:SMXL7DP-loop-VENUS 4 N 4 3s678 max2 SMXL7pro:SMXL7DEAR-VENUS 2 Y 6 5s678 max2 SMXL7pro:SMXL7alaEAR-VENUS 2 N 4 4s678 max2 SMXL7pro:SMXL7DP-loop,DEAR-VENUS 2 N 4 3Col-0 SMXL7pro:SMXL7d53,alaEAR-VENUS 4 N 3 3s678 SMXL7pro:SMXL7d53,alaEAR-VENUS 2 N 2 2Col-0 35Spro:SMXL7-YFP 4 Y 2 2s678 max2 35Spro:SMXL7DNLS-YFP 2 N 5 5Col-0 35Spro:SMXL7d53-YFP 4 N 6 2Col-0 35Spro:SMXL7DP-loop-YFP 4 N 5 2s678 max2 35Spro:SMXL7DEAR-YFP 2 N 5 3s678 max2 35Spro:SMXL7alaEAR-YFP 2 N 5 2Col-0 35Spro:SMXL7DP-loop,DEAR-YFP 4 N 3 3

List of stably transformed SMXL7 protein fusion lines in Arabidopsis used in this study. Constructs were transformed into the indicated geneticbackgrounds; the total number of T1 lines analyzed is indicated, along with the number of lines showing the phenotypes discussed here. The copynumber of SMXL7 in these lines and whether the transgenic whether the transgenic SMXL7 protein can be degraded in response to rac-GR24 are alsoindicated.

1588 The Plant Cell

Figure 4. SMXL7 Responds to SL Treatment in Leaves and Stems.

Red signal is chloroplast fluorescence. Yellow signal is SMXL7-YFP/VENUS.(A) to (H)ResponseofSMXL7variants in leaf petioles to20min treatmentwith5mM rac-GR24:SMXL7-VENUS in theCol-0 ([A]and [B]) andmax2-1 ([C]and[D]) backgrounds; SMXL7d53-VENUS inCol-0 ([E]and [F]) andSMXL7d53-YFP inCol-0 ([G]and [H]). Expression from theSMXL7promoter ([A] to [F]) or35Spromoter ([G] and [H]).

can be rescued by expression of intact SMXL7-VENUS (Figures8K to 8N). Interestingly, max2 and SL biosynthetic mutants alsohave reduced interfascicular cambium compared with Col-0, andtreatment with SL can promote interfascicular cambium de-velopment (Agusti et al., 2011). It is thus very difficult to assessthe ability of the SMXL variants to restore the smxl678 max2phenotype toward the max2 phenotype. We therefore did notattempt detailed quantification. Nevertheless, it is clear thatboth SMXL7alaEAR and SMXL7DEAR are able to promote the de-velopment of cambium in a smxl678 max2 background (Figures8O and 8P). A similar result is observed for the diameter of theprimary inflorescence,which likely reflects cambiumactivity. Bothmax2 and smxl678 max2 have thinner stems than the wild type(Supplemental Figure 5). Here again, the increase in stem thick-ness in smxl678 max2 plants expressing SMXL7alaEAR andSMXL7DEARdemonstrates that these proteins are able to increasestem diameter.

We also tested the ability of 35Spro:SMXL7alaEAR-YFP to re-store the smxl678 max2 mutant phenotype toward the max2phenotype relative to a control 35Spro:SMXL7-YFP line(Supplemental Figure 6). We found no significant differencesbetween SMXL7alaEAR and intact SMXL7 in their modest butstatistically significant ability to reduce blade length, petiolelength, or branch angle and to increase shoot branching to-ward max2 branch numbers (ANOVA + Tukey HSD, P > 0.05)(Supplemental Figure 6). Neither line shows a clear reduction inplant height relative to smxl678 max2 (Supplemental Figure 6).These data demonstrate that in general, SMXL7alaEAR-YFP is asactive as SMXL7-YFP when expressed from the 35S promoter.Although these data seem somewhat inconsistent with thosepresented for expression from the SMXL7 promoter, the rescue by35Spro:SMXL7-YFP is not complete, and sowhen expressed fromthe 35S promoter, SMXL7alaEAR is not being tested across the fullrange of SMXL7 activity.

Loss of the EAR Motif Counteracts SMXL7 Stabilization

We next tested whether the EAR motif is required for the moreextreme phenotypes associated with expression of stabilizedSMXL7 (Figure 6) by comparing the effects of SMXL7pro:SMXL7d53,alaEAR-VENUS toSMXL7pro:SMXL7d53-VENUS in bothCol-0 and smxl6-4 smxl7-3 smxl8-1 (smxl678) backgrounds(Figure 9; Supplemental Figure 7). Strikingly, mutation of the EARmotif completely abolishes the d14-like phenotypes causedby stabilized SMXL7d53 in a wild-type background, resulting inplants with essentially wild-type leaf shape, branch angle, branchnumbers, and primary stemheight and thickness (Figures 9A to 9Cand 9G to 9K; Supplemental Figure 7). In isolation, this result couldbe interpreted as demonstrating that the protein lacking both

a functionaldegronandEARmotif issimplynonfunctional, resultingin nophenotypic effects.However, analysis of the sameconstructsin the smxl678 background shows that this is not the case, and theprotein is still functional, albeit with reduced activity. AlthoughSMXL7d53,alaEAR does not induce strong d14-like phenotypes ina smxl678 background, it is still able to rescue the smxl678 phe-notype toward a wild-type phenotype, or in some cases beyondwild-typeandtowardad14-likephenotype(Figures9Dto9Fand9Lto 9N). The leaves of smxl678 SMXL7pro:SMXL7d53,alaEAR-VENUShave wild-type dimensions and full rescue of the reduced width ofsmxl678 leaves (Figure 9G). Both the increased branch angle andreduced stem width of smxl678 are restored to wild-type(Supplemental Figure 7). The number of branches is slightly in-creased (Figure 9H) and height of the primary stem is slightly de-creased (Supplemental Figure 7) relative to both smx678 and thewild type, such that theplantshaveamored14-likephenotype.Thelack of interfascicular cambium in smxl678 is also at least partiallyrescued by SMXL7d53,alaEAR (Figures 9R to 9T). These data showthat the SMXL7d53,alaEAR protein is active and functional with re-spect to all the phenotypes assessed, but the protein does notappear to have the hypermorphic activity required for gain-of-function phenotypes. Indeed, based on its ability to restore thesmxl678mutant towild-type, theactivityof thisprotein seems tobeapproximately equivalent to wild-type SMXL7. We also observedsimilar effects in 35Spro:SMXL7Dp-loop,DEAR-YFP relative to35Spro:SMXL7Dp-loop-YFP, where the strong gain-of-functionphenotypescausedbystabilizationwerenegatedby the lossof theEAR motif (Supplemental Figure 8).

DISCUSSION

Strigolactone Signaling Perception IsPredominantly Nuclear

The past few years have seen a rapid increase in our un-derstanding of SL signaling, including the definition of a corepathwaymediatedbyD14 (reviewed inBennett andLeyser, 2014).The results presentedhere refine andelaborate upon the essentialdetails previously established and lend support to key concepts inplanta. We show SL-dependent physical association of D14 andSMXL7 in planta, as predicted by previous analyses (Jiang et al.,2013; Zhou et al., 2013; Umehara et al., 2015; Wang et al., 2015).Furthermore, we confirm that D14 also interacts with MAX2, butthat there is no evidence of direct interaction between SMXL7 andMAX2. Previous reports have provided evidence for the nuclearlocalization for SMXL7-family proteins (Jiang et al., 2013; Wanget al., 2015; Soundappan et al., 2015), andwe have demonstratednot only that this is the case in a wide variety of tissues, but also

Figure 4. (continued).

(I) Very low levels of SMXL7-VENUS in longitudinal hand sections through Col-0 stems.(J) to (P)ResponseofSMXL7variants expressed in longitudinal handsections of stems to treatmentwith5mM rac-GR24 (treatment time indicated inpanel);SMXL7-VENUS in smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2) (J) and max1-1 ([K] and [L]) backgrounds; SMXL7d53-VENUS in Col-0 ([M] and [N]);SMXL7d53-YFP inCol-0 (O)andSMXL7DP-loop-YFP inCol-0 (P). Expression from theSMXL7promoter ([I] to [N]) or35Spromoter ([O]and [P]).White arrowsindicate SMXL7 expressing nuclei before and after treatment with rac-GR24.

1590 The Plant Cell

Figure 5. Effect of SMXL7 Dose on Development.

(A) to (F)Rosette leafmorphology inCol-0 ([A]and [B]),smxl6-4smxl7-3smxl8-1max2-1 (s678m2) ([C]and [D]), andmax2-1 ([E]and [F]) untransformed ([A],[C], and [E]) or homozygous for SMXL7pro:SMXL7-VENUS ([B], [D], and [F]).(G) to (L)Adult shootmorphology inCol-0 ([G] and [H]), smxl6-4 smxl7-3 smxl8-1max2-1 (s678m2) ([I]and [J]), andmax2-1 ([K]and [L]) untransformed ([G],[I], and [K]) or homozygous for SMXL7pro:SMXL7-VENUS ([H], [J], and [L]).(M) Leaf blade length, bladewidth, and petiole length in the 7th leaf of Col-0, smxl6-4 smxl7-3 smxl8-1max2-1 (s678m2), andmax2-1 untransformed (2) orhomozygous forSMXL7pro:SMXL7-VENUS (+). n = 11 to 16; error bars indicate SE; bars with the same letter are not significantly different from one another(ANOVA + Tukey HSD, P < 0.05).(N) Number of cauline, rosette and total primary branches in Col-0, smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2), and max2-1 untransformed (2) ortransformed with SMXL7pro:SMXL7-VENUS (+), measured at proliferative arrest. n = 20 to 24; error bars indicate SE; bars with the same letter are notsignificantly different from one another (ANOVA + Tukey HSD, P < 0.05).

Shoot Developmental Regulation by SMXL7 1591

that this localization is required for both full SMXL7 function and itsefficient degradation. While SMXL7 localization is difficult todetect outside the nucleus, we find that D14 and MAX2 are lo-calized to both the nucleus and cytoplasm. However, our datasuggest that, at least in the caseofD14, it is thenuclear pool that is

responsible for D14 function. The relevance of the cytoplasmicpool is unclear.Our results also suggest that SMXL proteins may act as

heterodimers, though again, whether this if of functional rele-vance is currently unclear. Another intriguing aspect suggested

Figure 6. Effect of SMXL7 Stabilization on Development.

(A) to (H) Rosette leaf morphology in Col-0 untransformed (A) or transformed with SMXL7pro:SMXL7-VENUS (B), SMXL7pro:SMXL7d53-VENUS (C),SMXL7pro:SMXL7DP-loop-VENUS (D), 35Spro:SMXL7d53-YFP (F), 35Spro:SMXL7DP-loop-YFP ([G], hemizygous; [H], homozygous), and in d14-1 (E).(I) to (M)Adult shootmorphology (mainpicture) and reproductivemorphology (inset) inCol-0 transformedwithSMXL7pro:SMXL7d53-VENUS (I),SMXL7pro:SMXL7DP-loop-VENUS (J), 35Spro:SMXL7d53-YFP (K), and 35Spro:SMXL7DP-loop-YFP ([L], hemizygous; [M], homozygous).(N) Leaf blade length, bladewidth andpetiole length in the 7th leaf of untransformedCol-0, Col-0 transformedwithSMXL7pro:SMXL7-VENUS,SMXL7pro:SMXL7d53-VENUS, SMXL7pro:SMXL7DP-loop-VENUS, 35Spro:SMXL7d53-YFP, 35Spro:SMXL7DP-loop-YFP (hemizygous), and in d14-1. n = 9 to 16; errorbars indicate SE; bars with the same letter are not significantly different from one another (ANOVA + Tukey HSD, P < 0.05).(O) Number of cauline, rosette, and total primary branches of untransformed Col-0, Col-0 transformed with SMXL7pro:SMXL7-VENUS, SMXL7pro:SMXL7d53-VENUS, SMXL7pro:SMXL7DP-loop-VENUS, 35Spro:SMXL7d53-YFP, 35Spro:SMXL7DP-loop-YFP (hemizygous), and in d14-1. n = 10 to 13; errorbars indicate SE; bars with the same letter are not significantly different from one another (ANOVA + Tukey HSD, P < 0.05).

1592 The Plant Cell

Figure 7. SMXL7 Influences PIN1 and Auxin Transport.

(A) to (D) Expression of SMXL7pro:GUS ([A] and [B]) and PIN1pro:GUS ([C] and [D]) in transverse ([A] and [D]) and longitudinal ([B] and [C]) sections ofArabidopsis inflorescence stems. Small bold letters indicate tissues within the stem: C, cambium; XP, xylem parenchyma.(E) Coexpression of SMXL7-VENUS (red, arrowheads) and PIN1-GFP (green) in xylem parenchyma cells of the Arabidopsis inflorescence stem in a linedoubly homozygous for the fusion proteins expressed from their respective native promoters. The bright-field image is superimposed, showing cellboundaries and spiral thickening on the adjacent xylem. Blue indicates chloroplast autofluorescence.(F) and (G) Expression of PIN1pro:PIN1-GFP in Col-0 and Col-0 homozygous for SMXL7pro:SMXL7d53-VENUS.(H) Quantification of PIN1-GFP levels at the basal plasma membrane in xylem parenchyma cells of Arabidopsis inflorescence stems in Col-0 and Col-0homozygous forSMXL7pro:SMXL7d53-VENUS.n=8plants per genotype, fivemembranesmeasured in eachplant. Error bars indicate SE; asterisks indicatesignificant difference from the wild type at the P < 0.005 level (t test).(I) Transport of radiolabeled IAA through 18-mm stem segments of Col-0 and Col-0 homozygous for SMXL7pro:SMXL7d53-VENUS over a 6-h timeframe.n = 17 to 19; error bars indicate SE. Asterisks indicate significant difference from the wild type at the P < 0.005 level (t test).

Shoot Developmental Regulation by SMXL7 1593

Figure 8. Phenotypic Effects of SMXL7 Are Partially EAR-Independent.

(A) to (F) Rosette leaf morphology in Col-0 (A), max2-1 (B), untransformed smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2) (C), or s678m2 homozygous forSMXL7pro:SMXL7-VENUS (D), SMXL7pro:SMXL7alaEAR-VENUS (E), and SMXL7pro:SMXL7DEAR-VENUS (F).(G) Leaf blade length, bladewidth, andpetiole length in the 7th leaf of Col-0,max2-1, untransformed smxl6-4 smxl7-3 smxl8-1max2-1 (s678m2), or s678m2homozygous forSMXL7pro:SMXL7-VENUS,SMXL7pro:SMXL7alaEAR-VENUS, andSMXL7pro:SMXL7DEAR-VENUS. n=10; error bars indicate SE; barswiththe same letter are not significantly different from one another (ANOVA + Tukey HSD, P < 0.05).(H) Number of cauline, rosette, and total primary branches of Col-0, max2-1, untransformed smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2), or s678m2homozygous forSMXL7pro:SMXL7-VENUS,SMXL7pro:SMXL7alaEAR-VENUS, andSMXL7pro:SMXL7DEAR-VENUS. n=10; error bars indicate SE; barswiththe same letter are not significantly different from one another (ANOVA + Tukey HSD, P < 0.05).(I) Adult shoot morphology in (left to right) Col-0, max2-1, untransformed smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2), or s678m2 homozygous forSMXL7pro:SMXL7-VENUS, SMXL7pro:SMXL7DEAR–VENUS, and SMXL7pro:SMXL7alaEAR-VENUS.(J) Height of the primary inflorescence stem in Col-0, max2-1, untransformed smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2), or s678m2 homozygous forSMXL7pro:SMXL7-VENUS, SMXL7pro:SMXL7alaEAR-VENUS, and SMXL7pro:SMXL7DEAR-VENUS, measured at proliferative arrest. n = 10; error barsindicate SE; bars with the same letter are not significantly different from one another (ANOVA + Tukey HSD, P < 0.05).(K) to (P) Stem anatomy in Col-0 (K),max2-1 (L), untransformed smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2) (M), or s678m2 homozygous for SMXL7pro:SMXL7-VENUS (N),SMXL7pro:SMXL7alaEAR-VENUS (O), andSMXL7pro:SMXL7DEAR-VENUS (P) plants stainedwith toluidine blue. Red bar illustrates thedepth of the interfascicular cambium layer. Images are representative of multiple independent samples.

1594 The Plant Cell

Figure 9. Loss of the EAR Motif Counteracts SMXL7 Stabilization.

(A) to (C) Rosette leaf morphology in untransformed Col-0 (A) or Col-0 transformed with SMXL7pro:SMXL7d53-VENUS (B) or with SMXL7pro:SMXL7d53,alaEAR-VENUS (C).(D) to (F)Rosette leaf morphology in untransformed smxl6-4 smxl7-3 smxl8-1 (smxl678) (D) or smxl678 homozygous for SMXL7pro:SMXL7d53-VENUS (E)or SMXL7pro:SMXL7d53,alaEAR-VENUS (F).(G) Leaf blade length, blade width, and petiole length in the 7th leaf of Col-0 and smxl678, either untransformed or homozygous for SMXL7pro:SMXL7d53-VENUS or SMXL7pro:SMXL7d53,alaEAR-VENUS. n = 10; error bars indicate SE; bars with the same letter are not significantly different from one another(ANOVA + Tukey HSD, P < 0.05).(H)Number of cauline, rosette and total primary branches of Col-0 and smxl678, either untransformed or homozygous forSMXL7pro:SMXL7d53-VENUS orSMXL7pro:SMXL7d53,alaEAR-VENUS. n= 10; error bars indicate SE; barswith the same letter are not significantly different fromone another (ANOVA+TukeyHSD, P < 0.05).(I) to (K)Adult shootmorphology in untransformedCol-0 (I)orCol-0 homozygous forSMXL7pro:SMXL7d53-VENUS (J)orwithSMXL7pro:SMXL7d53,alaEAR-VENUS (K).

Shoot Developmental Regulation by SMXL7 1595

by our data is the apparent subnuclear localization of SMXL7 indistinct bodies or “speckles.” The SL-dependent association ofD14 with SMXL7 involves recruitment of D14 to the speckles, asindicated by a clear FRET signal for the D14-SMXL7 interactionfocused in these sites. In contrast, the association of MAX2 andD14 does not alter their subnuclear localization and occursthroughout the nucleus, suggesting that this interaction mayoccur independently of SMXL7. Nuclear speckles are generallyviewedas storage andassembly areas for splicing factors,whichare associated with transcriptionally active sites (Reddy et al.,2012). This could be interpreted as an association betweenSMXL7and transcriptional regulation; however, evidence for thisis currently lacking, and current models for transcriptional reg-ulation by SMXL7 involve repression rather than activation. Themolecular mode of action of SMXL7 is unclear. SMXL7 is a largeprotein with no obvious homology to transcription factors, yet itcan bind to TPR proteins through its EAR motif (Soundappanet al., 2015; Wang et al., 2015), and so could potentially act asscaffold protein in chromatin assemblies. Further investigation isrequired in this respect, althoughasdiscussed further below, it isclear that the EAR motif is not absolutely required for SMXL7activity.

Strigolactone Triggers SMXL7 Degradation across thePlant Body

The principal components of SL signaling are strongly expressedin vascular-associated tissues in both the shoot and root system(Stirnberg et al., 2007; Chevalier et al., 2014; Soundappan et al.,2015). However, results presented here also suggest that MAX2and D14 are more broadly active than these expression patternssuperficially suggest. For instance, we see rapid turnover ofSMXL7 in all cells of the Arabidopsis root meristem in 35Spro:SMXL7-YFP lines in response to rac-GR24 treatment. This turn-over is demonstrably D14- and MAX2-dependent (Soundappanet al., 2015), but expressionofD14andMAX2 ismuchhigher in thestele than elsewhere (Stirnberg et al., 2007; Chevalier et al., 2014).The weak expression of MAX2 and D14 detected in other roottissues in transcriptomic studies (Brady et al., 2007) is apparentlysufficient tomediate SMXL7 degradation. Theremay also be non-cell-autonomousaccumulationofSLsignalingproteins (Chevalieret al., 2014), but the addition of a strong nuclear localization signalto D14, which may limit its cell-to-cell movement, has apparentlyno effect on its function.

The results presented here show that SL-triggered MAX2-dependent SMXL7 degradation is also detectable in the shoot inpetioles and stems. We have previously shown that SL signaling

regulates the abundance of the PIN1 protein at the basal plasmamembrane of xylem parenchyma cells (Bennett et al., 2006;Crawford et al., 2010; Shinohara et al., 2013). Here, we show thatSMXL7 and PIN1 are expressed in the same vascular cambiumand xylem parenchyma cells in the stem; furthermore, we haveobserveddirect coexpressionofSMXL7andPIN1within the samexylemparenchyma cell. The toolswe have developed in this studywill be useful in dissecting the relationship between SMXL7degradation, PIN1 depletion from the plasmamembrane, and thediverse SL-regulated shoot morphological phenotypes in moredetail.

Plant Tissues Respond to a Wide Range of SMXL7 Doses

Our results suggest that there is broad competence to respond toSMXL7 in the shoot, even in tissues where SMXL7 is not normallyhighly expressed. For instance, overexpression of stabilizedSMXL7 results in dramatic phenotypes, such as defective flowerdevelopment, that do not occur when the same protein isexpressed from itsnativepromoter. It isnotcurrentlyclearwhetherthis is due to off-target effects or because the cellular activitiestargeted by SMXL7 occur outside the normal range of SMXL7expression and are thus ectopically affected when SMXL7 isoverexpressed.Apart from these responses to ectopic expression of SMXL7,

our data suggest that tissuesare sensitive toSMXL7protein levelsover a verywide concentration range that extendsboth above andbelow the typical wild-type range. Expression of the wild-typeSMXL7 protein is unable to take plants out of the wild-typephenotypic range, presumably because of the rapid degradationof the protein by the D14-MAX2 system. This idea is supported bythe very low levels of detectable SMXL7-VENUS in SMXL7pro:SMXL7-VENUS lines in all tissues examined. In contrast, dramaticphenotypes result from expression of SMXL7when it is stabilized,either by expression in a SL-insensitive or SL-deficient mutantbackground, or by deletion of the P-loop of its first NTPase do-main. In these situations, there is a clearSMXL7dose response forthe shoot phenotypes we analyzed. For example, expressingSMXL7pro:SMXL7-VENUS in the max2-1, d14-1, or max1-1backgrounds results in strong phenotypic effects that exceedthose seen in SMXL7pro:SMXL7d53-VENUS. In both cases, thetransgenically expressed SMX7 is stabilized, but max2-1SMXL7pro:SMXL7-VENUS effectively has at least four stabilizedSMXL proteins, SMXL6, SXML8, and endogenous and transgenicSMXL7, while SMXLpro:SMXL7d53-VENUS has only one stabi-lized protein, plus three labile SMXL proteins. Consistent withthis dose sensitivity, the effect of SMXLpro7:SMXL7-VENUS in

Figure 9. (continued).

(L) to (N) Adult shoot morphology in untransformed smxl678 (L) or smxl678 homozygous for SMXL7pro:SMXL7d53-VENUS (M) or with SMXL7pro:SMXL7d53,alaEAR-VENUS (N).(O) to (Q)Stemanatomy in untransformedCol-0 (O) or Col-0 homozygous forSMXL7pro:SMXL7d53-VENUS (P) orSMXL7pro:SMXL7d53,alaEAR-VENUS (Q)plants stained with toluidine blue. Red bar illustrates the depth of the interfascicular cambium layer. Images are representative of multiple independentsamples.(R) to (T)Stemanatomy inuntransformed smxl678 (R)orsmxl678homozygous forSMXL7pro:SMXL7d53-VENUS (S)orSMXL7pro:SMXL7d53,alaEAR-VENUS(T) plants stained with toluidine blue. Red bar illustrates the depth of the interfascicular cambium layer.

1596 The Plant Cell

smxl678max2 ismuchweaker than inmax2, because in the formerbackground, theSMXL7 transgene is the only copy of aSMXL6/7/8-type gene present. One interpretation of these data is thatSMXL6 and SMXL8 act in a qualitatively similar manner to SMXL7in the regulation of development, although they may makequantitatively smaller contributions, consistent with their re-spective loss-of-function phenotypes (Soundappan et al., 2015).Certainly, overexpression of stabilized SMXL6 generates at leastsome of the same phenotypic effects as we show for SMXL7(Wang et al., 2015). Thus, the opposite end of this dose–responserange occurs with the complete loss of SMXL6, SMXL7, andSMXL8, which also causes dramatic phenotypes that are inseveral cases quantitatively opposite to the effect of increasingSMXL7 activity.

Altering SMXL7 activity can thus be conceived as shiftingdevelopment along various phenotypic continua, as illustrated inFigure 10. These phenotypes can be categorized into four broadclasses. In class I, branching angle appears to be sensitiveacross the full range of SMXL levels, with smxl678 mutantshaving thewidest branch angles, while very high accumulation ofSMXLs results in the lowest branchangles. In class II, bladewidthis dose-sensitive across the lower end of the range, but theresponse hits a maximum, with very high SMXL levels unable toincreasebladewidth further. In class III, branchnumbers, primarystem height, petiole length, and blade length are dose-sensitiveat the higher endof theSMXL range.Reductions inSMXL level donot strongly affect these phenotypes, but effects are pro-gressively stronger with increasing SMXL levels above those ofwild-type plants. For shoot branching, maximum levels arereached at lower SMXL levels than, for example, for petiolelength, but this is because all nodes have an actively growingbranch, so further increases in primary branching levels are notpossible. Phenotypes only observed at very high SMXL levels,such as leaf curling, can also be included in this category. Lastly,in class IV, stemwidth and interfascicular cambiumdevelopmentshow a bell-shaped relationship with SMXL7 dose, with both lowand high doses resulting in reductions compared with the wildtype.

There are many possible explanations for the different dose–response curves for these phenotypes. It could be that all thephenotypes are mediated by the same targets downstream ofSMXL7, but the resulting morphological effects are differentlybufferedagainst,or sensitive to, variations in theactivityof targets.For example, it is possible that changes in branch angle andchanges in plant height are both caused by SMXL7-mediatedchanges in PIN1 accumulation and, therefore, changes in auxindistribution, but that branch angle is more sensitive to thesechanges than height. Alternatively, there may be multiple down-stream SMXL7 targets that are themselves differentially sensitiveto SMXL7 levels. Our analysis of the EAR sensitivity of thesephenotypes provides some insight on these alternatives.

Signaling Downstream of SMXL7: One or Many Targets?

SLs have very clearly defined effects on multiple shoot de-velopmental traits. An intriguing question is whether these traitsare all regulated through the same immediate target of SMXLs orwhether there are multiple SMXL targets. There is good evidence

that both quantitatively and qualitatively, most SL signaling in theshoot involves targeted degradation of SMXL6, SMXL7, andSMXL8 (Soundappan et al., 2015; Wang et al., 2015). However,events downstream of SMXL7 are currently much less clear.Our analysis of the role of the EAR motif in different shoot

phenotypes reveals clear differential sensitivity to the loss of theEAR motif among SL-regulated shoot phenotypes. For example,SMXL7 effects on leafmorphology and branch angle are relativelyunaffected by removal of the EARmotif. In contrast, the effects onbranch number and plant height are particularly sensitive to EARmutation. These observations suggest the attractive hypothesisthat branching and height are regulated by an EAR-dependentSMXL7 interaction, such as a transcriptional response mediated

Figure 10. SMXL7 Activity and EAR Dependency.

Summary diagram illustrating the effects of modulating SMXL7 activity ondifferent shoot architectural traits. The smxl678 triple mutant representsa very low level of SMXL7-like activity relative to the wild type, whilestabilizedSMXL7 representsmuchhigheractivity than thewild type, furtherenhanced when SMXL7 is also overexpressed (orange triangle). Differentphenotypes show different sensitivity to modulation of SMXL7 activity.Class I traits, for instance branch angle, are sensitive to SMXL7 across thewhole range of activity. Class II traits, for instance blade width, are es-pecially sensitive to the lower range of SMX7 activity, but not to the higherrange. Class III traits, including shoot branching and leaf blade length andheight, are especially sensitive to the higher range of SMXL7 activity, butnot to the lower end of the range. Class IV traits do not show a linear re-lationship with SMXL7 activity. Phenotypic effects that occur at the higherrange of SMXL7 activity are dependent on the presence of the EAR motif,but those at the lower end of the range do not require the EARmotif (purpletriangle).

Shoot Developmental Regulation by SMXL7 1597

by TPL (or homologs). In contrast, SMXL7 effects on leaf mor-phology and branch angle could be mediated by interaction be-tween SMLX7 and a partner other than TPL, which could result innontranscriptional responses. However, despite differentialsensitivity to EAR mutation, the EAR-mutated SMXL7 variantscan still affect all the phenotypes tested to at least some extent.Furthermore, there is a strong correlation between those phe-notypes that can be rescued by EAR-mutatedSMXL7 and thosephenotypes that are sensitive to low SMXL doses (Figure 10).For instance, the twomostEAR-dependent phenotypes, branchnumber and primary stem height, are also the least affected bycomplete loss of SMXL6, SMXL7, and SMXL8. Conversely, theleast EAR-dependent phenotypes, such as leaf width andbranch angle, are sensitive to variation in SMXL levels at the lowend of the range. This suggests that SMXL7 with a mutant EARmotif simply has low activity, and so is unable to induce phe-notypes that depend on high levels of SMXL7 activity, such asincreasedshoot branchnumber, but they canaffect phenotypesthat can respond to low levels of SMXL7 activity, such as branchangle.

In this context, petiole length provides an interesting exceptionto the pattern described above. Like shoot branch number andplant height, petiole length is wild-type in the smxl678 back-ground, but increasing levels of SMXL7 activity above wild-typeresult in progressively shorter petioles. However, unlike shootbranching and plant height, the short petiole length phenotypesassociated with high SMXL7 activity in themax2 background arealmost completely insensitive to EAR mutation. This suggeststhat not all the variation in the sensitivity of shoot phenotypes toEAR deletion can be explained simply by quantitative variation inthe requirement for a single unified SMXL7 activity. It also arguesagainst the possibility that deletion of the EAR motif has somerather nonspecific effect on SMXL7 abundance or activity.Rather, it supports the hypothesis that SMXL7 might regulatedifferent downstream processes through different immediatetargets. In this respect, SMXL7 and homologs can be consideredas conceptually similar to DELLA proteins, which can act throughvarious targets to regulate growth (Hauvermale et al., 2012).Indeed, this is a useful comparison in more general contexts.Although DELLAs were originally discovered as targets for gib-berellin signaling, it is nowclear that they act as growth regulatoryhubs, and it is perhaps more useful to conceptualize them as theendpointof theGAsignalingpathway, rather thanan intermediaryin it. In a similar way, the emerging picture for SMXL7 and ho-mologs is that theseproteins have functions that are independentof SL. This might explain why both the smxl678 triple mutant andlines with stabilized SMXL7 have reduced interfascicular cam-bium development and stem width. It is possible that cambiumdevelopment requires SMXL7 expression in tissues in which SLsignaling does not occur but is inhibited by SMXL7 activity intissues where there is SL signaling.

While the core signaling mechanism for SL is increasingly wellcharacterized, we are only just beginning to understand thedownstream pathways that lead to the diverse effects of SL onplant development. A clear priority will be to test the ability ofSMXL7 variants to regulate specific immediate downstreamtargets such as PIN1 or potential transcriptional targets in theshoot.

METHODS

Plant Materials

The Arabidopsis thaliana max1-1,max2-1 (Stirnberg et al., 2002),max4-5,PIN1:GUS (Bennett et al., 2006), d14-1 (Waters et al., 2012), smxl6-4smxl7-3 smxl8-1 max2-1, 35Spro:SMXL7-YFP, 35Spro:SMXL7DP-loop-YFP(Soundappan et al., 2015), and PIN1pro:PIN1-GFP (Xu et al., 2006) lineshave been described previously. Where new genotypes were assembledby crossing relevant existing genotypes, the required homozygous lineswere identified using visible, fluorescent, or selectable markers or usingPCR genotyping.

Cloning

Supplemental Table 2 shows all of the constructs made in this study. TheSMXL7, SMXL6, D14, and MAX2 coding sequences were amplified fromcDNA and cloned into pDONR 221 using Gateway reactions using theprimers listed in Supplemental Table 3. Variants of SMXL7 and D14 werecreated from pENTR 221 SMXL7/D14 with the Q5 Site-Directed Muta-genesis Kit (New England Biolabs), as described in Supplemental Table 1.TheSMXL7,D14, andMAX2 promoters were amplified fromgenomic DNAand cloned into pDONR 1R4 using Gateway reactions, using the primerslisted in Supplemental Table 3. CERULEAN and CITRINE tags were am-plified from non-Gateway vectors and cloned in pDONR 2R3, using theprimers listed inSupplemental Table 3. Existing entry vectorswere used for35Spro, GFP, CFP, YFP, VENUS, N7-VENUS (=NLS),mCherry, and GUS.Final assembly of constructs was performed using multisite Gatewayreactions. Binary destination vectors for these reactions are listed inSupplemental Table 2.

MAX2:MAX2-GFP was assembled using conventional cloning.pBI101.3 was digested with XmaI/SacI to drop out GUS, which was re-placed with GFP. The MAX2 promoter was amplified and cloned into thepBI101.3 GFP vector via SalI/BamHI digest. The MAX2 cDNA was thenamplified and cloned into the pBI101.3 MAX2:GFP vector via a BamHI/XmaI digest. Primers are listed in Supplemental Table 3.

To express constructs transiently in Nicotiana benthamiana epidermalcells,Agrobacterium tumefaciens strain EHA105carrying the constructs ofinterest, grown toanOD600of0.5,was infiltrated into leavesof4- to5-week-old plants (Bozkurt et al., 2011). Constructs were stably transformed intovarious Arabidopsis genetic backgrounds using the Agrobacterium floraldip method (Clough and Bent, 1998).

Plant Growth Conditions

Mature Arabidopsis plants for analysis were grown on Levington’s F2compost, under a standard 16-h/8-h light/dark cycle (22°C/18°C) incontrolled environment rooms with light provided by white fluorescenttubes (intensity ;150 mmol m22 s21). Mature N. benthamiana plants fortransient transformation were grown on Levington’s F2 compost, undera standard 16-h/8-h light/dark cycle in a greenhouse for 4 to 5 weeks. Foraxenic growth of seedlings, seeds were sterilized and stratified at 4°C forseveral days. Seedlings were grown on Arabidopsis thaliana salts (ATS)medium (Wilson et al., 1990) with 1% sucrose, solidified with 0.8% agar.Plates were oriented vertically in growth chambers with a 16-h/8-hlight/dark cycle (22°C/18°C), with light provided bywhite fluorescent tubes(intensity ;150 mmol m22 s21).

Leaf Measurements

The 7th leaf of each plant wasmarked with indelible marker at;4 weeksafter germination. These leaves were provisionally measured at 35 dafter germination and then measured again at 37 d after germination toconfirm that growth of these leaves was arrested. The length of the

1598 The Plant Cell

petiole and the blade, and the maximum width of the blade weremeasured.

Branching, Height, and Stem Measurements

Branch angle was measured as the angle formed between branch andprimary stem at the axil by imaging branches, and the angles in the imageswere quantified using ImageJ (Schneider et al., 2012). Plant height and thenumber of primary cauline and rosette inflorescences were measured atglobal proliferative arrest (;7weeks after germination). The diameter of thebasal internode of the primary inflorescence was also measured at globalproliferative arrest using a Keyence digital microscope.

Imaging of Reporter Fusions

Fluorescent reporter proteins in leaves and roots were imaged usingconfocal laser scanningmicroscopywith a Zeiss LSM780 imaging system.Excitation was performed using a 514-nm laser for YFP and VENUS, anda 405-nm laser for CFP and CER. For Arabidopsis leaves, samples weremounted on glass slides with or without 5 mMGR24 in liquid ATS solution,then imaged with a 203 lens. For N. benthamiana, leaves were infiltratedwith Agrobacterium carrying the relevant reporter line (see above), alongwith 5mM rac-GR24 (LeadGenLabs) ormocksolution. After 2 h, leaf pieceswere immobilized on Petri dishes with high-strength adhesive and imagedusing a 203 water-immersion lens. For Arabidopsis roots, 3- to 5-d-oldseedlings were mounted on slides with 10 mM propidium iodide (65 mMrac-GR24) and then imaged using a 203 lens.

For imaging of fluorescent reporters in stems, handsectionsweremadethrough the vascular bundles of basal internodal stem segments of6-week-old plants, and the slices were then embedded in agar plates. Thesamples were then covered with ATS medium (65 mMGR24) and imagedby confocal laser scanning microscopy using a Zeiss LSM700 imagingsystemwith 203water immersion lenses. Excitation was performed usinga 514-nm laser for YFP, 488-nm laser for GFP, and 405-nm laser for CFPand CER. PIN1-GFP quantification was performed on nonsaturated im-ages, usingZeissZENsoftware. Fluorescence intensity in theGFPchannelwas measured in four or five basal plasma membranes per sample, in atleast eight independent samples.

Staining for GUSactivity was performed by incubating tissue in stainingsolution (0.1 M Na2HPO4, 5 mM K3[Fe(CN)6], 5 mM K4[Fe(CN)6], 0.05%Triton-X-100, and 0.5 mg/mL X-gluc [5-bromo-4-chloro-3-indolyl-b-D-glucuronide]) overnight at 37°C. The tissue was the destained in 70%ethanol and imaged using a Keyence digital microscope.

FRET Detection

To detect the interaction between SMXL7, D14, and MAX2, FRET wasmeasured by FLIM using a Leica TCS SP8 confocal laser scanning mi-croscopeequippedwitha time-correlated single-photoncountingmodule.Apulseddiode laser (405nm) tunedat 20MHzwasused toexcite thedonoralone (CFP) and the donor in the presence of acceptor (CFP +YFP). Theemission from 450 to 490 nm was collected by a HyD detector in 256 3

256 pixel format. The acquired FLIM decay curve from regions of interestwas fitted by two-exponential theoretical models using SymPhoTimesoftware. The mean CFP lifetimes were calculated as the mean values ofthe fit function and analyzed using SymPhoTime software. At least 10 cellswere tested for each FRET experiment.

The heterodimerization of SMXL7-YFP/SMXL6-CFP was tested inFRET assays by spectral imaging using a Zeiss LSM 780 confocal mi-croscope with a 203 water immersion lens. To detect the fluorescence ofthe CFP donor and the YFP acceptor, excitation with a 405-nm argon laserlinewasperformed, and lambda serieswere collectedwith the ZeissMETAdetector.The lambdastackof imageswascollected inanemission rangeof415 to 620 nm with a 24-channel (9-nm interval) detector. The regions of

interest in the nucleus were selected randomly. The normalized fluores-cence intensity in each channel from different regions of interest wascollected and processed using Zeiss LSM image Examiner software tocreate the spectral profiles.

Histology

Staining for GUS activity was performed as described above. Resinsectionswere thenmadeofGUSstained tissuesbyfixing inFAAsolutionand dehydrating through an ethanol series prior to infiltration andsubsequent embedding with embedding media (Leica HistoResinStandard Kit). The tissues were sliced into 7-mm sections with a LeicaRM2255 microtome. For analysis of cambial activity, hand sectionsthrough the basal internode of the primary inflorescence were stainedwith toluidine blue O. Images were taken with a Keyence digitalmicroscope.

Auxin Transport Assays

Auxin transport assays were modified from those described by Crawfordet al. (2010). The 18-mm stem segments from basal internodes were ex-cised, and the apical end was submerged in 30 mL ATS without sucrose(pH = 5.6), containing 1mM14C-IAA (American Radiolabeled Chemicals) inanEppendorf tube.Stemswere incubated for 6h, and thebasal 5mmof thesegmentwas thenexcised,placed in200mLscintillation liquid, andshakenovernight at 400 rpm prior to scintillation counting.

Accession Numbers

Sequence data from this article can be found in TAIR under the fol-lowing accession numbers:D14 (At3g03990),MAX2 (At2g42620),PIN1(At1g73590), SMXL6 (At1g07200), SMXL7 (At2g29970), and SMXL8(At2g40130).

Supplemental Data

Supplemental Figure 1. SL signaling components function in thenucleus.

Supplemental Figure 2. SMXL7 physically interacts with D14 ina SL-dependent manner.

Supplemental Figure 3. Effect of SMXL7 dose on development.

Supplemental Figure 4. Effect of SMXL7 stabilization on development.

Supplemental Figure 5. Effects of SMXL7 are partially EAR-independent.

Supplemental Figure 6. Effects of SMXL7 are partially EAR-independent.

Supplemental Figure 7. Loss of the EAR motif counteracts SMXL7stabilization.

Supplemental Figure 8. Loss of the EAR motif counteracts SMXL7stabilization.

Supplemental Table 1. Constructs made in this study.

Supplemental Table 2. Cloning primers used in this study.

Supplemental Table 3. Protein variants made in this study.

ACKNOWLEDGMENTS

This work was funded by the European Research Council (No. 294514–EnCoDe), by the Gatsby Foundation (GAT3272C), and by the ChineseGovernment Scholarship PhD Program (Sichuan Agriculture University) toY.L. We thank David Nelson for very helpful discussions.

Shoot Developmental Regulation by SMXL7 1599

AUTHOR CONTRIBUTIONS

T.B., P.L., and O.L. designed the research. Y.L., S.W., and T.B. performedresearch.Y.L., S.W., andT.B. analyzeddata. T.B. andO.L.wrote thearticle.

Received April 11, 2016; revised June 6, 2016; accepted June 14, 2016;published June 17, 2016.

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Shoot Developmental Regulation by SMXL7 1601

DOI 10.1105/tpc.16.00286; originally published online June 17, 2016; 2016;28;1581-1601Plant Cell

Yueyang Liang, Sally Ward, Ping Li, Tom Bennett and Ottoline LeyserPartially EAR Motif-Independent Mechanisms

SMAX1-LIKE7 Signals from the Nucleus to Regulate Shoot Development in Arabidopsis via

 This information is current as of August 18, 2020

 

Supplemental Data

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