Evolutionarily conserved repressive activity of WOXproteins mediates leaf blade outgrowth and floralorgan development in plantsHao Lina,1, Lifang Niua,1, Neil A. McHaleb, Masaru Ohme-Takagic, Kirankumar S. Mysored, and Million Tadegea,2

aDepartment of Plant and Soil Sciences, Institute for Agricultural Biosciences, Oklahoma State University, Ardmore, OK 73401; bDepartment of Biochemistryand Genetics, Connecticut Agricultural Experiment Station, New Haven, CT 06504; cBioproduction Research Institute, National Institute of Advanced IndustrialScience and Technology, Tsukuba 305-8562, Japan; and dPlant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73401

Edited by Sarah Hake, University of California, Berkeley, CA, and approved November 15, 2012 (received for review September 4, 2012)

The WUSCHEL related homeobox (WOX) genes play key roles instem cell maintenance, embryonic patterning, and lateral organdevelopment. WOX genes have been categorized into threeclades—ancient, intermediate, and modern/WUS—based on phy-logenetic analysis, but a functional basis for this classification hasnot been established. Using the classical bladeless lam1 mutant ofNicotiana sylvestris as a genetic tool, we examined the function oftheMedicago truncatula WOX gene, STENOFOLIA (STF), in control-ling leaf blade outgrowth. STF and LAM1 are functional orthologs.We found that the introduction of mutations into the WUS-box ofSTF (STFm1) reduces its ability to complement the lam1 mutant.Fusion of an exogenous repressor domain to STFm1 restores com-plementation, whereas fusion of an exogenous activator domainto STFm1 enhances the narrow leaf phenotype. These results in-dicate that transcriptional repressor activity mediated by the WUS-box of STF acts to promote blade outgrowth. With the exceptionof WOX7, the WUS-box is conserved in the modern clade WOXgenes, but is not found in members of the intermediate or ancientclades. Consistent with this, all members of the modern clade ex-cept WOX7 can complement the lam1 mutant when expressedusing the STF promoter, but members of the intermediate andancient clades cannot. Furthermore, we found that fusion of eitherthe WUS-box or an exogenous repressor domain to WOX7 or tomembers of intermediate and ancient WOX clades results ina gain-of-function ability to complement lam1 blade outgrowth.These results suggest that modern clade WOX genes have evolvedfor repressor activity through acquisition of the WUS-box.

The WUSCHEL related homeobox (WOX) genes form a plant-specific family of the eukaryotic homeobox transcription

factor superfamily (1, 2). In Arabidopsis, the WOX family con-sists of 15 members, including the founding member WUSCHEL(WUS) and WOX1–WOX14 (3), which are involved in the regu-lation of key developmental processes, including stem cellmaintenance in shoot and root meristems, embryo apical-basalpolarity patterning, and development of lateral organs (1, 4–7).Based on phylogenetic analysis and their distribution in the plantkingdom, WOX genes have been classified into three clades:modern/WUS (found in seed plants), intermediate (found invascular plants including lycophytes), and ancient (found invascular and nonvascular plants, including mosses and greenalgae) (1, 8). WOX genes have been proposed to have a commonmechanism of action, as demonstrated by complementation of thewox5 and pressed flower (prs/wox3) mutant phenotypes byWUS, aswell as by the partial complementation of prs with WOX4 (5, 9,10). However, the extent to which the functions of WOX familymembers are conserved remains unclear.The critical requirement of WOX genes for the development

of lateral organs, including leaves and flowers, has become in-creasingly apparent from the identification of several mutantphenotypes in angiosperms. In maize, the narrowsheath1 and 2(ns1/ns2) double mutant exhibits a leaf margin deletion pheno-type (11, 12). NS1 and NS2 are homologs of the Arabidopsis genePRS/WOX3 involved in the development of lateral sepals and

stipules (11, 13). Recent work has identified narrow leaf mutantsin the WOX1 homologs maewest (maw) in Petunia x hybrida (14),stenofolia (stf) in Medicago truncatula (15), lam1 in Nicotianasylvestris (15), and wox1/prs double mutant in Arabidopsis thaliana(14, 16). These mutants produce narrow leaf blades owing todefects in lateral cell proliferation affecting leaf width, whereasleaf length is virtually unaffected. In leaf primordia, STF/MAW/WOX1 and PRS/WOX3 are expressed at the adaxial–abaxialboundary layer both in the middle mesophyll and at the leafmargin (14–16), indicating that this region may be important forblade outgrowth. Arabidopsis WOX1 and PRS are proposed tocoordinate adaxial/abaxial patterning through interaction withadaxial and abaxial domain-specific polarity factors (16). How-ever, the details of this interaction and its quantitative significanceto blade outgrowth remain to be established.In M. truncatula and N. sylvestris, loss-of-function mutations

in STF and LAM1, respectively, are sufficient to arrest lateralgrowth, leading to severe defects in lamina outgrowth, veinpatterning, petal lobe expansion, and ovary fusion (15). Thelamina and flower phenotypes of lam1 mutants can be com-plemented by Arabidopsis WUS when expressed under the con-trol of the STF promoter, suggesting that a WUS-like function isrequired for cell proliferation in determinate lateral organs (15,17). We conducted the present study to determine the mecha-nism of STF/LAM1 function and establish the molecular basis ofWUS-mediated complementation of mutant phenotypes in lat-eral organs. We found that the regulation of leaf blade out-growth by STF is dependent on the WUS-box, which is essentialfor transcriptional repressor activity. In addition, we found thatall members of the Arabidopsis modern clade WOX genes thatcontain a WUS-box can substitute for STF/LAM1, whereas theintermediate and ancient clades cannot, owing to a lack of therepressor WUS-box motif. Our findings establish the minimumrequirement for the functional conservation of WOX genes, andprovide mechanistic support to the phylogenetic classification ofWOX family proteins.

ResultsSTF Regulates Leaf Blade Outgrowth and Floral Organ Developmentin the lam1 Mutant Background in an Expression Level-DependentManner. In the classical bladeless lam1 mutant of N. sylvestris,leaf blades are reduced to vestigial strips that lack mesophylldifferentiation, and mutant plants are locked in a vegetativestage without apparent stem elongation (18). The narrow

Author contributions: H.L., L.N., and M.T. designed research; H.L. and L.N. performedresearch; N.A.M., M.O.-T., and K.S.M. contributed new reagents/analytic tools; H.L., L.N.,and M.T. analyzed data; and H.L., L.N., and M.T. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1H.L. and L.N. contributed equally to this work.2To whom correspondence should be addressed. E-mail: million.tadege@okstate.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1215376110/-/DCSupplemental.

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phenotype is recognizable in cotyledons, but severe inhibition oflamina outgrowth becomes apparent in the first true leaf (Fig. 1A–H). Rarely, lam1 plants can spontaneously bolt and initiateflowers under high temperature, but they form narrow petalswith partially fused corollas and open ovaries, leading to femalesterility (Fig. S1). Both the leaf blade and flower phenotypes oflam1 can be complemented by the genomic fragment of STFgene, which includes 2.6 kb upstream of the translation start and∼300 bp downstream of the translation stop (15). The severelam1 phenotype provides an ideal system for investigating thedegree to which other factors can contribute to the developmentof leaf blade outgrowth.To test whether the coding region of STF confers identical

function to its genomic fragment, we transformed the lam1mutant with a gene construct consisting of the same 2.6-kbpromoter region driving the STF CDS. Three phenotypic classeswere observed in transgenic plants: well complemented (7 of 21plants), with fully formed leaf blades and fertile flowers (Fig. 1 I,L, and O); partially complemented (10 of 21 plants), with nar-rowed leaves and formation of capsules but most without seeds(Fig. 1 J, M, and P); and poorly complemented (4 of 21 plants),with little blade development and flowers that are female-sterileand make no capsules or seeds (Fig. 1 K, N, and Q). Real-timePCR analysis of STF expression in these three classes showedthat the well-complemented classes have the highest transcriptlevels, whereas the partially complemented classes have inter-mediate levels, and the poorly complemented classes have the

lowest levels (Fig. 1R). These results suggest that STF regulationof blade outgrowth and floral organ development in the lam1background is dependent on the level of STF expression.

STF Acts as a Transcriptional Repressor in Regulating Leaf BladeOutgrowth and Floral Organ Development. To determine whetherSTF acts as a transcriptional activator or repressor, we examinedits transcriptional activity by luciferase transient expressionassays in Arabidopsis protoplasts. The effector plasmid containedSTF fused to the GAL4 DNA-binding domain, and the reporterplasmid contained the luciferase (LUC) gene fused to a 5×GAL4-binding site (Fig. 2A). Bioluminescence measurementsrevealed a more than twofold reduction in luciferase activity inthe presence of STF protein, indicating strong repressive activity(Fig. 2C). Mutation of two amino acids (leucine-to-alanine ex-change) in the WUS-box of STF (STFm1), similar to thatreported by Ikeda et al. (19), partially relieved this repression(Fig. 2 A–C), suggesting that STF is a transcriptional repressorand that its WUS-box is at least partially responsible for medi-ating the repressive activity.To confirm the role of the WUS-box in leaf blade outgrowth,

we performed complementation analysis in lam1 mutant plants.In these experiments, STF and STFm1 were expressed under theSTF promoter (Fig. 2D). Compared with STF::STF transgenicplants, mutation in WUS-box (STF::STFm1) diminished theability of STF to complement the lam1mutant (Fig. 2 E, F, I, andJ). Most of the STF::STFm1-complemented plants can bolt andinitiate flowers, but poorly complement the leaf and floral phe-notypes of lam1, indicating that the WUS-box is essential to thefunction of STF in both leaf blade outgrowth and floral organdevelopment. To further ascertain the requirement of transcrip-tional repression activity in STF function, we fused the exogenousEAR motif repression domain SRDX (20) to the N terminus ofSTFm1 (SRDX-STFm1) and transformed this construct into lam1(Fig. 2D). STF::SRDX-STFm1 transgenic plants exhibited fullcomplementation of lam1 that was indistinguishable from WT orthe well-complemented class of STF-complemented lam1 plants(Fig. 2 G and K). In contrast, fusion of the activation domain ofthe herpes simplex virus VP16 protein (21) to STFm1 (STFm1-VP16) resulted in enhanced narrow leaf phenotype comparedwith the STF::STFm1 transgenic plants (Fig. 2 D, H, and L).Taken together, these results suggest that STF acts as a tran-scriptional repressor primarily through its WUS-box in organizingcell proliferation during leaf blade morphogenesis, as well asduring petal and carpel development.

Conservation of WOX Proteins in Regulating Blade Outgrowth. Wehave previously reported that the Arabidopsis WUS under controlof the STF promoter can complement the lam1 phenotypes (15),and that STF::WUS can also rescue the M. truncatula stf mutant(Fig. S2). Similarly, other transgenic analyses have found thatWUS can substitute for WOX5 function in root meristem main-tenance (5) and for PRS function in lateral floral organ devel-opment (10) when expressed under the control of appropriatepromoters. These complementation data prompted us to testwhether other WOX genes can substitute for the STF/LAM1function in leaf blade outgrowth, and to examine whether thesame repressive mechanism is used by other WOX family mem-bers to complement lam1. Accordingly, we expressed 11 of the 15Arabidopsis WOX genes, including all of the modern/WUS clademembers (WUS, WOX1–WOX7), and representatives of the in-termediate (WOX9,WOX11) and ancient (WOX13) clades, undercontrol of the STF promoter in lam1 plants. We expressed STF::GUS in lam1 as the control. Our results revealed that althoughcomplementation by WOX5 is weak, all members of the modernWOX clade exceptWOX7 can complement lam1 (Fig. 3 A–H, Fig.S3A–H, and Table S1), whereas members of the intermediate andancientWOX clades cannot (Fig. 3 I–K, Fig. S3 I–K, and Table S1).Interestingly, STF::WOX9 transgenic plants exhibited an enhancedlam1 blade phenotype with thinner and shorter leaf blades com-pared with lam1 (Fig. 3 I and L). These results indicate that

Fig. 1. Phenotypes of lam1 mutant and expression level-dependent com-plementation by STF. (A–H) Phenotypes of tobacco WT and lam1 plants atdifferent developmental stages. (A and B) One-wk-old WT and lam1 seed-lings showing cotyledons and the emerging first leaves. (Scale bars: 1 mm.) (Cand D) Cross-sections of first leaves from One-wk-old WT and lam1 plants.(Scale bars: 100 μm.) (E and F) Six-wk-old vegetative-stage WT and lam1plants. (Scale bars: 1 cm.) (G and H) Flowering-stage WT and lam1 plants.(Scale bars: 10 cm.) (I–K) Leaf phenotypes of different STF::STF-com-plemented lam1 lines. (Scale bars: 1 cm.) (L–N) Petal phenotypes of trans-genic lines corresponding to those in I–K. (Scale bars: 1 cm.) (O–Q) Femalefloral organs of transgenic lines corresponding to those in I–K. (Scale bars:1 cm.) (R) Quantitative RT-PCR showing expression levels of STF in transgeniclines corresponding to I–K, normalized to the expression of Ubiquitin. Errorbars represent mean ± SD (n = 3). *P < 0.05; **P < 0.01 (one-way ANOVA,Dunnett’s test).

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functional conservation among WOX members with regard toblade outgrowth extends only to members of the modern/WUSclade, suggesting that the ability to confer blade outgrowth is arecent evolutionary acquisition by a specific group ofWOX genes.We next tested whether the ability or inability of WOX mem-

bers to complement lam1 is associated with the presence or ab-sence of repressive activity by introducing mutations in the WUS-box and fusing exogenous repressor and activator domains (Fig. 3M and N). Analogous to STFm1, introduction of WUS (WUSm1)mutations into the WUS-box reduced its ability to complementlam1 (Fig. 3O), indicating that the WUS-box is required forWUS to act as a functional substitute of STF. Consistent with thisinterpretation, fusion of the repressor domain SRDX to WUSm1(SRDX-WUSm1) restored complementation of lam1 (Fig. 3P),whereas fusion of the VP16 activator domain toWUSm1 (WUSm1-VP16) worsened the WUSm1 phenotype (Fig. 3Q). In fact, STF::WUSm1-VP16 transgenic plants showed a more severe phenotypecompared with the original lam1 mutant, forming a more radialblade with added proximodistal defects similar to leaves of STF::WOX9-expressing lam1 transgenic plants. Microscopic examina-tion of transverse sections through the leaf blades also confirmedthat the vestigial blade strips in lam1 were lost in leaves of both

WUSm1-VP16– andWOX9- expressing lam1 plants, and the bladesbecame completely radialized in both cases (Fig. 3 R–T). Thesefindings suggest two points: (i) Transcriptional repressive activity iscritical for blade outgrowth, and by analogy, all of themodernWOXclade members except WOX7 must have repressive activity, butproteins of the intermediate and ancient clades, which lack theWUS-box, do not; and (ii) transcriptional activation function in theSTF expression domain is antagonistic to blade outgrowth, andWOX9 may have an activator role that counterbalances re-pression to modulate growth in both mediolateral and prox-imodistal planes during leaf morphogenesis.To test these assumptions directly, we examined the transcrip-

tional activity of WUS, WOX7, WOX9, and WOX13 using lucif-erase expression assays and domain-swap lam1 complementationexperiments. Consistent with a previous report (19) and ourcomplementation results, WUS repressed luciferase activity bymore than twofold, whereas luciferase activity in WOX7-, WOX9-,and WOX13-expressing protoplasts was somewhat stronger com-pared with the control reporter plasmid (Fig. 4C). WOX9 exhibi-ted the greatest activation activity among these three, and mightaccount for the enhanced narrow leaf phenotype and proximodistaldefects in WOX9-expressing lam1 plants. WOX7 is the sole mem-ber of the modern WOX clade that failed to complement lam1(Fig. 3H), likely owing to the absence of repressive activity.

WUS-Box Has Evolutionary Significance in the Function of WOXProteins. Phylogenetic analysis indicated that the WOX familyhomeobox proteins can be divided into three clades based on thetime of their first appearance in the plant kingdom: ancient, in-termediate, and modern/WUS (1, 8). A close examination of thehomeodomain and WUS-box amino acid sequences of MedicagoSTF and the 15 Arabidopsis WOX proteins revealed that, in ad-dition to sequence variations in the homeodomain regions, thereis a clear divergence in the organization of the WUS-box re-pressor domain (Fig. 4A). The core WUS-box motif, TLXLFP, isinvariably conserved in the modern/WUS clade ofWOX proteins,with the exception of WOX7. However, the initial TL sequence inthis core motif is absent, and the subsequent LFP element isvariable in both the intermediate and ancient clade WOX pro-teins (Fig. 4 A and B). These observations are in agreement withour hypothesis that WOX function in leaf blade outgrowth islikely conferred by the repressive activity of the WUS-box. Al-though the homeodomain sequence of WOX7 is more similar tothat of the modern clade than to that of the intermediate andancient clades (Fig. 4 A and B), the analyzed WOX7 sequence,based on the National Center for Biotechnology Information andArabidopsis Resource Center gene model AT5G05770 and threeESTs from GenBank, encodes a truncated 122-aa protein lackingthe 3′ region including the WUS-box. These observations are inagreement with our hypothesis that WOX function in leaf bladeoutgrowth is achieved by the repressive activity of the WUS-box,and suggest that the WUS-box was acquired relatively late in theevolution of the WOX gene family.To determine whether repression activity and leaf blade out-

growth regulatory function can be conferred to activator proteinsby domain swapping, we fused the SRDX or the WUS-box toWOX7, WOX9, and WOX13 (Fig. 4D) and examined lam1complementation in transgenic plants. Both SRDX and WUS-box fusion constructs rescued lam1 plants in all cases, includingthe strongest activator WOX9, although the WUS-box appearedto be a weaker repressor than SRDX (Fig. 4 E–K and Fig. S4).The conversion of the strong activator protein WOX9 intoa repressor simply by fusion with a repressor domain suggeststhat repression is dominant over activation, which is consistentwith acquisition of the WUS-box later in evolution. Taken to-gether, these results demonstrate a conserved repression mech-anism that plays a central role in organizing cell proliferation formeristem maintenance and lateral organ development conferredon the evolutionarily dynamic WOX gene family by the acquisi-tion of one or more transcriptional repressor domains.

Fig. 2. STF acts mainly as a repressor in blade outgrowth. (A) Constructsused in transient expression assay. (B) Amino acid sequence of the WUS-boxin STF and mutations introduced into the WUS box (STFm1), indicated byframes. (C) Relative luciferase activities using STF or STFm1 as effectorcompared with GAL4-DB control. Error bars indicate SD (n = 3). **P < 0.01(one-way ANOVA, Dunnett’s test). (D) Constructs used for complementationof lam1mutant. (E–L) Transgenic lam1 plants complemented with STF::STF (Eand I), STF::STFm1 (F and J), STF:: SRDX-STFm1 (G and K), and STF::STFm1-VP16 (H and L). (Scale bars: 5 cm.)

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DiscussionThe M. truncatula WOX gene STF, along with the orthologousLAM1 in N. sylvestris, MAW in petunia, and WOX1 in Arabi-dopsis, are key regulators of the lateral outgrowth of leaf bladesand floral organs. These genes play important roles in deter-mining the broad appearance of dicot leaves (14–16). STFmodulates blade outgrowth by promoting cell proliferation at theadaxial–abaxial junction of leaf primordia (15), but the mecha-nism of this action is unknown. Here we demonstrate thattranscriptional repressive activity conferred by the WUS-box ofthe STF protein underlies this mechanism. Interestingly, theWUS-box of WUS, the founding member of the WOX family(22), has been identified as a repressor domain required forWUSfunction in shoot apical meristem maintenance (19). This sug-gests the existence of a conserved meristem-like function indeterminate lateral organ primordia orchestrated by the sameprotein motif of the WOX gene family. In fact, the WUS geneitself can substitute for STF function if expressed in the correctdomain, as demonstrated by complementation of the lam1 andstf mutants in N. sylvestris and M. truncatula, respectively. TheWUS-box is a conserved domain with a core TLXLFP motiffound near the C terminus region of STF and WUS clade pro-teins (3). Our site-directed mutagenesis analysis in this motifindicated that the two leucine residues in “LXL” are critical forrepressive activity (Fig. 2 B, C, F, and J), but that the mutatedSTF protein in these residues, STFm1, retains some residualrepressive activity that can be abolished by fusing it to the VP16activator domain (Fig. 2 F, H, J, and L), indicating the possibilityof additional repressor domain(s) in the STF protein. In light ofthis, Ikeda et al. (19) reported that an EAR-like motif at the Cterminus of the WUS protein provides additional repressiveactivity. The corresponding region in the STF protein awaitsfurther investigation.

Importantly, the WUS-box exhibits a key element in themechanistic conservation of WOX gene functions and providesa functional basis for the phylogenetic classification of the evo-lutionarily dynamic plant-specific WOX family proteins. It isknown that WUS can substitute for WOX5 and PRS functions inthe root and shoot, respectively (5, 10). Characterization of theWUS-box not only uncovered the mechanism for such conserva-tion of functions, but also revealed the extent of functional con-servation existing among WOX genes. The modern clade WOXgenes, including WUS and WOX1–WOX6, contain intact WUS-box elements and can complement the lam1 mutant in a mannersimilar to STF (Fig. 3 A–G and Fig. S3 A–G). WOX5 comple-mentation is weaker, although it is most similar in sequence toWUS, perhaps because WOX5 is shorter, and the missing regionmay stabilize the protein or facilitate protein–protein interactionsin the shoot. WOX7 is the only member in this clade that fails tocomplement lam1, which is attributed to loss of the WUS-box.Interestingly, 3′ RACE revealed a longer splice variant of WOX7(WOX7-L) with an intact WUS box, which was not present in theannotation based on existing ESTs (Fig. S5). Although the in-termediate and ancient clade WOX genes lack the WUS-box andact as transcriptional activators in transient expression assays, theycan gain competence for lam1 complementation when fused withthe WUS box or SRDX repressor domain.Taken together, the foregoing observations suggest that the

original or default function of WOX genes was transcriptionalactivation, whereas repressive function was acquired later inthe evolution of modern clade WOX genes through acquisitionof the WUS-box as a major functional module. Because thedirect targets of STF required for leaf blade outgrowth functionhave not yet been identified, whether STF, WUS, and othermodern clade WOX genes repress the same target gene(s) tocarry out their functions is unclear. Nonetheless, it is interestingto note that the transcriptional repression functions of both

Fig. 3. Functional complementation of lam1 by WOX family genes. (A–L) Complementation of the lam1 mutant with Arabidopsis WOX family genes. Six-wk-oldwhole plants and leaves of lam1 mutant after transformation with STF::WUS (A), STF::WOX1 (B), STF::WOX2 (C), STF::WOX3 (D), STF::WOX4 (E), STF::WOX5 (F), STF::WOX6 (G), STF::WOX7 (H), STF::WOX9 (I), STF::WOX11 (J), STF::WOX13 (K), and STF::GUS (L). (Scale bars: 5 cm.) (M) Amino acid sequence of the WUS-box in WUS.Mutations (L to A amino acid substitutions) introduced into the WUS box (WUSm1) are indicated in red. (N) Schematic representation of the chimeric constructs usedfor transformation. (O–Q) Plant and leaf phenotypes of lam1 complemented with STF::WUSm1 (O), STF::SRDX-WUSm1 (P), and STF::WUSm1-VP16 (Q). (Scale bars:5 cm.) (R–T) Transverse sections through the leaves of transgenic lam1 transformed with STF::GUS (R), STF::WUSm1-VP16 (S), and STF::WOX9 (T). (Scale bars: 100 μm.)

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STF and WUS are required to activate cell proliferation. In theArabidopsis shoot apical meristem, WUS has several directtargets (23), including the well-studied CLAVATA regulatoryfeedback loop (7), in which WUS directly represses CLV1 (23),and cytokinin signaling, in which WUS directly represses A-typetwo-component response regulators (24). Whether STF sharesany of these regulatory modules to promote cell proliferation inleaf primordia is not known. On the other hand, genes impor-tant for leaf development, such as ASYMMETRIC LEAVES1and 2 (AS1 and AS2), form a repression complex that includeschromatin remodeling factors to prevent meristematic activityorchestrated by class I KNOTTED1-LIKE HOMEOBOX(KNOX) genes in determinate leaf primordia (25, 26). Dis-secting the relationship between the STF-mediated WUS-like

function and the AS1/AS2 repression complex will providefurther insight into the mechanistic understanding of cell pro-liferation regulation in the indeterminate meristem and de-terminate leaf primordia.Given that WOX9/STIMPY acts as a strong transcriptional acti-

vator in luciferase assays and enhances the lam1 phenotype withadditional proximodistal defects, it is tempting to speculate thatmeristematic activity and lateral organ development in higherplants requires a balance between the activation and repressionfunctions of WOX genes. WOX9 has been proposed to coordinatecytokinin- and sugar-mediated signaling in shoot meristem de-velopment (27–29). In addition, STF modulation of robust hor-mone and sugar homeostasis in leaf blade development has beenproposed (15). However,WOX9 function in leaf primordia has not

Fig. 4. WUS-box and repressive activity required for leaf blade outgrowth are conserved only in the modern clade WOX family members. (A) Sequencealignment of the homeodomain and WUS-box of STF and Arabidopsis WOX proteins. The conserved WUS-box core sequence is shown below the alignment.(B) Phylogenetic analysis of STF and Arabidopsis WOX proteins using homeodomain sequences. The three evolutionary clades are statistically supported bybootstrap values. (C) Relative luciferase activities in Arabidopsis leaf protoplasts using the indicated genes driven by the 35S promoter as effector plasmids.Error bars indicate SD (n = 3). **P < 0.01 (one-way ANOVA, Dunnett’s test). (D) Schematic representation of the chimeric constructs used for transformation.(E–K) Phenotypes of transgenic lam1 plants and leaves complemented with STF::WOX7-WB (E), STF::SRDX-WOX7 (F), STF::WOX9-WB (G), STF::SRDX-WOX9(H), STF::WOX13-WB (I), STF::SRDX-WOX13 (J), and STF::GUS (K). WB represents WUS-box. (Scale bars: 5 cm.)

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been established, and it remains to be shown whether STF andWOX9 antagonistically regulate the same target or set of targetsduring leaf blade morphogenesis.The relatively recent emergence of modern/WUS clade genes in

seed plants and the widespread requirement for WOX function inthe leaves and meristems are fascinating phenomena. In the mer-istems of angiosperms,WUS performs an internal function essentialto the maintenance of a stratified tunica-corpus design. Likewise,WOX1,MAW, STF, andLAM1 perform internal functions essentialto the formation of stratified leaf blades. Conservation of the DNAbinding domain and C-terminal WUS box in all of these genes wasthe initial foundation for the idea that leaves and meristems mightshare a common mechanism. This concept received strong supportfrom complementation studies showing thatWUS can substitute forthe STF, LAM1, and PRS functions. Most remarkable, however, isthe demonstration in the present study that this capacity is sharedby WUS clade genes operating in such diverse structures as em-bryos, ovules, and the vascular cambium, in addition to shoot androot meristems. Interestingly, the fossil record indicates that whenvascular plants diverged approximately 400 Mya, primitive plantshad branched stems with sporangia but no leaves, and leaf-likeorgans evolved independently in lycophytes and euphyllophytes,and seed plants rose uniquely from leafless progymnospermancestors (30–32). The roles of ancient or intermediateWOX genesin the evolution of extant plants remain to be clarified, but it is clearfrom the seed plants that the emergence of the WUS clade hasplayed a prominent role in the evolution of modern plant form.

Materials and MethodsPlant Materials and Growth Conditions. Plants were grown in 24 °C/16-h (day)and 20 °C/8-h (night) photoperiods at 70–80% relative humidity and 150μmol·m2·s light intensity.

Plasmid Construction and Plant Transformation. Plasmid construction andplant transformations were performed as described previously (15). Detailsare provided in SI Materials and Methods.

RNA Extraction, RACE-PCR and Real-Time PCR Analysis. Total RNA isolation,cDNA synthesis, and real-time PCR were conducted as described previously(15); 3′ RACE of WOX7 was performed using an Invitrogen 3′ RACE system,in accordance with the manufacturer’s instructions. Primers used are listedin Table S2.

Transient Expression Assay. Transient expression assays were performed asdescribed in SI Materials and Methods.

Histological Analysis. Histological analyses were performed as describedpreviously (33). Details are provided in SI Materials and Methods.

Sequence Alignment and Phylogenetic Analysis. Amino acid sequences ofWOX homeodomain and WUS-box regions were aligned using Clustal W,and a neighbor-joining phylogenetic tree was constructed for the homeo-domain using MEGA 4 software. The most parsimonious tree with bootstrapvalues from 1,000 trials was used.

Accession Numbers. Accession numbers for the sequence data used in thisstudy are listed in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Richard Dixon for providing p2GW7 andpRLC plasmids; Huan-zhong Wang for assisting with the luciferase transientassays; Michael Scanlon for providing plasmids harboring WOX3/PRS; JinNakashima for helping with histological analysis; and Fei Zhang and RandyAllen for providing stimulating discussion and critical comments on the man-uscript. This work was supported by Oklahoma Center for the Advancementof Science and Technology Grant PBS11-002 and National Science Founda-tion Grant EPS-0814361.

1. van der Graaff E, Laux T, Rensing SA (2009) The WUS homeobox-containing (WOX)protein family. Genome Biol 10(12):248.

2. Mukherjee K, Brocchieri L, Bürglin TR (2009) A comprehensive classification andevolutionary analysis of plant homeobox genes. Mol Biol Evol 26(12):2775–2794.

3. Haecker A, et al. (2004) Expression dynamics of WOX genes mark cell fate decisionsduring early embryonic patterning in Arabidopsis thaliana. Development 131(3):657–668.

4. Ueda M, Zhang Z, Laux T (2011) Transcriptional activation of Arabidopsis axis pat-terning genes WOX8/9 links zygote polarity to embryo development. Dev Cell 20(2):264–270.

5. Sarkar AK, et al. (2007) Conserved factors regulate signalling in Arabidopsis thalianashoot and root stem cell organizers. Nature 446(7137):811–814.

6. LenhardM, Bohnert A, Jürgens G, Laux T (2001) Termination of stem cell maintenancein Arabidopsis floral meristems by interactions between WUSCHEL and AGAMOUS.Cell 105(6):805–814.

7. Schoof H, et al. (2000) The stem cell population of Arabidopsis shoot meristems inmaintained by a regulatory loop between the CLAVATA and WUSCHEL genes. Cell100(6):635–644.

8. Nardmann J, Reisewitz P, Werr W (2009) Discrete shoot and root stem cell-promotingWUS/WOX5 functions are an evolutionary innovation of angiosperms. Mol Biol Evol26(8):1745–1755.

9. Ji J, Shimizu R, Sinha N, Scanlon MJ (2010) Analyses of WOX4 transgenics providefurther evidence for the evolution of the WOX gene family during the regulation ofdiverse stem cell functions. Plant Signal Behav 5(7):916–920.

10. Shimizu R, et al. (2009) Tissue specificity and evolution of meristematic WOX3 func-tion. Plant Physiol 149(2):841–850.

11. Nardmann J, Ji J, Werr W, Scanlon MJ (2004) The maize duplicate genes narrowsheath1 and narrow sheath2 encode a conserved homeobox gene function in a lat-eral domain of shoot apical meristems. Development 131(12):2827–2839.

12. Scanlon MJ, Schneeberger RG, Freeling M (1996) The maize mutant narrow sheathfails to establish leaf margin identity in a meristematic domain. Development 122(6):1683–1691.

13. Matsumoto N, Okada K (2001) A homeobox gene, PRESSED FLOWER, regulates lateralaxis-dependent development of Arabidopsis flowers. Genes Dev 15(24):3355–3364.

14. Vandenbussche M, et al. (2009) Differential recruitment of WOX transcription factorsfor lateral development and organ fusion in Petunia and Arabidopsis. Plant Cell 21(8):2269–2283.

15. Tadege M, et al. (2011) STENOFOLIA regulates blade outgrowth and leaf vascularpatterning inMedicago truncatula andNicotiana sylvestris. Plant Cell 23(6):2125–2142.

16. Nakata M, et al. (2012) Roles of the middle domain-specific WUSCHEL-RELATED HO-MEOBOX genes in early development of leaves in Arabidopsis. Plant Cell 24(2):519–535.

17. Tadege M, Lin H, Niu L, Mysore KS (2011) Control of dicot leaf blade expansion bya WOX gene, STF. Plant Signal Behav 6(11):1861–1864.

18. McHale NA (1992) A nuclear mutation blocking initiation of the lamina in leaves ofNicotiana sylvestris. Planta 186:355–360.

19. Ikeda M, Mitsuda N, Ohme-Takagi M (2009) Arabidopsis WUSCHEL is a bifunctionaltranscription factor that acts as a repressor in stem cell regulation and as an activatorin floral patterning. Plant Cell 21(11):3493–3505.

20. Hiratsu K, Matsui K, Koyama T, Ohme-Takagi M (2003) Dominant repression of targetgenes by chimeric repressors that include the EAR motif, a repression domain, inArabidopsis. Plant J 34(5):733–739.

21. Sadowski I, Ma J, Triezenberg S, Ptashne M (1988) GAL4-VP16 is an unusually potenttranscriptional activator. Nature 335(6190):563–564.

22. Mayer KF, et al. (1998) Role of WUSCHEL in regulating stem cell fate in the Arabi-dopsis shoot meristem. Cell 95(6):805–815.

23. Busch W, et al. (2010) Transcriptional control of a plant stem cell niche. Dev Cell 18(5):849–861.

24. Leibfried A, et al. (2005) WUSCHEL controls meristem function by direct regulation ofcytokinin-inducible response regulators. Nature 438(7071):1172–1175.

25. Moon J, Hake S (2011) How a leaf gets its shape. Curr Opin Plant Biol 14(1):24–30.26. Guo M, Thomas J, Collins G, Timmermans MC (2008) Direct repression of KNOX loci by

the ASYMMETRIC LEAVES1 complex of Arabidopsis. Plant Cell 20(1):48–58.27. Skylar A, Sung F, Hong F, Chory J, Wu X (2011) Metabolic sugar signal promotes

Arabidopsis meristematic proliferation via G2. Dev Biol 351(1):82–89.28. Skylar A, Hong F, Chory J, Weigel D, Wu X (2010) STIMPYmediates cytokinin signaling

during shoot meristem establishment in Arabidopsis seedlings. Development 137(4):541–549.

29. Wu X, Dabi T, Weigel D (2005) Requirement of homeobox gene STIMPY/WOX9 forArabidopsis meristem growth and maintenance. Curr Biol 15(5):436–440.

30. Harrison CJ, et al. (2005) Independent recruitment of a conserved developmentalmechanism during leaf evolution. Nature 434(7032):509–514.

31. Floyd SK, Bowman JL (2006) Distinct developmental mechanisms reflect the in-dependent origins of leaves in vascular plants. Curr Biol 16(19):1911–1917.

32. Tomescu AM (2009) Megaphylls, microphylls and the evolution of leaf development.Trends Plant Sci 14(1):5–12.

33. Lin H, et al. (2009) DWARF27, an iron-containing protein required for the biosynthesisof strigolactones, regulates rice tiller bud outgrowth. Plant Cell 21(5):1512–1525.

6 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1215376110 Lin et al.

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