The TCP4 Transcription Factor Directly ActivatesTRICHOMELESS1 and 2 and SuppressesTrichome Initiation1[OPEN]

Batthula Vijaya Lakshmi Vadde, Krishna Reddy Challa, Preethi Sunkara, Anjana S. Hegde, andUtpal Nath2,3

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012 Karnataka, India

ORCID IDs: 0000-0001-8448-5575 (B.V.L.V.); 0000-0001-8832-6985 (K.R.C.); 0000-0002-3600-2089 (P.S.); 0000-0002-6697-4567 (A.S.H.);0000-0002-5537-5876 (U.N.).

Trichomes are the first line of defense on the outer surface of plants against biotic and abiotic stresses. Because trichomes on leafsurfaces originate from the common epidermal progenitor cells that also give rise to pavement cells and stomata, their densityand distribution are under strict genetic control. Regulators of trichome initiation have been identified and incorporated into abiochemical pathway wherein an initiator complex promotes trichome fate in an epidermal progenitor cell, while an inhibitorcomplex suppresses it in the neighboring cells. However, it is unclear how these regulator proteins, especially the negativeregulators, are induced by upstream transcription factors and integrated with leaf morphogenesis. Here, we show that theArabidopsis (Arabidopsis thaliana) class II TCP proteins activate TRICHOMELESS1 (TCL1) and TCL2, the two establishednegative regulators of trichome initiation, and reduce trichome density on leaves. Loss-of-function of these TCP proteinsincreased trichome density whereas TCP4 gain-of-function reduced trichome number. TCP4 binds to the upstream regulatoryelements of both TCL1 and TCL2 and directly promotes their transcription. Further, the TCP-induced trichome suppression isindependent of the SQUAMOSA PROMOTER BINDING PROTEIN LIKE family of transcription factors, proteins that alsoreduce trichome density at later stages of plant development. Our work demonstrates that the class II TCP proteins coupleleaf morphogenesis with epidermal cell fate determination.

Trichomes are specialized epidermal cells that groworthogonal to the tissue surface to form hair-like projec-tions. Even though trichomeless mutants are viable withno other abnormalities, trichomes are thought to pro-vide mechanical and chemical protections to the plantsunder biotic and abiotic stresses (Hauser, 2014). Tri-chomes arise at an early stage of organ morphogenesis

out of the epidermal progenitor cells that also give riseto other cell types such as stomata and pavement cells.Trichomes constitute a small fraction of epidermal cellsand their distribution is fairly uniform in amature organ.The density and distribution of trichomes are regulatedby genetic factors (Hülskamp, 2004; Balkunde et al.,2010), mutation of which results in thinner or densertrichome population.Trichome development includes two primary

processes—trichome patterning and trichome differ-entiation. Trichome patterning requires the decision-making of the epidermal progenitor cells to adopttrichome cell identity, a process that involves the an-tagonistic action of two protein complexes known as the“initiator complex” and the “inhibitor complex.” Theinitiator complex includes GLABRA1 (GL1; Koornneef,1981; Oppenheimer et al., 1991), GL3 (Hülskamp et al.,1994; Payne et al., 2000; Esch et al., 2003), ENHANCEROFGL3 (EGL3; Zhang et al., 2003), and TRANSPARENTTESTA GLABRA1 (TTG1; Koornneef, 1981; Galwayet al., 1994), while the inhibitor complex consists ofall the components of the initiator complex exceptGL1, which is replaced by any of the following mole-cules with inhibitory function: TRIPTYCHON (TRY;Hülskamp et al., 1994; Schellmann et al., 2002), CA-PRICE (CPC; Wada et al., 1997), ENHANCER OF TRYAND CPC1 (ETC1), ETC2, and ETC3 (Kirik et al.,2004a, 2004b), and TRICHOMELESS1, 2 (TCL1, 2;Wang et al., 2007, 2008b; Gan et al., 2011). The initiator

1This work was supported by the Ministry of Human ResourceDevelopment, Government of India (fellowships to B.V.L.V.,K.R.C., and P.S.), the Council of Scientific & Industrial Research, Gov-ernment of India (fellowship to A.S.H.), the Department of Science &Technology-For Improvement of S&T Infrastructure (DST-FIST), theUniversity Grants Commission Centre for Advanced Studies, and theDepartment of Biotechnology (DBT)-IISc Partnership Program Phase-IIat IISc (sanction No. BT/PR27952/INF/22/212/2018 to U.N.).

2Author for contact: utpalnath@iisc.ac.in.3Senior author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Utpal Nath (utpalnath@iisc.ac.in).

K.R.C. initiated the project; B.V.L.V. designed and performedmostexperiments, analyzed data, finalized figures, and wrote the firstdraft of the article, with inputs from K.R.C.; P.S. designed and per-formed luciferase assays, site-directed mutagenesis, and part ofEMSA; A.S.H. repeated several experiments including all trichomecounting experiments; U.N. contributed in designing the experi-ments, guided other authors, and finalized the article.

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complex consisting of GL1, GL3/EGL3, and TTG1activates the downstream molecule GL2, whose pro-tein product initiates trichome morphogenesis. Bind-ing of the inhibitors such as TRY, CPC, and TCL resultsin the replacement of GL1 and renders the initiatorcomplex inactive, leading to the failure ofGL2 activation.

The negative regulators play an important role intrichome patterning, spacing, and density, which can beappreciated from the higher order mutant combina-tions of these negative regulators (Hülskamp, 2004;Wang et al., 2008b). Inactivation of these negativeregulators results in an increased trichome densitywhereas their overexpression leads to fewer trichomes(Schellmann et al., 2002; Kirik et al., 2004a, 2004b;Wanget al., 2007). The expression of many of these nega-tive regulators is activated by the trichome initiationcomplex itself, except for ETC2, TCL1, and TCL2(Morohashi et al., 2007). Among these, TCL1 andTCL2 have been shown as negative regulators of tri-chome initiation mainly on inflorescence stems (Wanget al., 2007; Gan et al., 2011). There are no studies im-plicating the role of TCL1 and TCL2 in trichome de-velopment on leaves.

No upstream regulators of TCL1 and TCL2 wereknown until recently (Yu et al., 2010; Tian et al., 2017).NTM1 LIKE8, a membrane-associated NAC tran-scription factor, has been shown to activate TCL1 andTRY directly (Tian et al., 2017). It has also been shownthat the miR156-targeted SQUAMOSA PROMOTERBINDING PROTEIN LIKE (SPL; SPL2, SPL3, SPL4,SPL5, SPL6, SPL9, SPL10, SPL11, SPL13, SPL15) familyof transcription factors, primarily SPL9, activates TCL1on inflorescence epidermis (Yu et al., 2010). The in-volvement of SPLs in regulating trichome density atlater phases of plant life is linked to their expressionlevel, which progressively increases as the abundanceof miR156 declines with plant maturity (Xu et al., 2016).The upstream transcriptional regulators of TCLs atearly stages of plant development, especially in rosetteleaves, are not known, even though TCL1 and TCL2transcripts are detected in rosette leaves of Arabi-dopsis (Arabidopsis thaliana; Wang et al., 2007; Ganet al., 2011).

We therefore reasoned that TCL1 and TCL2 wouldbe activated by transcription factors that are presentthroughout leaf morphogenesis. The CIN–TCP tran-scription factors are expressed in young leaf primordiawhere they regulate leaf shape and size, modulatingthe cell proliferation potential by directly activating theKIP-related cell division inhibitors (Nath et al., 2003;Palatnik et al., 2003; Schommer et al., 2014). In addition,they also play important roles in hypocotyl cell elon-gation and trichome branching by directly activatingdiverse target proteins (Challa et al., 2016; Vadde et al.,2018). Considering their wide-ranging roles in organand cell morphogenesis (Sarvepalli et al., 2019), andthe temporal and spatial overlap between trichome in-itiation and CIN–TCP expression (Efroni et al., 2008;Alvarez et al., 2016), we examined the role of theseproteins in trichome initiation.

By combining loss and gain-of-function analysis ofTCP proteins with biochemical experiments, we hereshow that TCP4 suppresses trichome initiation in Ara-bidopsis leaves by directly activating the transcriptionof TCL1 and TCL2. Thus, the CIN–TCP proteins act asregulators of trichome development from the juvenilephase of Arabidopsis and integrate organ morpho-genesis with cellular patterning.

RESULTS

CIN–TCPs Inhibit Trichome Initiation

Gain-of-function of TCP4 consistently showed fewertrichomes on leaf surfaces compared to the wild type(Fig. 1A; Efroni et al., 2008, 2013), suggesting a linkbetween class II TCP action and trichome initiation. Toexamine the role of these proteins in trichome devel-opment in more detail, we compared the number anddensity of trichomes on the leaf surface of wild-typeArabidopsis and two loss-of-function mutant lines,tcp2;tcp4;tcp10 (Karidas et al., 2015) and jaw-D, where allfive target transcripts of miR319—TCP2, TCP3, TCP4,TCP10, and TCP24—are substantially downregulated(Palatnik et al., 2003). The total number of trichomesand their density (expressed as number per unit area)on the mature first leaf pair in both the mutant linesappeared to be higher than in Col-0 (Table 1; Fig. 1, Aand B; Supplemental Fig. S1), implying that TCPs in-hibit trichome initiation. Consistent with this, the den-sity of trichomes was reduced (Table 1; Fig. 1, C and D;Supplemental Fig. S1) on pTCP4::TCP4:VP16 leaf sur-faces, where TCP4 activity was enhanced due to thefusion of a strong viral activation domain (Sarvepalliand Nath, 2011), and in pBLS::rTCP4:GFP leaves,where a miR319-resistant form of TCP4 is expressedunder the At3g49950 promoter throughout the youngleaf primordia (Lifschitz et al., 2006; Efroni et al.,2013). Because the TCP proteins have been shown topromote pavement cell growth as well (Das Guptaet al., 2014; Challa et al., 2019), we expressed trichomedensity as a fraction of pavement cells and found thattrichome number per pavement cell is also reducedin pTCP4::TCP4:VP16 and in pBLS::rTCP4:GFP leavescompared to Col-0 (Fig. 1E). In addition, trichome den-sity was also reduced when a miR319-resistant form ofTCP4 (mTCP4)was activated in its endogenous domainby mobilizing it from cytoplasm to nucleus upon con-stitutive dexamethasone (DEX) treatment (Fig. 1, D, F,and G) in the inducible lines jaw-D pTCP4::mTCP4:GR(henceforth jaw-D;GR) and Col-0 pTCP4::mTCP4:GR(henceforth Col-0;GR; Supplemental Fig. S2A; Challaet al., 2016). The relative reduction in trichome den-sity upon TCP4 activation was estimated to be morein jaw-D;GR compared to the Col-0;GR background(Fig. 1F; Supplemental Fig. S2B), possibly because theinitial trichome density (the denominator in calculatingthe relative trichome density) is higher in jaw-D;GRcompared to Col-0;GR (Fig. 1B) and because TCP4

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alone can compensate for the loss of its putativeparalogs in the jaw-D mutant (Challa et al., 2019). To-gether, these results provide genetic evidence that themiR319-targeted TCP proteins inhibit trichome initia-tion on leaf surface.

Transcript Levels of Trichome Initiation Genes AreAltered in tcp Mutants

To test if there is a correlation between the regula-tors of trichome initiation and class II TCP function,we analyzed the transcript levels of trichome pat-terning genes in mutants with altered class II TCP ex-pression using the database “Genevestigator” (https://

genevestigator.com/gv/). Among the seven negativeregulators tested (CPC, TRY, ETC1, ETC2, ETC3, TCL1,and TCL2), five (CPC, TRY, ETC1, ETC2, and ETC3)were detected in the database and none of them showeda substantial change in their transcript level except CPC(Fig. 2A). Among the five positive regulators tested(GL1, GL2, GL3, EGL3, and TTG1), three of the probes(GL1, GL2, and TTG1) were detected in the databaseand GL1 and GL2 showed substantial change in ex-pression (Fig. 2A). Both the transcripts were upregulatedin TCP loss-of-functionmutants and downregulated inthe gain-of-function lines, suggesting a negative rela-tionship between TCP and GL1/ GL2. Results of thisin silico analysis were validated by a reverse tran-scription quantitative PCR (RT-qPCR) experiment,

Figure 1. Trichome density in class II TCP mutant lines. A, Top-view of 9-d–old seedlings (from left to right) of Col-0,tcp2;tcp4;tcp10, jaw-D, TCP4:VP16 (pTCP4::TCP4:VP16), and pBLS::rTCP4:GFP to highlight the trichomes on the first pair ofleaves. Scale bars5 1mm. B toD and F, Trichome density as expressed in number in unit area on the adaxial surface ofmature first(B, C, and F) or eighth (D) leaf in TCP loss (B) and gain-of-function (C, D, and F) mutants. Averages of four to seven leaf pairs (B, C,and F; total 60–300 trichomes) or three leaves (D; 250–1000 trichomes) are shown. “Mock” and “DEX” indicate the absence andpresence of 12-mM DEX, respectively. E and G, Trichome density as expressed in number of trichomes per pavement cell on theadaxial surface of first pair of leaves of indicated genotypes (E) and in the absence (Mock) and presence (DEX) of 12-mM DEX (G).Sample number is four to five leaves for (E) and (G). jaw-D;GR indicates pTCP4::mTCP4:GR in jaw-D backgrounds. Error bars in(B–G) indicate SD. *P # 0.05; unpaired Student’s t test was performed.

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where GL1/ GL2 transcripts were reduced upon TCP4activation in the jaw-D;GR seedlings (Fig. 2B). In addi-tion, GUS activity in the pGL2::GUS jaw-D;GR seedlingswas reduced when TCP4 was induced by continuousDEX treatment (Fig. 2, C and D). However, no changewas observed in the transcript level of TRY, a knownnegative regulator of trichome initiation (Fig. 2B;Schellmann et al., 2002), or in the GUS activity inpTRY::GUS;jaw-D;GR plants upon TCP4 activation

(Fig. 2, E and F). Taken together, these results showthat TCP4 activation suppresses certain positive reg-ulators of trichome initiation.

During the initiation of trichomes, their spacingmechanism is regulated by a group of functionallyredundant genes encoding small, single-repeat MYBtranscription factors that include TRY, CPC, TCL1,TCL2, ETC1, ETC2, and ETC3. Even though the ETC3transcript level increased in the jaw-D;GR plants upon

Table 1. Trichome number per leaf and leaf area of the indicated genotypes

An unpaired Student’s t test was performed, and P values were calculated for total number of trichomesper leaf in relation with Col-0.

GenotypeTotal No. of Trichomes on First Leaf

(Mean 6 SD)

First Leaf Area (mm2)

(Mean 6 SD)

Sample

No.P Value

Col-0 17.28 6 1.98 21.2 6 1.98 7 —tcp2;tcp4;tcp10 45.43 6 8.77 50.12 6 8.68 5 ,0.001jaw-D;GR Mock 47.55 6 4.19 46.31 6 3.06 9 ,0.001jaw-D;GR DEX 2.8 6 0.84 6.73 6 1.37 5 ,0.001pTCP4::TCP4:VP16 6.83 6 1.94 15.73 6 1.73 6 ,0.001pBLS::rTCP4:GFP 6.75 6 1.5 14.86 6 1.68 4 ,0.001

Figure 2. Expression of trichome initiation genes in TCPmutants. A, “Genevestigator” (https://genevestigator.com/gv/) analysis ofthe trichome initiation genes. GL1 expression levels are highlighted with a red outline. B, Heat map representation of the ex-pression levels of trichome initiation genes, estimated by RT-qPCR, in 12-d–old jaw-D;GR seedlings grownwithout (Mock) or with(DEX) 12-mM DEX. Averages of biological triplicates are shown. C and E, Differential interference contrast microscopic images of14-d–old jaw-D;GR;pGL2::GUS (C) and jaw-D;GR;pTRY::GUS (E) homozygous seedlings, grown in the absence (Mock) orpresence (DEX) of 12-mM DEX, to show GUS activity. Scale bars5 200 mm. D and F, Quantification of GUS activity in 14-d–oldjaw-D;GR;pGL2::GUS (D) and jaw-D;GR;pTRY::GUS (F) seedlings, grown in the absence (Mock) or presence (DEX) of 12-mMDEX.Averages of biological triplicates are shown. G, Percentage of trichome cluster on the adaxial surface of the first pair of matureleaves of indicated genotypes. Average of five leaf pairs are plotted (total 50–230 trichomes). jaw-D;GR indicatespTCP4::mTCP4:GR in the jaw-D background. Error bars indicate mean6 SD. *P# 0.05; unpaired Student’s t test was performed.ns, not significant.

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TCP4 activation (Fig. 2B), we did not observe any tri-chome clusters in the mutants of class II TCP genes.To examine this further, we first perturbed the tri-chome spacing in the jaw-D;GR plants by crossing themto the try mutant where trichomes appear in clusters(Schnittger et al., 1998), and then tested the effect ofTCP4 induction on cluster formation. No change in theextent of clustering was observedwhen TCP transcriptswere reduced in the jaw-D;GR;try leaves compared tothe trymutant, or when TCP4 was activated in this lineby continuous DEX treatment (Fig. 2G), suggesting thatTCPs are not involved in trichome clustering.

TCP4 Activates TCL1 and TCL2

The class II TCP proteins function as transcriptionalactivators (Efroni et al., 2013; Challa et al., 2016), andtherefore the downregulation of GL1/GL2 by TCP4 is

likely to be an indirect effect and is perhaps mediatedby upstream regulators. In an attempt to identify theseregulators, we first compared the transcript levels of 12major trichome initiation genes in pTCP4::TCP4:VP16plants with their levels in Col-0. While the transcriptlevels of most genes remained unaltered, those of ETC2,ETC3, TCL1, and TCL2 increased in this TCP4 gain-of-function line (Fig. 3A). GIS, a known target of TCP4 intrichome branching (Vadde et al., 2018) and LOX2,another TCP4 target involved in jasmonic acid biosyn-thesis (Schommer et al., 2008), were used as positivecontrols. When we analyzed their levels in an earliermicroarray experiment that was performed after in-ducing TCP4 in the jaw-D;GR seedlings (Challa et al.,2016), we noticed that only TCL1 and TCL2 tran-scripts were upregulated at both 2 h and 4 h of TCP4induction (Fig. 3B; Supplemental Fig. S3A). Activa-tion of TCL1, 2was also confirmed by RT-qPCR, wherewe found that these transcripts were upregulated in

Figure 3. Expression analyses of trichome genes in TCP mutants. A, Relative transcript levels of genes involved in trichomeinitiation as determined by RT-qPCR analysis in 12-d–old Col-0 and pTCP4::TCP4:VP16 seedlings. GIS and LOX2 were used aspositive controls. Averages of biological triplicates are shown. B, Transcript level of trichome initiation genes detected in themicroarray experiment of the jaw-D;GR seedlings upon TCP4 induction for 2 h and 4 h (Challa et al., 2016). C, Relative transcriptlevels of TCL1 and TCL2 in 12-d–old jaw-D;GR seedlings grown without (Mock) or with (DEX) 12-mM DEX. GIS was used as apositive control (Vadde et al., 2018). D, Relative transcript levels of TCL1, TCL2 andGL1 in 12-d–old p35S::mTCP4:GR seedlingsafter 0, 2, 4, and 8 h of TCP4 induction by DEX. Averages of biological triplicates are shown. y axis label for (C) and (D) are thesame as that of (B). E and F, Relative transcript levels of TCL1 and TCL2 in TCP gain (E) and loss-of-function (F) lines compared toCol-0. Averages of biological triplicates are shown.GISwas used as a positive control in (E; Vadde et al., 2018).G, Relative levelsof TCL1 and TCL2 transcripts determined by RT-qPCR analysis in 12-d–old jaw-D;GR seedlings in the absence (Mock) or presence(DEX) of 20-mMDEX alongwith (CHX1DEX) or (CHX) 40-mM CHX for the 4-h y axis label for (F) and (G) are the same as that of (E).GIS was used as a positive control. Averages of biological triplicates are shown. jaw-D;GR indicates pTCP4::mTCP4:GR in thejaw-D background. Error bars indicate mean 6 SD.

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jaw-D;GR (Fig. 3C) and in p35S::mTCP4:GR seedlings(Challa et al., 2016; Supplemental Fig. S3B) grown withconstitutive TCP4 induction. Time-course RT-qPCR anal-ysis also demonstrated that TCL1/ TCL2 are activated asearly as 2 h of TCP4 induction, after which the TCL2 levelstabilized while that of TCL1 continued to increase for 8-hpostinduction (Fig. 3D; Supplemental Fig. S3C). A corre-sponding decrease in the GL1 transcript, a cognate targetof the TCL proteins, was also noticed (Fig. 3D). Gain-of-function of TCP4 elevated the TCL1 transcript level inanother dominant line, pBLS::rTCP4:GFP, aswell (Fig. 3E).Importantly, TCL1/ TCL2 transcripts were much reducedin the loss-of-function lines of class II TCP genes, jaw-Dand tcp2;tcp4;tcp10 (Fig. 3F). These results together pro-vide evidence that the class II TCP proteins activate TCL1and TCL2 in wild-type leaves. Further, activation of TCL1,2 is associatedwith a concomitant downregulation ofGL1(Figs. 2, A and B, and 3D), a trichome promoting genewhose expression is suppressed by the TCL transcriptionfactors (Wang et al., 2007).

TCL1, 2 Are Direct Targets of TCP4

To examine whether TCP4 directly activates TCL1and TCL2, we estimated their transcript levels uponTCP4 induction in the absence of protein synthesis.When TCP4was induced by 4 h of DEX treatment in thejaw-D;GR seedlings grown in the presence of cyclohex-imide (CHX), a general inhibitor of protein synthesis(Schneider-Poetsch et al., 2010), transcripts of TCL1 andTCL2were elevated by 2- to 4-fold (Fig. 3G), suggestingthat TCL1 and TCL2 are directly activated by the TCP4transcription factor. CHX alone failed to cause any in-duction over the mock level, suggesting that the in-crease in TCL transcripts is specifically caused by TCP4induction by DEX treatment. It is possible that TCP4activates transcription of TCLs by binding to their ge-nomic regions. To test this, we analyzed TCL1/TCL2loci for the presence of TCP4 consensus binding sites(Schommer et al., 2008) and identified one sequencemotif in the TCL1 upstream regulatory region (BS1.1)and two in TCL2 (BS2.1 and BS2.2) that contained pu-tative TCP4 core binding sequence (Fig. 4, A and B).Recombinant His6-TCP4 protein (Aggarwal et al., 2010)bound to the oligonucleotides (BS1.1 and BS2.1) corre-sponding to these motifs and retarded their mobilityin electrophoretic mobility shift assay (EMSA) experi-ments (Fig. 4C). Addition of anti-His6 antibody to thereaction mixture resulted in further retardation ofthe DNA–protein complex, thus demonstrating that theretardation of the oligonucleotide was indeed causedby the His6-TCP4 protein. The intensity of the DNA–protein band reduced with increasing amounts of unla-beled wild-type oligonucleotide, but not when mutatedoligonucleotide, where TCP4-binding nucleotides werealtered (Supplemental Fig. S4A), was added to the re-action mixture, suggesting that the in vitro interaction ofTCP4 is specific to these sequence motifs. Chromatinimmuno-precipitation (ChIP) experiments using the

genomic DNA of p35S::TCP4-3F6H seedlings (Kubotaet al., 2017) with anti-FLAG antibody showed that theTCP4 protein is recruited to the upstream regulatoryregions of TCL1 and TCL2 near the TCP4-binding mo-tifs (Fig. 4D), but not to the genomic region of the ret-rotransposon TA3, which was used as a negativecontrol (Han et al., 2012). The known TCP4 target LOX2was used as a positive control (Schommer et al., 2008).

To test whether TCP4-binding to these cis-elementsresults in a change in the chromatin conformation atthe TCL1 and TCL2 loci, we performed a formaldehyde-assisted isolation of regulatory elements (FAIRE) ex-periment, which monitors the level of DNA-associatedproteins as a readout of chromatin conformation (Simonet al., 2012). We observed increased DNA accessibility atthe TCL1 and TCL2 chromatins compared to the mockcontrol when TCP4 was induced in the p35S::mTCP4:GRseedlings by DEX treatment (Fig. 4, E and F) or whenTCP4 activity was enhanced in the pTCP4::TCP4:VP16seedlings (Supplemental Fig. S4, B and C).

To examine if TCP4 is capable of activating the TCLpromoters in vivo, we carried out a transient expres-sion assay using the firefly luciferase reporter driven bywild-type and mutant TCL1 upstream regulatory re-gions in isolated Arabidopsis protoplasts (Fig. 4, G andH). The presence of the 35S::mTCP4:GR effector con-struct resulted in a massive increase in luciferase ac-tivity of the pTCL1::LUC reporter when treated withDEX as compared to the mock (Fig. 4H). However,35S::mTCP4:GR failed to induce luciferase activity inthe inductive condition from a mutant pTCL1::LUCconstruct where the TCP4-binding site was mutated.Taken together, these results strongly suggest that TCP4binds to the upstream cis-element of TCL1 and activatesits transcription.

To examine whether TCP4 binding activates the en-dogenous TCL transcription in planta, we estimatedpTCL1::GUS (Xue et al., 2014) activity upon TCP4 in-duction. While weak GUS activity was detected in thetrichomes on the rosette leaves of 14-d–old Col-0 3pTCL1::GUS seedlings, the reporter activity increased inthe pTCP4::TCP4:VP163 pTCL1::GUS line (Fig. 5, A andB) and in the jaw-D;GR 3 pTCL1::GUS line when TCP4was activated (Fig. 5C). TCP4 activation, however, didnot increase the reporter activity in Col-0 3 pCPC::GUSseedlings (Supplemental Fig. S5), used to monitor thepromoter activity ofCPC (Wada et al., 1997), whose levelremained unaltered in the pTCP4::TCP4:VP16 seedlingscompared to the wild type (Fig. 3A).

Taken together, these results suggest that TCP4 bindsto the upstream regulatory regions of TCL1 and TCL2loci and activates their expression.

TCL1 Is Required for TCP4-Mediated Suppression ofTrichome Density

To test whether the TCP-mediated suppression oftrichome initiation requires functional TCL genes, weexamined the effect of TCP4 activation on trichome

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density in the tcl1-1 single mutant background, becausetranscript analysis suggested that TCL1 is more re-sponsive to TCP activity than TCL2 (Fig. 3). Besides, noloss-of-function allele of TCL2 has been reported thusfar (Wang et al., 2007; Gan et al., 2011). Use of TCP4activation byDEX in the jaw-D;GR line posed a technicalproblem in this experiment because the jaw-D allele wassuppressed in the tcl1-1 3 jaw-D;GR seedlings. As an

alternative to jaw-D;GR, we attempted to establishthe tcp2;tcp4;tcp10;pTCP4::mTCP4:GR line by geneticcross, but we failed to establish the tcl1;tcp2;tcp4;tcp10;pTCP4::mTCP4:GR plants due to a strong linkage be-tween the TCL1 (At2G30432) locus and the TCP10 locus(AT2G31070). Therefore,we used the pTCP4::TCP4:VP16and the pBLS::rTCP4:GFP gain-of-function lines to testthe dependence of TCP4 on TCL1. The trichome density

Figure 4. TCL1 and TCL2 are direct targets of TCP4. A and B, Schematic representation of TCL1 (A) and TCL2 (B) genomic locishowing the TCP4 consensus binding sequences. BS1.1, BS2.1, and BS2.2 represent oligonucleotides used in the EMSA ex-periments described below to test TCP4 binding. Exons are indicated by black solid boxes with intervening introns and upstreamregions are represented by black lines. Arrows indicate the translation start site. Gray solid boxes (R1.1, R1.2, R2.1, R2.2, andR2.3) indicate the regions used for PCR amplification in ChIP and FAIRE experiments. C, EMSA autoradiogram showing the re-tardation of the radio-labeled oligonucleotides corresponding to BS1.1 and BS2.1, but not of BS2.2, by recombinant His6-TCP4fusion protein. Bands corresponding to free oligonucleotides and DNA–protein complex are indicated by an arrow and an as-terisk, respectively. A 150- to 300-fold higher concentration of unlabeled BS1.1 and BS2.1 oligonucleotides were used forcompetition assay (autoradiograms in the middle and on the right), respectively. Anti-His6 antibody was used for the supershiftexperiment. The translucent, horizontal white strip seen in themiddle was a consequence of gel processing, but does not hide anybands underneath. A replicate of the gel is shown in Supplemental Figure S4. D, ChIPanalysis to test the binding of TCP4 proteinto the putative TCP4-binding sites marked in (A) and (B). qPCR analysis results from two biological replicates for the enriched cis-elements in Col-0 and p35S::TCP4-3F6H seedlings are shown. TA3 and LOX2 were used as negative and positive controls, re-spectively. *P # 0.05. ns 5 no significance. E and F, qPCR analysis of FAIRE samples showing the fold enrichment of variousgenomic regions shown in (A) and (B) upon 4 h of DEX induction in p35S::mTCP4;GR seedlings. Averages of biological triplicateswere used. G and H, Schematics of various constructs used in the transient protoplast assay (G) and the relative luciferase activity(H) driven by the wild-type (WT Mock, WT DEX) or mutant (SDM Mock, SDM DEX) pTCL1::LUC construct without (WT Mock,SDMMock) or with (WT DEX, SDM DEX) TCP4 induction by 12 mM of DEX. The 35S::mTCP4:GR effector construct was used todrive TCP4. “WT-Effector” indicates pTCL1::LUC construct alone without 35S::mTCP4:GR. 35S::GUS was used as transfec-tion control. For (H), averages of biological duplicates are shown. Error bars indicate mean 6 SD. Unpaired Student’s t test wasperformed.

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on the tcl1 single mutant leaves was similar to that ofCol-0 leaves (Fig. 5D), perhaps because of functionalredundancy between TCL1 and TCL2 (Gan et al., 2011).Besides, the effect of TCL genes on trichome initiation ismore pronounced at the later part of plant growth, es-pecially on the inflorescence stem (Wang et al., 2007). Asshown earlier (Fig. 1, A, C, and E), trichome density onthe surface of pTCP4::TCP4:VP16 and pBLS::rTCP4:GFPleaves was reduced compared to Col-0 (Fig. 5, D andE). However, TCP4 gain-of-function failed to reducetrichome density on pTCP4::TCP4:VP16 tcl1-1 andpBLS::rTCP4:GFP tcl1-1 leaves (Fig. 5, D and E), suggest-ing a dependence of TCP4 on TCL1, and possibly also onTCL2, in suppressing trichome density on rosette leaves.

Activation of TCLs by the TCP Proteins Is Independentof SPLs

Apart from the class II TCP genes, the miR156-targeted SPL genes are also involved in reducing tri-chome density, primarily on the inflorescence stem, bydirectly activating TCL1 (Yu et al., 2010). However,these SPL proteins on their own are inadequate in ac-tivating TCL1 (Gan et al., 2011), suggesting the in-volvement of additional factors. The demonstrationthat TCP4 and SPL9 proteins form hetero-dimer com-plexes in planta (Rubio-Somoza et al., 2014) raises thepossibility that TCPs activate TCL1 transcription in aSPL-dependent manner. To test this, we analyzed the

level of TCL1 and TCL2 in seedlings where both TCPand SPL transcripts are downregulated. Transcripts ofTCL1 and TCL2were highly reduced in the TCP loss-of-function lines jaw-D and tcp2;tcp4;tcp10 compared tothe Col-0 control (Fig. 3F). When both TCP genes andmiR156-targeted SPL genes were downregulated inthe mock-treated jaw-D;GR 3 miR156 seedlings (heremiR156 indicates miR156-overexpressing line), thetranscript level of TCL1, but not of TCL2, was reduced2-fold compared to the jaw-D;GR3Col-0 control (Fig. 6,A and B). This additive effect of TCP and SPL genes onTCL1 transcript levels implies that the TCP proteinsactivate TCL1 independent of SPL.

To further test the dependence of TCP on SPL,we induced TCP4 by DEX treatment in the jaw-D;GRseedlingswith reduced SPL levels (jaw-D;GR3miR156),or with increased SPL9 activity due to the expression ofa miR156-resistant form of SPL9 under the endogenouspromoter (Yu et al., 2010). Induction of TCP4 increasedthe level of TCL1 and TCL2 transcripts in the Col-0 3jaw-D;GR seedlings by $2-fold (Fig. 6), as also reportedearlier (Fig. 3, B and C). The TCL genes were also ac-tivated when TCP4 was induced in the absence ofmiR156-regulated SPLs in the jaw-D:GR 3 miR156plants, suggesting that TCP proteins do not requireSPL activity to promote TCL transcription. In a com-plementary experiment, a high level of TCL1 tran-script, but not of TCL2 transcript, was detected in themock-treated jaw-D;GR 3 rSPL9 seedlings (Fig. 6, Aand B), where SPL9 is elevated and class II TCP level is

Figure 5. pTCL1::GUS expression in TCP4 gain of function lines. A, Differential interference contrast microscopic images of thirdrosette leaves from 14-d–old Col-03 pTCL1::GUS (Col-0) and pTCP4::TCP4:VP163 pTCL1::GUS (TCP4:VP16) seedlings. Scalebars5 100 mm. B and C, Quantification of pTCL1::GUS expression by 4-methylumbelliferyl-b-D-glucuronide assay in the Col-03 pTCL1::GUS (Col-0) and pTCP4::TCP4:VP16 3 pTCL1::GUS (TCP4:VP16) seedlings (B) and in jaw-D;GR 3 pTCL1::GUSseedlings (C) grown in the absence (Mock) or presence (DEX) of 12-mMDEX. Averages of biological triplicates are shown. D and E,Trichome density on the adaxial surface of first pair of leaves. Averages of trichomes from four to seven leaf pairs are shown. jaw-D;GR indicates pTCP4::mTCP4:GR in the jaw-D background. Error bars indicate mean 6 SD. Unpaired Student’s t test was per-formed. *P # 0.05 and **P # 0.01.

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reduced, suggesting that SPLs activate TCL1 inde-pendent of TCPs.A curious observation in this experiment is that TCP4

induction resulted in a higher fold-increase of TCL1transcript in the absence of SPL activity (in jaw-D;GR 3miR156) than in the presence of SPLs (in jaw-D;GR3Col-0; Fig. 6C). On the contrary, when TCP4 was induced inthe presence of higher SPL9 activity (in jaw-D;GR 3rSPL9), TCL1 transcript was reduced compared to whatwas seen in the presence of normal SPL levels (Fig. 6C).This negative effect of SPL proteins on TCP4-dependentTCL1 activation can be explained by the sequestration ofTCP4 by SPL, possibly mediated by a direct protein–protein interaction between them (Rubio-Somoza et al.,2014). The presence of SPL, however, did not affect TCL2activation by TCP4 (Fig. 6, B and D).

DISCUSSION

Leaf development is broadly classified into threestages; primordia initiation, primary morphogenesis,

and secondary morphogenesis. Primary morphogene-sis involves mainly cell division and the formation ofspecialized cell types on the epidermis such as tri-chomes and stomata, whereas secondary morphogen-esis primarily involves cell expansion and maturation.On the leaf epidermis, how the three diverse cell types(trichomes, stomata, and pavement cells) arise from theepidermal progenitor cells and are patterned have beenstudied independently (Balkunde et al., 2010; Dong andBergmann, 2010; Schommer et al., 2014), yet whetherand how these processes are coordinated during theprimary morphogenesis is less clear. The CIN–TCPclade members of the TCP proteins are known for theirrole in temporal coordination between primary andsecondary morphogenesis (Efroni et al., 2008). Loss ofthese TCP proteins prolongs the primary leaf mor-phogenesis without affecting the entry into secondarymorphogenesis, thus allowing the leaves to remain inthe proliferation phase for a longer duration. The majorcell type affected by mutations in these genes is the leafpavement cell (Nath et al., 2003; Schommer et al., 2014).

Figure 6. TCPactivates TCL1 independent of SPL.A and B, Relative transcript levels of TCL1 (A) andTCL2 (B) determined by RT-qPCR in 12-d–old F1seedlings of indicated genotypes. Averages of bi-ological triplicates are shown. Error bars indicatemean 6 SD. Unpaired Student’s t test was per-formed. *P # 0.05. C and D, Levels of TCL1 andTCL2 transcripts upon DEX induction relative tomock-induction calculated from the data shownin (A) and (B). Averages of biological triplicates areshown. E, A proposed model for the regulation oftrichome initiation by TCP4. TCP4 binds to theupstream regulatory region of TCLs and activatestheir transcription. The TCL proteins bind to GL3(Gan et al., 2011), as do the other single-repeatMYB-transcription factors such as TRY, CPC, andETC1, ETC2, and ETC3, and dislocate GL1 fromthe trichome initiation complex (TTG1-GL3/EG3-GL1), thus rendering it inactive. TCL1 also binds tothe GL1 locus and represses its transcription,thereby reducing the level of GL1, which is re-quired for trichome initiation (Wang et al., 2007).

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However, recent reports have identified a role for theCIN–TCP proteins in the differentiation of trichome andhypocotyl epidermal cells as well (Challa et al., 2016;Vadde et al., 2018). Here, we show that the CIN–TCPproteins reduce the density of trichomes in Arabi-dopsis leaves by suppressing their initiation via directtranscriptional activation of TCL1 and TCL2 (Fig. 6E).The TCL1 and TCL2 proteins are proposed to diffuseto the neighboring cells and destabilize the initiatorcomplex (Wang et al., 2008b; Gan et al., 2011), therebyretaining their pavement cell identity. Thus, class IITCP proteins are upstream regulators of multiple celltypes and couple proliferation with patterning.

The molecular basis of trichome initiation and spac-ing has been studied in detail and an elegant mecha-nism has been put forth (Pesch and Hülskamp, 2009;Balkunde et al., 2010). According to this, a stochasticdifference in the level of a trichome initiation complex isamplified beyond a threshold by autoactivation, lead-ing to the activation of the key trichome differentiationmolecule, GL2. When a few epidermal progenitor cellsin the leaf primordium are randomly committed totrichome fate, they also produce mobile factors thatdiffuse to the neighboring cells and inactivate the ini-tiation complex, thereby retaining their default pave-ment cell identity (Digiuni et al., 2008; Zhao et al., 2008).Therefore, the level of these trichome-repressing mole-cules determine trichome density in mature leaves. Wehave limited knowledge on the genetic factors thattranscriptionally promote these repressors, especiallyTCL1 andTCL2. Ourwork demonstrates that, in additionto controlling primary leaf morphogenesis (Sarvepalliand Nath, 2011; Schommer et al., 2014), the class II TCPfactors limit trichome initiation by activating TCL1 andTCL2. Because TCL1 and TCL2 proteins are thought todiffuse to neighboring cells, this work implicates the classII TCP factors in cell-cell communication within the leafepidermis.

There is a spatial difference among the transcrip-tional activations of the negative regulators of tri-chome initiation. TRY, CPC, and ETC are activated bythe initiator complex itself (Digiuni et al., 2008; Zhaoet al., 2008), whereas TCLs are activated by the het-erochronic developmental factors such as TCPs andSPLs (Yu et al., 2010; Tian et al., 2017) and this work).Because the initiator complex possesses autoactiva-tion property, it is likely that TRY, CPC, and ETC areactivated in discrete cells throughout the leaf pri-mordium, concomitant to acquiring the trichomefate by the pavement progenitor cells. The activationof other negative regulators of trichome initiation,notably ETC2, TCL1, and TCL2, is independent ofthe initiation complex (Morohashi et al., 2007; Wanget al., 2008b). Activation of the TCL genes by SPL9(Yu et al., 2010) and TCP4 (this study) suggests thattheir transcripts appear in all pavement progeni-tor cells before trichome fate determination. Becausethe CIN–TCP genes are abundant within the transi-tion zone of a leaf primordium (Nath et al., 2003,Das Gupta et al., 2014), their inhibitory function on

trichome initiation is likely to be initiated withinthis zone.

There is a temporal difference between TCL acti-vation by the miR319-regulated TCP proteins and themiR156-regulated SPL proteins. The SPL expressionprogressively increases with the developmental ma-turity of plants as the abundance of miR156 declines(Wu et al., 2009; Xu et al., 2016) and consequently theypromote TCL expression primarily in later organs suchas inflorescence (Xu et al., 2016). By contrast, the CIN–TCP genes are expressed in lateral organ primordiafrom the embryonic stage onward (Palatnik et al., 2003)and therefore promote TCL expression in early rosetteleaves. This temporal difference between TCP and SPLfactors suggest that their action on the TCL genes isindependent, an observation that is supported by ourgenetic interaction analysis (Fig. 6).

Genetic interaction studies between TCP4 gain-of-function lines and tcl1-1 showed that TCP4 requiresTCLs to suppress trichome initiation. The CIN–TCPproteins interact with BRAHMA (BRM), a chromatinremodeler, in regulating leaf maturation (Efroni et al.,2013). When a miR319-resistant rTCP4 is expressed inyoung leaf primordia under the BLS promoter, tri-chome density is reduced on the leaf surface. How-ever, rTCP4 expression under BLS promoter in thebrm-106 mutant background fails to reduce trichomedensity. The recruitment of BRM protein in the TCL2locus has been demonstrated (Li et al., 2016). Thissuggests that TCL activation by TCP4 is dependent onchromatin remodelers, indicating a layer of epigeneticregulation on TCL expression.

The class II TCP proteins are primarily known fortheir role in inducing pavement cell maturity in leavesby activating KIP-related cell cycle inhibitor-encodinggenes (Schommer et al., 2014) and ARABIDOPSISRESPONSE REGULATOR16 (ARR16), a negative reg-ulator of cytokinin signaling (Efroni et al., 2013).ARR16has recently been shown to reduce stomatal density inleaves (Vatén et al., 2018). Our previous and currentwork show that these TCP proteins also reduce tri-chome density (Fig. 1) and branching (Vadde et al.,2018) in rosette leaves by recruiting independent tran-scriptional targets. Thus, the TCP proteins act as gen-eral regulators of epidermal cell morphogenesis bydirect promotion of cell-type–specific target genes.

CONCLUSION

In summary, we have identified the CIN–TCP pro-teins as direct activators of TCL1 and TCL2, the twogenes that redundantly suppress trichome initiation onrosette leaf surface. TCP4 binds to the upstream regu-latory elements of TCL1 and TCL2 and induces changesin chromatin conformation leading to TCL transcription.This TCP-mediated TCL activation is independent ofSPL transcription factors, which also activate TCL genes.Thus, the miR319-regulated class II TCP proteins coupleleaf morphogenesis with epidermal cell patterning.

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MATERIALS AND METHODS

Growth Conditions and Plant Materials

All Arabidopsis (Arabidopsis thaliana) transgenic andmutant lines are in Col-0 accession. Seeds were surface-sterilized with 0.05% (w/v) SDS dissolved in70% (v/v) ethanol for 10 min, washed two to three times with 100% (v/v)ethanol, air-dried, and sown on 13 Murashige and Skoog (MS) medium(Himedia) plates. For DEX experiments, a 25-mM DEX stock was prepared in100% (v/v) ethanol and a final concentration of 12-mM DEX was added to MSmedium and 100% (v/v) ethanol was used as a Mock control. Seeds were firststratified for 3 d in dark at 4°C and then shifted to a growth chamber (22°C to23°C, 16-h/8-h light/dark cycle regime) for germination. After 12 to 14 d ofpostgermination, seeds were transplanted to soil and kept under the samegrowth conditions (22°C to 23°C, 16-h/8-h light/dark cycle regime) until seedswere collected.

The jaw-D, pTCP4::TCP4:VP16, pTCP4::mTCP4:GR, p35S::mTCP4:GR, tcp2tcp4 tcp10, pBLS::rTCP4:GFP, p35S::miR156, and pSPL9::rSPL9:GFP lines werereported in Palatnik et al. (2003), Wang et al. (2008a, 2009), Sarvepalli and Nath(2011), Efroni et al. (2013), Karidas et al. (2015), and Challa et al. (2016). All GUSreporter lines used in this study (pGL2::GUS, pTRY::GUS, pTCL1::GUS) werereported by Szymanski et al. (1998) and Yu et al. (2010). The try mutant lineswere obtained from the Arabidopsis Stock Center (http://arabidopsis.org/)and the insertions were confirmed by PCR using gene- and T-DNA–specificprimers. The combinations of TCP4:VP16;tcl1-1, pBLS::rTCP4;tcl1-1, and tcp2tcp4 tcp10 pTCP4::mTCP4:GR mutant lines were generated by crossing the re-spective parents and selecting in F3 or F4 generation by genotyping.

Trichome Counting and Trichome Density

Trichome counting was performed on the adaxial side of the first pair ofleaves, or the eighth leaf, under a lightmicroscope. For determining the trichomedensity, mature adaxial trichomes were observed under a model no. M3Z LightMicroscope (Wild Heerbrugg), the number of trichomes was noted, and thedensity was expressed as number of trichomes per unit area.

For measuring the fraction of trichomes per unit pavement cell, we firstdetermined the epidermal cell size on the adaxial surface of thefirst pair of leavesby measuring the areas of 100 to 150 cells per leaf and averaging them. Thenumber of pavement cells per leaf was derived by dividing the leaf area by theaverage pavement cell area, as reported by Karidas et al. (2015). After countingthe total number of trichomes on the adaxial surface, we derived the fraction ofepidermal cells forming trichomes using the formula “number of trichomes/number of pavement cells.” Stomata were ignored for this analysis. We carriedout this analysis for all genotypes with at least four biological samples.

DEX-Induction Experiment

For continuous DEX induction, 12-d–old seedlings grown on 12 mM ofDEX-containing MS plates were transplanted to DEX or ethanol-treated soil,and a solution containing DEX (12 mM) or ethanol (Mock) was sprayed on theplants on alternate days until the experiment was complete (Aggarwal et al.,2018). For CHX treatment, 12-d–old jaw-D;pTCP4:mTCP4:GR seedlings weretreated with either 40 mM of CHX alone or a combination of CHX (40 mM) andDEX (20 mM) for 4 h.

RNA Isolation and cDNA Synthesis

Total RNAwas isolated from plant samples using the trizol method (Sigma-Aldrich), treated with DNase (Fermentas) for 2 h, and RNA was precipitatedfor further use. RNA concentration was estimated using a NanoDrop 1000Spectrophotometer (Thermo Fisher Scientific) and 2 mg of RNA was reverse-transcribed to cDNA using Revert Aid M-MuLV reverse transcriptase (Fer-mentas) according to the manufacturer’s instructions. PCR reactions wereperformed in a 20-mL total volume with the synthesized cDNA as templates inthe presence of required primer combinations. PCR products after 30 to 33cycles were visualized on an ethidium bromide-stained 1% (w/v) agarose gel.

RT-qPCR Analysis

Twenty-five nanograms of cDNA was used for all quantification studies.RT-qPCR was performed using SYBR Green RT-qPCR kit (Kapa SYBR Fast

qPCR Kits; Kapa Biosystems) in a 10-mL reaction volume according to themanufacturer’s protocol. Data analysis was performed using the ABI Prism7900HT Sequence Detection System (Applied Biosystems). Expression ofPROTEIN PHOSPHATASE 2A was used for normalization. Primers usedfor RT-qPCR analysis are listed in Supplemental Table S1.

EMSA

The hexa-His-TCP4 fusion protein used for EMSA in this study wasexpressed from the vector pRSET-C-TCP4 (Aggarwal et al., 2010). Oligonu-cleotides were end-labeled with [g-32P]-ATP using T4 polynucleotide kinase(Thermo Fisher Scientific). The DNA–protein binding reaction was per-formed in a 15-mL reaction volume containing an oligonucleotide probe, 13binding buffer (20 mM of HEPES-KOH at pH 7.8, 100 mM of KCl, 1 mM ofEDTA, 0.1% [w/v] bovine serum albumin, 10 ng of herring sperm DNA, and10% [v/v] glycerol), and ;2 mg of crude bacterial lysate with recombinantprotein. The binding reaction mixture was incubated for 30 min at 25°C andloaded on a 9% native polyacrylamide gel. Electrophoresis was conducted at4 V/cm for 1 h in 1 3 Tris-borate-EDTA buffer at room temperature. The gelwas autoradiographed using a phosphor-image cassette for 4–6 h and ana-lyzed using a phosphor-imager (BAS-2000; Fuji Film). The sequences of theEMSA probes are listed in Supplemental Table S2.

ChIP

ChIPwas performed according toGendrel et al. (2005). Fourteen-d–oldCol-0and p35S::TCP4-3F6H (Kubota et al., 2017) seedlings were fixed with 1% (v/v)formaldehyde in ChIP buffer 1 under vacuum for 10 min and the reaction wasquenched by adding 2 M of Gly. Chromatin was isolated and sheared using aBioruptor (Diagenode), with 20 s on/30 s off, for 40 cycles at high frequency.Sheared chromatin was diluted 10 times in ChIP Dilution buffer and incubatedwith anti-FLAG antibody (cat. no. F1804; Sigma-Aldrich) andwithout antibody(as a no-antibody control) overnight. Immunocomplexes were pulled down byProtein G Dynabeads (cat. no. 10003D; Thermo Fisher Scientific) and eluted inTris-EDTA buffer. The immune complexes were de-cross-linked and DNA wasprecipitated by the sodium acetate method (McCullough et al., 2017). Theprecipitated DNAwas directly used for qPCR analysis. The list of primers usedin this experiment is listed in Supplemental Table S3.

FAIRE

FAIRE protocol was followed as mentioned in Omidbakhshfard et al. (2014)and Challa et al. (2016) with slight modifications. Two grams of 14-d–oldp35S::mTCP4:GR seedlings was treated with Mock and DEX (20 mM) solutionfor 4 h and cross-linked with 1% (v/v) formaldehyde solution. The genotypesTCP4:VP16 and Col-0 without any treatment were fixed in 1% (v/v) for-maldehyde and used for downstream processing. The regulatory elementswere isolated as mentioned in Omidbakhshfard et al. (2014). TA3 was used asan internal control. A list of all primers used in this experiment is given inSupplemental Table S3.

Transient Luciferase Assay

For generating the reporter constructs, 1.6 Kb of TCL1 promoter was am-plified using primers with overhangs of BamHI and NcoI sites and cloned intothe pARR6::LUC construct (Hwang and Sheen, 2001) by replacing the ARR6promoter. The primers used in this experiment are listed in Supplemental TableS4. For the mutant reporter construct, the TCP4-binding GTGGCCA sequencein the BS1.1 cis-element was mutated to GTATCCA by standard site-directedmutagenesis protocol (Shenoy and Visweswariah, 2003). Complementaryoverlapping primers with the mutated nucleotides were used for inverse PCRon the wild-type pTCL1::LUC construct as a template. The effector construct35S::mTCP4:GR has been by Challa et al. (2016). The internal control used fortransfection was the pCAMBIA 1301 plasmid expressing the GUS reporterunder the 35S cauliflower mosaic virus promoter.

The transient protoplast assay was carried out as mentioned by Yoo et al.(2007). Arabidopsis mesophyll protoplastswere isolated from the fifth and sixthleaves of 4-week–old Col-0 plants grown under long-day conditions (16-h/8-h,light/dark cycle). Polyethylene glycol-mediated transfection was carried outwith 20 mg of plasmid DNA containing effector plasmid, reporter plasmid, andinternal control in a ratio of 5:4:1 in a 10-mL reaction volume for 30min followed

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by incubation in dark at 22°C for 24 h. A luciferase reporter assay system(Promega) was used for assaying luciferase activity. Luminescence was mea-sured in a TD-20/20 Luminometer (Turner Designs). GUS assaywas performedon the protoplast lysatesmixedwith the substrate 1mM of 4-methylumbelliferylglucuronide and 2mM of CaCl2 in 10mM of Tris (pH 8). The reactionwas carriedout at 37°C for 60min and then 0.2 M of sodium carbonate was added to stop thereaction. The fluorescence of the product methylumbelliferone was measuredwith excitation at 365 nm and emission at 455 nm using an Infinite M200 PlateReader (Tecan Trading).

GUS Staining and Quantification

GUS assays on plant tissue were performed as described in Sessions et al.(1999) and Karidas et al. (2015) with slight modifications. Samples were col-lected in 90% (v/v) acetone, incubated at room temperature for 30 min withgentle shaking, followed by washing with staining buffer (50 mM of sodiumphosphate at pH 7.0, 0.2% [v/v] Triton X-100, 5 mM of potassium ferrocyanide,and 5 mM of potassium ferricyanide). Fresh staining buffer was added alongwith 2mMX-Gluc and incubated at 37°C overnight. Next day, the samples werewashed with 70% (v/v) ethanol twice and left in the same solution until itcleared, after which they were observed under a model no. BX51DIC micro-scope (Olympus). GUS quantification was done as described in Weigel andGlazebrook (2002).

Statistical Analysis

Statistical analysis was performed using unpaired Student’s t test in the soft-ware GraphPad Prism V (GraphPad, https://www.graphpad.com/scientific-software/prism/). The sample numbers and replicates are provided in the figurelegends.

Accession Numbers

The accession numbers of the genes mentioned in this article are givenhere and their sequence data can be found in Arabidopsis Genome Initiative(www.arabidopsis.org). At3g15030 (TCP4), At2g30432 (TCL1), At2g30424(TCL2), At2g42200 (SPL9), At5g24520 (TTG1), At3g27920 (GL1), At1g79840(GL2), At5g41315 (GL3), At1g63650 (EGL3), At5g53200 (TRY), At2g46410(CPC), At1g01380 (ETC1), At2g30420 (ETC2), At4g01060 (ETC3), At3g58070(GIS), At3g45140 (LOX2).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Trichome density phenotype in the mutant linesof TCP genes.

Supplemental Figure S2. Trichome density upon TCP4 induction in theCol-0 background.

Supplemental Figure S3. Expression levels of TCL1, 2 in inducible linesof TCP4.

Supplemental Figure S4. FAIRE experiment to determine open or closestate of TCL chromatin.

Supplemental Figure S5. pCPC::GUS expression pattern in TCP4:VP16seedlings.

Supplemental Table S1. List of primers used for RT-qPCR analysis.

Supplemental Table S2. List of primers used for EMSA experiment.

Supplemental Table S3. List of primers used for ChIP and FAIRE.

Supplemental Table S4. List of primers used for transient luciferase assay.

ACKNOWLEDGMENTS

We thank Xiao-Ya Chen (Chinese Academy of Sciences, Beijing, China),Detlef Weigel (Max Planck Institute, Tübingen, Germany), and Yuval Eshed

(Weizmann Institute of Science, Israel) for pTCL1::GUS, jaw-D, andpBLS::rTCP4:GFP lines, respectively; Arabidopsis Biological Resource Cen-ter (www.arabidopsis.org) for the T-DNA insertion lines; and NottinghamArabidopsis Stock Centre (http://arabidopsis.info/BasicForm) for pCPC::GUS line.

Received February 14, 2019; accepted September 9, 2019; published October 1,2019.

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