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• W2991283314 abstract "•Identification of genome-wide target genes for the RCO transcription factor•RCO delimits its own expression through autorepression by low-affinity binding•RCO represses local leaf growth via regulating multiple cytokinin (CK)-related genes•RCO negative autorepression fine-tunes CK activity and regulates leaf shape Mechanisms through which the evolution of gene regulation causes morphological diversity are largely unclear. The tremendous shape variation among plant leaves offers attractive opportunities to address this question. In cruciferous plants, the REDUCED COMPLEXITY (RCO) homeodomain protein evolved via gene duplication and acquired a novel expression domain that contributed to leaf shape diversity. However, the molecular pathways through which RCO regulates leaf growth are unknown. A key question is to identify genome-wide transcriptional targets of RCO and the DNA sequences to which RCO binds. We investigate this question using Cardamine hirsuta, which has complex leaves, and its relative Arabidopsis thaliana, which evolved simple leaves through loss of RCO. We demonstrate that RCO directly regulates genes controlling homeostasis of the hormone cytokinin to repress growth at the leaf base. Elevating cytokinin signaling in the RCO expression domain is sufficient to both transform A. thaliana simple leaves into complex ones and partially bypass the requirement for RCO in C. hirsuta complex leaf development. We also identify RCO as its own target gene. RCO directly represses its own transcription via an array of low-affinity binding sites, which evolved after RCO duplicated from its progenitor sequence. This autorepression is required to limit RCO expression. Thus, evolution of low-affinity binding sites created a negative autoregulatory loop that facilitated leaf shape evolution by defining RCO expression and fine-tuning cytokinin activity. In summary, we identify a transcriptional mechanism through which conflicts between novelty and pleiotropy are resolved during evolution and lead to morphological differences between species. Mechanisms through which the evolution of gene regulation causes morphological diversity are largely unclear. The tremendous shape variation among plant leaves offers attractive opportunities to address this question. In cruciferous plants, the REDUCED COMPLEXITY (RCO) homeodomain protein evolved via gene duplication and acquired a novel expression domain that contributed to leaf shape diversity. However, the molecular pathways through which RCO regulates leaf growth are unknown. A key question is to identify genome-wide transcriptional targets of RCO and the DNA sequences to which RCO binds. We investigate this question using Cardamine hirsuta, which has complex leaves, and its relative Arabidopsis thaliana, which evolved simple leaves through loss of RCO. We demonstrate that RCO directly regulates genes controlling homeostasis of the hormone cytokinin to repress growth at the leaf base. Elevating cytokinin signaling in the RCO expression domain is sufficient to both transform A. thaliana simple leaves into complex ones and partially bypass the requirement for RCO in C. hirsuta complex leaf development. We also identify RCO as its own target gene. RCO directly represses its own transcription via an array of low-affinity binding sites, which evolved after RCO duplicated from its progenitor sequence. This autorepression is required to limit RCO expression. Thus, evolution of low-affinity binding sites created a negative autoregulatory loop that facilitated leaf shape evolution by defining RCO expression and fine-tuning cytokinin activity. In summary, we identify a transcriptional mechanism through which conflicts between novelty and pleiotropy are resolved during evolution and lead to morphological differences between species. cis-regulatory variation of developmental genes plays a pivotal role in morphological evolution of plants and animals and often involves diversification of transcriptional enhancers [1Carroll S.B. Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution.Cell. 2008; 134: 25-36Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar, 2Chan Y.F. Marks M.E. Jones F.C. Villarreal Jr., G. Shapiro M.D. Brady S.D. Southwick A.M. Absher D.M. Grimwood J. Schmutz J. et al.Adaptive evolution of pelvic reduction in sticklebacks by recurrent deletion of a Pitx1 enhancer.Science. 2010; 327: 302-305Crossref PubMed Scopus (685) Google Scholar, 3Frankel N. Erezyilmaz D.F. McGregor A.P. Wang S. Payre F. Stern D.L. Morphological evolution caused by many subtle-effect substitutions in regulatory DNA.Nature. 2011; 474: 598-603Crossref PubMed Scopus (142) Google Scholar, 4Gompel N. Prud’homme B. Wittkopp P.J. Kassner V.A. Carroll S.B. Chance caught on the wing: cis-regulatory evolution and the origin of pigment patterns in Drosophila.Nature. 2005; 433: 481-487Crossref PubMed Scopus (487) Google Scholar, 5Hay A. Tsiantis M. The genetic basis for differences in leaf form between Arabidopsis thaliana and its wild relative Cardamine hirsuta.Nat. Genet. 2006; 38: 942-947Crossref PubMed Scopus (288) Google Scholar, 6Indjeian V.B. Kingman G.A. Jones F.C. Guenther C.A. Grimwood J. Schmutz J. Myers R.M. Kingsley D.M. Evolving new skeletal traits by cis-regulatory changes in bone morphogenetic proteins.Cell. 2016; 164: 45-56Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 7Rast-Somssich M.I. Broholm S. Jenkins H. Canales C. Vlad D. Kwantes M. Bilsborough G. Dello Ioio R. Ewing R.M. Laufs P. et al.Alternate wiring of a KNOXI genetic network underlies differences in leaf development of A. thaliana and C. hirsuta.Genes Dev. 2015; 29: 2391-2404Crossref PubMed Scopus (52) Google Scholar, 8Studer A. Zhao Q. Ross-Ibarra J. Doebley J. Identification of a functional transposon insertion in the maize domestication gene tb1.Nat. Genet. 2011; 43: 1160-1163Crossref PubMed Scopus (447) Google Scholar]. Regulatory sequence variation is believed to facilitate morphological change while minimizing the potentially adverse effects of pleiotropy—the phenomenon by which a single gene influences multiple aspects of development [1Carroll S.B. Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution.Cell. 2008; 134: 25-36Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar, 9Doebley J. Lukens L. Transcriptional regulators and the evolution of plant form.Plant Cell. 1998; 10: 1075-1082Crossref PubMed Scopus (353) Google Scholar, 10Stern D.L. Orgogozo V. Is genetic evolution predictable?.Science. 2009; 323: 746-751Crossref PubMed Scopus (357) Google Scholar]. However, the precise mechanisms that link cis-regulatory changes to morphological diversity remain poorly understood [11Rebeiz M. Patel N.H. Hinman V.F. Unraveling the tangled skein: the evolution of transcriptional regulatory networks in development.Annu. Rev. Genomics Hum. Genet. 2015; 16: 103-131Crossref PubMed Scopus (49) Google Scholar, 12Rebeiz M. Tsiantis M. Enhancer evolution and the origins of morphological novelty.Curr. Opin. Genet. Dev. 2017; 45: 115-123Crossref PubMed Scopus (48) Google Scholar]. For example, do cis-regulatory changes at transcription factor loci cause specific effects on downstream gene expression or global transcriptome remodeling? Do these transcriptional changes affect few genes with large effects on development or a multitude of downstream processes with small effect? And how do cis-regulatory changes circumvent pleiotropy, given that transcriptional enhancers can show considerable pleiotropy despite their modularity [13Preger-Ben Noon E. Davis F.P. Stern D.L. Evolved repression overcomes enhancer robustness.Dev. Cell. 2016; 39: 572-584Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar]? Recent work indicates that low-affinity transcription factor binding sites play a vital role in fine-tuning developmental gene expression, thus ensuring its specificity and robustness [14Crocker J. Abe N. Rinaldi L. McGregor A.P. Frankel N. Wang S. Alsawadi A. Valenti P. Plaza S. Payre F. et al.Low affinity binding site clusters confer hox specificity and regulatory robustness.Cell. 2015; 160: 191-203Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar]. These sites can evolve rapidly and therefore might also have important yet undiscovered roles in morphological evolution [15Crocker J. Noon E.P. Stern D.L. The soft touch: low-affinity transcription factor binding sites in development and evolution.Curr. Top. Dev. Biol. 2016; 117: 455-469Crossref PubMed Scopus (63) Google Scholar]. Plant leaves provide a powerful model in which to explore such questions on the mechanistic basis of evolutionary change because they show substantial, heritable morphological variation at different evolutionary scales and are ecophysiologically important because they fix CO2 in terrestrial ecosystems [16Bar M. Ori N. Leaf development and morphogenesis.Development. 2014; 141: 4219-4230Crossref PubMed Scopus (153) Google Scholar, 17Maugarny-Calès A. Laufs P. Getting leaves into shape: a molecular, cellular, environmental and evolutionary view.Development. 2018; 145 (dev161646)Crossref PubMed Scopus (40) Google Scholar]. Considerable insights into the genetic basis for diversification of leaf shape have come from comparative studies of simple leaves of the reference plant A. thaliana versus complex leaves of its relative Cardamine hirsuta, where leaves are divided into distinct units called leaflets. The RCO gene was discovered in C. hirsuta on the basis of its simplified mutant phenotype, and RCO-type genes support development of leaf marginal outgrowths of different sizes in different crucifer species. RCO arose by duplication of its ancestral paralog LATE MERISTEM IDENTITY 1 (LMI1), which is conserved in seed plants, and its emergence promoted evolution of leaf complexity in crucifers [18Vlad D. Kierzkowski D. Rast M.I. Vuolo F. Dello Ioio R. Galinha C. Gan X. Hajheidari M. Hay A. Smith R.S. et al.Leaf shape evolution through duplication, regulatory diversification, and loss of a homeobox gene.Science. 2014; 343: 780-783Crossref PubMed Scopus (184) Google Scholar]. Neofunctionalization of an RCO enhancer element (RCOenh500) altered leaf shape by changing RCO expression from the distal leaf blade to the leaf base [19Vuolo F. Mentink R.A. Hajheidari M. Bailey C.D. Filatov D.A. Tsiantis M. Coupled enhancer and coding sequence evolution of a homeobox gene shaped leaf diversity.Genes Dev. 2016; 30: 2370-2375Crossref PubMed Scopus (39) Google Scholar]. In this domain, RCO represses growth in a series of foci along the leaf margin, allowing the outgrowth of lobes or leaflets between flanking regions of RCO expression [20Kierzkowski D. Runions A. Vuolo F. Strauss S. Lymbouridou R. Routier-Kierzkowska A.-L. Wilson-Sánchez D. Jenke H. Galinha C. Mosca G. et al.A growth-based framework for leaf shape development and diversity.Cell. 2019; 177: 1405-1418.e17Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar]. RCO was secondarily lost from the A. thaliana genome, leading to leaf simplification. However, its reintroduction in A. thaliana as a transgene was sufficient to increase leaf complexity, demonstrating that RCO is a large-effect gene underlying morphological evolution. However, the downstream effector genes through which RCO acts to repress growth and the upstream transcriptional inputs that delimit RCO expression are both unknown. To address these issues, we identified genome-wide RCO target genes by combining chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) experiments. We further conducted functional validation of our findings using genetics, molecular biology, microscopy, and hormone measurements. In this way, we show that RCO coordinates homeostasis of the hormone cytokinin (CK) through direct regulation of multiple genes involved in CK biosynthesis and catabolism and provide evidence that this RCO/CK module is required for complex leaf development. In parallel, we show that RCO directly delimits its own expression through binding to clusters of low-affinity repressive sites in its 5′ upstream regulatory region and gene body and that this autoregulatory loop also shapes CK activity in the leaf. Thus, a paradigm emerges whereby low-affinity binding sites facilitated morphological evolution by dampening the effects of cis-regulatory divergence in a potent transcription factor. We propose that this regulatory architecture allowed regulatory evolution to fine-tune levels of hormonal homeostasis and circumvent pleiotropy. To understand the molecular basis of RCO’s action in C. hirsuta, we performed ChIP-seq profiling in three biological replicates of C. hirsuta rco mutant plants expressing pRCO::RCOgenomic-VENUS using an anti-GFP antibody (see STAR Methods for details). We identified 598 binding peaks for 592 potential target genes, which showed a consistent binding pattern across all biological replicates (Data S1A). RCO is among the top 10 potential target genes of RCO, according to ranking of peaks by p value, and RCO associates with chromatin at its own promoter and gene body in a single broad peak (Figures 1A and S1; Data S1A). By contrast, a single narrow peak was detected at the 3′ end of LMI1—the ancestral paralog of RCO (Figures 1A and S1; Data S1A). To test whether RCO regulates its own expression, we compared RCO expression in transgenic rco mutant lines complemented with either the native version of RCO or the stabilized form of RCO (RCOA48D) [19Vuolo F. Mentink R.A. Hajheidari M. Bailey C.D. Filatov D.A. Tsiantis M. Coupled enhancer and coding sequence evolution of a homeobox gene shaped leaf diversity.Genes Dev. 2016; 30: 2370-2375Crossref PubMed Scopus (39) Google Scholar]. In the RCOA48D lines where protein levels are elevated relative to native RCO [19Vuolo F. Mentink R.A. Hajheidari M. Bailey C.D. Filatov D.A. Tsiantis M. Coupled enhancer and coding sequence evolution of a homeobox gene shaped leaf diversity.Genes Dev. 2016; 30: 2370-2375Crossref PubMed Scopus (39) Google Scholar], we found reduced RCO transcript levels compared to rco mutant lines expressing the native RCO gene (Figure 1B). This indicates that RCO might undergo negative autoregulation, an idea further supported by the observation that a single-copy RCO::GUS reporter gene showed broader and stronger expression in rco leaves compared to wild type (Figures 1C and 1C′). Together, these results suggest that RCO represses its own expression. If RCO binds to the RCO locus to negatively regulate its own expression, then deletion or randomization of these binding sites should increase RCO expression. To test this prediction, we selected 4 binding candidate fragments (BCFs): three fragments with the highest read coverage in the promoter region and the first intron and another fragment covering the whole second intron (Figure S2A). We engineered RCO promoter fragments in which the BCF1 and BCF2 were deleted, randomized, or replaced by the corresponding LMI1 sequence, where RCO is not expected to bind. Each modified RCO promoter was fused to both RCO-coding sequence (RCO-CDS) lacking BCF3 and BCF4 and the genomic sequence (including BCF3 and BCF4), including introns that harbor RCO-binding sites (RCOgenomic), from the ATG-start to the stop codon (Figures S2A and S2G). All constructs, including controls bearing the native promoter sequence, were transformed into the rco mutant background. We observed that removing peaks or modifying them, as described above, increased RCO expression (Figure S2C). In rco mutant plants containing the modified RCO promoters fused to RCO-CDS, which caused higher RCO expression, leaf shape was significantly altered—leaf dissection increased while leaf area and seed mass decreased (Figures S2B–S2F). Notably, transgenic rco mutant plants that expressed RCOgenomic (ATG-stop) driven by modified RCO promoters produced either wild-type or partially rescued leaves (Figures S2G and S2H). This indicates that deleting or modifying BCFs 1 and 2 within the RCO promoter sequence is insufficient to alter leaf development if BCFs 3 and 4 are still present in the gene body. Nevertheless, these constructs still rescued the rco mutant phenotype more effectively than the control construct (Figures S2G and S2H). In summary, RCO autorepression depends on multiple sites in its genomic locus and perturbing this autorepression has detrimental effects on leaf development and plant performance. We next sought to understand the cis-regulatory logic that underlies RCO autorepression. We first used protein-binding microarrays (PBMs) to identify the DNA-binding motifs that RCO binds to with high affinity in vitro (Figure S3A; Data S1B). We then constructed position weight matrices (PWMs) using these sequences, which we refer to as canonical binding sites [22Franco-Zorrilla J.M. López-Vidriero I. Carrasco J.L. Godoy M. Vera P. Solano R. DNA-binding specificities of plant transcription factors and their potential to define target genes.Proc. Natl. Acad. Sci. USA. 2014; 111: 2367-2372Crossref PubMed Scopus (404) Google Scholar]. However, when we scanned the peak sequences at the RCO locus with the PBM-derived PWMs using FIMO (default settings), we did not find canonical RCO-binding sites [23Grant C.E. Bailey T.L. Noble W.S. FIMO: scanning for occurrences of a given motif.Bioinformatics. 2011; 27: 1017-1018Crossref PubMed Scopus (1869) Google Scholar, 24Ma W. Noble W.S. Bailey T.L. Motif-based analysis of large nucleotide data sets using MEME-ChIP.Nat. Protoc. 2014; 9: 1428-1450Crossref PubMed Scopus (115) Google Scholar]. Yet we found canonical sites in 64 of the genome-wide identified RCO-binding peaks. These observations suggest that RCO-DNA binding may be influenced by protein interactions and/or the chromatin context, as is the case for other transcription factors [25Barrilleaux B.L. Burow D. Lockwood S.H. Yu A. Segal D.J. Knoepfler P.S. Miz-1 activates gene expression via a novel consensus DNA binding motif.PLoS ONE. 2014; 9: e101151Crossref PubMed Scopus (15) Google Scholar, 26Inukai S. Kock K.H. Bulyk M.L. Transcription factor-DNA binding: beyond binding site motifs.Curr. Opin. Genet. Dev. 2017; 43: 110-119Crossref PubMed Scopus (122) Google Scholar, 27Slattery M. Ma L. Négre N. White K.P. Mann R.S. Genome-wide tissue-specific occupancy of the Hox protein Ultrabithorax and Hox cofactor Homothorax in Drosophila.PLoS ONE. 2011; 6: e14686Crossref PubMed Scopus (61) Google Scholar]. To identify RCO-binding sites within the RCO locus, we used electrophoretic mobility shift assays (EMSAs) with native and mutagenized oligonucleotides to screen the four BCFs selected previously. In this way, we identified 11 RCO-binding sites residing on nine 55-bp fragments (Figure 2). These sites are AT-rich, a feature shared with both the canonical binding sites identified in the PBM and a motif enriched in RCO-bound ChIP-seq peaks (Figures 2A, 2B, S3B, and S3C; Data S1B). These 11 RCO-binding sites in the RCO locus showed concentration-dependent binding to RCO (Figures 2A and 2B) with 2 of the 11 sites located within RCOenh500 (Figures 2A and S4A). All the 11 binding sites show considerable conservation in crucifers (Figure S4B). Statistical analysis (STAR Methods) indicated that evolution of an array of low-affinity, but not high-affinity, binding sites in RCO is unlikely to reflect chance (Pneut(X = 0) = 0.024, where Pneut is the probability of observing X high-affinity binding sites under a neutral evolutionary model for the RCO/LMI1 duplication). In turn, this suggests that selective processes likely prevented accumulation of high-affinity binding sites in RCO while permitting emergence of low-affinity ones. Overall, these findings suggest that RCOenh500 co-evolved with an array of low-affinity RCO-binding sites to provide specificity to RCO expression via negative autoregulation. To test this hypothesis and understand the mechanism of action of these native RCO-binding sites, we compared their binding affinity to that of the canonical RCO binding sites defined by PBMs. We did this by performing EMSAs with native oligonucleotide sequences and oligos containing the canonical binding site (CAATAATT). Oligos with the native sites were outcompeted and failed to bind to RCO in the presence of an equal amount of oligos containing the canonical binding sites, indicating that native sites bind to RCO with a lower affinity than does the canonical RCO-binding site (Figures 2C and S3D). We thus hypothesized that these low-affinity native binding sites may play a crucial role in shaping the distinctive expression pattern of RCO around emerging leaflets. To test this idea, we made an RCOg construct that contains the high-affinity canonical binding sites in place of the native sites and introduced it into the rco mutant. This construct could not rescue the rco mutant phenotype, likely due to low levels of RCO expression reflecting autorepression (Figure S3E). To assay the functionality of low-affinity RCO-binding sites in vivo, we engineered an RCO promoter fragment in which the low-affinity binding sites were mutagenized (termed pmutRCO). This mutagenized promoter was then used to drive the expression of RCOgenomic-VENUS (pmutRCO::RCOg-VENUS) and RCOcds-VENUS (pmutRCO::RCOcds-VENUS) (Figure 3A). The resulting constructs and the control constructs comprising the native, non-mutagenized RCO promoter fused to RCOgenomic-VENUS (pRCO::RCOg-VENUS) and RCOcds-VENUS (pRCO::RCOcds-VENUS) were transformed into the rco mutant. In transgenic plants expressing pmutRCO::RCOcds-VENUS (no RCO-binding sites), RCO-VENUS levels and leaf complexity were significantly increased, relative to transgenic plants that contain either the native construct pRCO::RCOg-VENUS, which has all the RCO-binding sites, or the pmutRCO::RCOg-VENUS construct, which also includes the introns and consequently additional RCO-binding sites (Figures 3B–3E). In addition, ectopic RCO expression was detectable at the boundaries of terminal leaflets in pmutRCO::RCOcds-VENUS-expressing transgenic plants (white arrow in Figure 3E). RCO expression was also higher in rco mutant lines expressing pmutRCO::RCOg-VENUS than in rco mutant plants expressing the pRCO::RCOg-VENUS construct (compare RCO-VENUS fluorescence in [1] and [2] in Figures 3D and 3E). However, we did not observe a significant difference in their leaf complexity (compare [1] and [2] in Figures 3B and 3C). Our results demonstrate that low-affinity RCO-binding sites act redundantly with each other to shape leaf development by defining the correct RCO gene expression pattern and dose, thereby preventing pleiotropic RCO effects. Our findings suggest that a cluster of low-affinity repressive binding sites evolved in concert with regulatory neofunctionalization of RCO to delimit its expression. However, it is not known through which target genes RCO represses growth to generate complex leaves and how RCO autorepression influences such RCO-dependent growth regulation. To help identify RCO target genes, we used RNA-seq in combination with hierarchical clustering to assay RCO-dependent gene expression 2–10 h after dexamethasone-induced RCO activation in the rco mutant at 2-h time intervals (Figures S5A and S5B). Several Gene Ontology (GO) terms were enriched in RCO-responsive genes that relate to response to stimulus and hormones, including cytokinin (CK), and to hormone-mediated signaling (Data S1C–S1E). For example, RCO induction was accompanied by increased expression of LONELY GUY 7 (LOG7) and HISTIDINE-CONTAINING PHOSPHOTRANSFER FACTOR 4 (AHP4) genes, which are involved in CK biosynthesis and signaling, respectively [28Kieber J.J. Schaller G.E. Cytokinin signaling in plant development.Development. 2018; 145 (dev149344)Crossref PubMed Scopus (174) Google Scholar]. Additionally, several CYTOKININ OXIDASE genes had reduced expression after RCO induction (such as CKX2, CKX3, and CKX6), as well as the URIDINE DIPHOSPHATE GLYCOSYLTRANSFERASE (UGT85A1) (Data S1C), which are involved in CK degradation and inactivation [28Kieber J.J. Schaller G.E. Cytokinin signaling in plant development.Development. 2018; 145 (dev149344)Crossref PubMed Scopus (174) Google Scholar, 29Schmülling T. Werner T. Riefler M. Krupková E. Bartrina y Manns I. Structure and function of cytokinin oxidase/dehydrogenase genes of maize, rice, Arabidopsis and other species.J. Plant Res. 2003; 116: 241-252Crossref PubMed Scopus (266) Google Scholar]. GO analysis of the ChIP-seq data also showed a significant enrichment for hormonal response terms, including response to CK (Data S1F). These findings suggest that RCO might act via the CK pathway. To identify CK-related genes that are direct targets of RCO, we compared differentially expressed genes in the ChIP-seq and RNA-seq datasets and found 31 overlapping genes (Figure S5C). A Fisher’s exact test confirmed that this number of overlapping genes is significantly higher than expected by chance (p < 0.005). LOG7 and AHP4 are included in these overlapping genes, and our ChIP results show that RCO associates with the promoter of these genes (Figure S5D; Table S1). Interestingly, LOG7 and AHP4 respond similarly to RCO and both are classified in cluster I (Figure S5B) and show continuously increasing expression upon RCO induction (Figure 4A). In contrast to LOG7 and AHP4, CKX3 is classified in cluster VIII and continuously decreases in expression upon RCO induction (Figure S5B). Taken together, these results suggest that RCO might enhance cytokinin biosynthesis and/or signaling. To test this hypothesis, we measured endogenous CK levels in wild-type C. hirsuta, the rco mutant, wild-type A. thaliana, ChRCOg-expressing A. thaliana, and in transgenic C. hirsuta rco mutant and A. thaliana expressing DEX-inducible RCO (pRPS5a≫RCO). The presence or induction of the RCO gene increased endogenous CK levels (Figures 4B and S6A). We also used the CK-response markers TCSn::TDT and TCSn::GFP (TCS) [30Zürcher E. Tavor-Deslex D. Lituiev D. Enkerli K. Tarr P.T. Müller B. A robust and sensitive synthetic sensor to monitor the transcriptional output of the cytokinin signaling network in planta.Plant Physiol. 2013; 161: 1066-1075Crossref PubMed Scopus (188) Google Scholar] to examine CK signaling activity in wild-type C. hirsuta, the rco mutant, ChRCOg-expressing A. thaliana, and wild-type A. thaliana, which lacks the RCO gene (Figures 4C–4F). In agreement with the above results, we found that presence of the RCO gene leads to increased TCS expression in leaves, especially adjacent to leaflet primordia (C. hirsuta) or serrations (A. thaliana), indicating higher CK activity. We also found that higher RCO expression levels, caused by mutation of RCO-binding sites, resulted in increased LOG7, AHP4, and ARR5 expression (Figure S6B). Together, these results indicate that RCO promotes CK activity and that RCO autorepression attenuates this function. Based on these results, we hypothesized that RCO increased leaf complexity by stimulating CK activity at the flanks of developing leaflets, thus contributing to their separation. If RCO is required for local CK activity, and if the reduced complexity of leaves in the rco mutant reflects decreased CK activity, we should be able to partially bypass the requirement of RCO for leaflet separation by activating CK signaling in the RCO domain. To test this idea, we enhanced CK signaling by expressing ARR1ΔDDK, a constitutively active form of the type-B cytokinin response regulator ARR1 [31Sakai H. Honma T. Aoyama T. Sato S. Kato T. Tabata S. Oka A. ARR1, a transcription factor for genes immediately responsive to cytokinins.Science. 2001; 294: 1519-1521Crossref PubMed Scopus (353) Google Scholar], from the RCO promoter in the rco mutant. Consistent with our hypothesis, this transgene suppressed the rco mutant leaf phenotype, making it more similar, although not identical, to wild type (Figures 4G and 4H). In addition, the expression of pRCO::ARR1ΔDDK in A. thaliana converted simple A. thaliana leaves into complex ones, mimicking the effect of expressing RCOg in A. thaliana (Figures 5A–5C) and indicating that locally elevated CK signaling in the RCO domain is sufficient to dramatically increase complexity in A. thaliana leaves. We next investigated whether similar cell-level effects underlie the increased leaf complexity caused by elevating either CK signaling or expressing RCO in the RCO domain in A. thaliana leaves, which should be the case if CK mediates RCO function (Figures 5 and 6). Consistent with this idea, we observed that both cell area and cell lobeyness—a differentiation measure—are reduced to a similar level relative to wild-type in pRCO::ARR1ΔDDK and pRCO::RCOg-VENUS expressing plants (Figures 6A–6D). These observations indicate that the repressive effect of RCO and CK on local leaf growth involves reduced cell size and is associated with slowing down differentiation. These findings are also in line with observations that increased cytokinin levels can cause a reduction in leaf cell size [32Craft J. Samalova M. Baroux C. Townley H. Martinez A. Jepson I. Tsiantis M. Moore I. New pOp/LhG4 vectors for stringent glucocorticoid-dependent transgene expression in Arabidopsis.Plant J. 2005; 41: 899-918Crossref PubMed Scopus (145) Google Scholar, 33Skalák J. Vercruyssen L. Claeys H. Hradilová J. Černý M. Novák O. Plačková L. Saiz-Fernández I. Skaláková P. Coppens F. et al.Multifaceted activity of cytokinin in leaf development shapes its size and structure in Arabidopsis.Plant J. 2019; 97: 805-824Crossref PubMed Scopus (37) Google Scholar]. In conclusion, RCO regulates leaf shape, at least in part, by reprogramming local CK homeostasis, thereby reducing cell growth (Figure 6E). Finally, we asked whether the large effects of the RCO/CK module on leaf form are mirrored by large effects on the transcriptome that distinguishes A. thaliana and C. hirsuta leaves. An alternative would be that this module alters morphology via more restricted effects on this evolutionary divergent transcriptome. We found that RCO-responsive genes, including CK-related ones, are strongly overrepresented in the transcriptome that is differentially expressed between A. thaliana and C. hirsuta leaf primordia [34Gan X. Hay A. Kwantes M. Haberer G. Hallab A. Ioio R.D. Hofhuis H. Pieper B. Cartolano M. Neumann U. et al.The Cardamine hirsuta genome offers insight into the evolution of morphological diversity.Nat. Plants. 2016; 2: 16167Crossref PubMed Scopus (52) Google Scholar] (Data S1G and S1H), supporting the above “large effect on both transcriptome and morphology” hypothesis. Taken together, our findings suggest that the effect of RCO in leaf shape evolution likely reflects the integration of two opposing processes: the first creates the possibility for morphological change through re-shaping the developing leaf transcriptome and CK response, and the second limits the potentially pleiotropic effects of RCO by negative autoregulation via low-affinity binding sites. It is of note that this regulatory logic is in line with RCO acting both as an activator (e.g., of CK-related genes) and a repressor of its own transcription. This dual action may have put it in a favored position for contributing to evolutionary change by allowing fine-grained control of organ development. In the future, it will be interesting to explore the precise significance of RCO autoregulation for CK-dependent cell growth control. For example, from a theoretical perspective, negative autoregulation can reduce the effects of noise on genetic network readouts, so it is possible that this RCO/CK regulatory module allows tighter control of growth during leaf primordium development [35Alon U. Network motifs: theory and experimental approaches.Nat. Rev. Genet. 2007; 8: 450-461Crossref PubMed Scopus (2093) Google Scholar, 36Crews S.T. Pearson J.C. Transcriptional autoregulation in development.Curr. Biol. 2009; 19: R241-R246Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar]. CK is also important for leaf complexity in tomato [37Bar M. Israeli A. Levy M. Ben Gera H. Jiménez-Gómez J.M. Kouril S. Tarkowski P. Ori N. CLAUSA is a MYB transcription factor that promotes leaf differentiation by attenuating cytokinin signaling.Plant Cell. 2016; 28: 1602-1615PubMed Google Scholar], where leaflets evolved without a contribution of RCO [18Vlad D. Kierzkowski D. Rast M.I. Vuolo F. Dello Ioio R. Galinha C. Gan X. Hajheidari M. Hay A. Smith R.S. et al.Leaf shape evolution through duplication, regulatory diversification, and loss of a homeobox gene.Science. 2014; 343: 780-783Crossref PubMed Scopus (184) Google Scholar]. It will thus be interesting to determine which genes fulfill a role similar to RCO to locally regulate CK in that system. It will also be important to understand how RCO effects on CK are integrated with those of KNOX proteins [16Bar M. Ori N. Leaf development and morphogenesis.Development. 2014; 141: 4219-4230Crossref PubMed Scopus (153) Google Scholar, 20Kierzkowski D. Runions A. Vuolo F. Strauss S. Lymbouridou R. Routier-Kierzkowska A.-L. Wilson-Sánchez D. Jenke H. Galinha C. Mosca G. et al.A growth-based framework for leaf shape development and diversity.Cell. 2019; 177: 1405-1418.e17Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar]. These transcription factors also promote leaf complexity in a CK-related pathway that appears distinct from RCO [16Bar M. Ori N. Leaf development and morphogenesis.Development. 2014; 141: 4219-4230Crossref PubMed Scopus (153) Google Scholar, 38Jasinski S. Piazza P. Craft J. Hay A. Woolley L. Rieu I. Phillips A. Hedden P. Tsiantis M. KNOX action in Arabidopsis is mediated by coordinate regulation of cytokinin and gibberellin activities.Curr. Biol. 2005; 15: 1560-1565Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar] as it has much broader effects on leaf primordium growth [20Kierzkowski D. Runions A. Vuolo F. Strauss S. Lymbouridou R. Routier-Kierzkowska A.-L. Wilson-Sánchez D. Jenke H. Galinha C. Mosca G. et al.A growth-based framework for leaf shape development and diversity.Cell. 2019; 177: 1405-1418.e17Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar]. CK effects on leaf cell development are multifaceted and include modulation of the rate of cell proliferation, the timing of its cessation, as well as regulation of post-mitotic growth [33Skalák J. Vercruyssen L. Claeys H. Hradilová J. Černý M. Novák O. Plačková L. Saiz-Fernández I. Skaláková P. Coppens F. et al.Multifaceted activity of cytokinin in leaf development shapes its size and structure in Arabidopsis.Plant J. 2019; 97: 805-824Crossref PubMed Scopus (37) Google Scholar, 39Hay A. Tsiantis M. KNOX genes: versatile regulators of plant development and diversity.Development. 2010; 137: 3153-3165Crossref PubMed Scopus (366) Google Scholar]. Consequently, answering this question will require cell-level dissection of CK pathways at different developmental stages. Previous studies demonstrated that low-affinity transcription factor binding sites play a crucial role in activating developmental genes, thereby ensuring developmental robustness and precise patterns of tissue development [14Crocker J. Abe N. Rinaldi L. McGregor A.P. Frankel N. Wang S. Alsawadi A. Valenti P. Plaza S. Payre F. et al.Low affinity binding site clusters confer hox specificity and regulatory robustness.Cell. 2015; 160: 191-203Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 40Gaudet J. Mango S.E. Regulation of organogenesis by the Caenorhabditis elegans FoxA protein PHA-4.Science. 2002; 295: 821-825Crossref PubMed Scopus (294) Google Scholar, 41Farley E.K. Olson K.M. Zhang W. Brandt A.J. Rokhsar D.S. Levine M.S. Suboptimization of developmental enhancers.Science. 2015; 350: 325-328Crossref PubMed Scopus (153) Google Scholar]. Here, using plant leaves and regulation of CK homeostasis as an example, we show that low-affinity repressive binding sites played a major role in the generation of morphological diversity. Specifically, our work indicates that these sites can help resolve conflicts between pleiotropy and novelty that emerge during evolution by dampening the effects of cis-regulatory changes that underlie novel gene expression patterns [1Carroll S.B. Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution.Cell. 2008; 134: 25-36Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar, 9Doebley J. Lukens L. Transcriptional regulators and the evolution of plant form.Plant Cell. 1998; 10: 1075-1082Crossref PubMed Scopus (353) Google Scholar, 15Crocker J. Noon E.P. Stern D.L. The soft touch: low-affinity transcription factor binding sites in development and evolution.Curr. Top. Dev. Biol. 2016; 117: 455-469Crossref PubMed Scopus (63) Google Scholar]." @default.
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