Angiosperm leaves show extensive shape diversity and are broadly divided into two forms; simple leaves with intact lamina and compound leaves with lamina dissected into leaflets. The mechanistic basis of margin dissection and leaflet initiation has been inferred primarily by analysing compound-leaf architecture, and thus whether the intact lamina of simple leaves has the potential to initiate leaflets upon endogenous gene inactivation remains unclear. Here, we show that the CINCINNATA-like TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTORS (CIN-TCP) transcription factors activate the class II KNOTTED1-LIKE (KNOX-II) genes and the CIN-TCP and KNOX-II proteins together redundantly suppress leaflet initiation in simple leaves. Simultaneous downregulation of CIN-TCP and KNOX-II in Arabidopsis leads to the reactivation of the stemness genes KNOX-I and CUPSHAPED COTYLEDON (CUC) and triggers ectopic organogenesis, eventually converting the simple lamina to a super-compound form that appears to initiate leaflets indefinitely. Thus, a conserved developmental mechanism promotes simple leaf architecture in which CIN-TCP–KNOX-II forms a strong differentiation module that suppresses the KNOX-I-CUC network and leaflet initiation.
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There are no restrictions on data availability. The transcriptomic raw data used in this study have been deposited to the National Centre for Biotechnology Information (NCBI) and Gene Expression Omnibus (GEO) database under the accession numbers GSE174702 and PRJNA734903. Source data are provided with this paper.
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We thank J. Bowman, T. Imaizumi, H. Tsukaya, V. Pautot, T. Jack, D. Weigel, Y. Eshed, P. Laufs and P. Aggarwal for plant material; C. Tian for raw-read counts of tissue-specific RNA-seq datasets; and Genotypic Technology for the cycloheximide and cycloheximide + dexamethasone microarray experiment. This work was supported by the Ministry of Human Resource Development, Government of India (fellowships to K.R.C., M.R. and A.N.S.), Department of Science & Technology for Improvement of S&T Infrastructure (DST-FIST), University Grants Commission Centre for Advanced Studies, and Department of Biotechnology (DBT)-IISc Partnership Program Phase-II at IISc (sanction no. BT/PR27952/INF/22/212/2018 to U.N.). A.K.B. and S.D. were supported by Shodhaka Life Sciences.
The authors declare no competing interests.
Peer review information Nature Plants thanks Naomi Ori and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 CIN-TCP and KNOX-II redundantly promote cotyledon maturation.
a,b, Cotyledons of 9-day old seedlings grown in the presence of 0 (Mock) or 12 µM (Dex) dexamethasone (a) and their abaxial epidermal cell images (b). Scale bar in (a) 2 mm and in (b) 50 µm. c, Abundance of CIN-TCP and KNOX transcripts in Arabidopsis tissue types (shown in cartoon) analyzed using the expression datasets available in the Genevestigator database (https://genevestigator.com). Sample numbers of microarray experiments are indicated. (d) pTCP4::GUS and pKNAT4::GUS expression in 5 and 9-day old seedlings.
Extended Data Fig. 2 Higher order leaflet emergence in jk-mock leaves.
a, 6th leaf from a 60 DAS jaw-D;pTCP4::mTCP4:GR x knat3;4;5-amiR (jk-mock) plant (shown in Fig. 1c) branches dissected from the main leaf. The order of visible leaflets on the branch is shown as numbers from 1–8. b, 32 DAS rosettes (left) of indicated genotypes grown in the presence of 0 (Mock) or 12 µM (Dex) dexamethasone and their leaves (right). Node numbers of the leaves are indicated at the bottom. Scale bar, 5 mm.
Extended Data Fig. 3 Suppression of ectopic leaflet emergence in jk-Mock leaves upon transient TCP4 induction.
a, A scheme of transient dexamethasone treatment experiment for 24 h in 14 or 16 DAS jk-Mock plants which were further growth till 40 DAS. b, Leaves (numbers indicate node positions) from 40 DAS jk-Mock plants grown without (Mock) or with 12 µM dexamethasone treatment for 24 h at 14 DAS or 16 DAS. Leaflet count of these leaves is shown in Fig. 1g. c, 2nd cauline leaf of jk-Mock plants at 75 DAS grown in the presence of 0 (Mock) or 12 (Dex) µM dexamethasone. d,e, Scanning electron micrographs corresponding to the boxed region in (c). f, Scanning electron micrograph of a jk-Mock 6th leaf (*) at 15 DAS. Scale bar, 5 mm (c), 150 µm (d) and 50 µm (e).
Extended Data Fig. 4 Transient TCP4 induction suppresses ectopic pCyclinB1;1 expression in jk-Mock leaves.
a, pCyclinB1;1::GUS activity in leaves (numbers denote node positions) from 25 DAS plants of indicated genotypes. Scale bar, 1 mm. b, pCyclinB1;1::GUS activity in 5th, 6th and 8th leaves from 32 DAS jk-Mock plants grown without dexamethasone (Mock), with transient dexamethasone treatment for 24 h at 16 DAS (16 DAS-24h Dex), or with continuous dexamethasone (Dex). Scale bar, 1 mm.
Extended Data Fig. 5 pCUC2::GUS expression analysis in CIN-TCP and KNOX-II knockdown leaves.
a, pCUC2::GUS activity in 6th leaves of indicated genotypes. b, pCUC2::GUS activity in the 5th, 6th and 8th leaves of jk-Mock plants grown without dexamethasone (Mock), with transient dexamethasone treatment for 24 h at 16 DAS (16 DAS-24h Dex), or with continuous dexamethasone (Dex). c, pDR5::GUS activity in the 5th leaf from 28 DAS plants. In jk-Mock;pDR5::GUS, the two panels on the right indicate leaflets from the leaf shown on the left. In jk-Dex;pDR5::GUS, a magnified image of the boxed region is shown in the inset. Arrowheads point to GUS expression at leaflet tips. Scale bar, 1 mm.
Extended Data Fig. 6 Leaflet phenotype and KNOX-I expression analysis in jk-Mock leaves.
a, Rosettes of indicated genotypes at 55 DAS. Scale bar, 1 cm. b, 6th leaf from 56 DAS jk-Mock plants grown without (jk-Mock) or with (jk-Dex) 12 µM dexamethasone or 80 µM L-kynurenine (L-Kyn). Scale bar, 5 mm. c, GUS reporter analysis at different developmental stages of leaves from 30 DAS jk-Mock plant. Leaf lengths are indicated. Punctate pKNAT2::GUS and pKNAT6::GUS signal is highlighted with asterisks and arrowheads, respectively. pCUC2::GUS signal is shown for comparison.
Extended Data Fig. 7 Analysis of meristem gene expression in jk-Mock leaves.
a-c, GUS reporter analysis of the 6th leaf from 25 DAS plants of indicated genotypes. Magnified images of the boxed regions are shown as insets on the right. Scale bar, 1 mm (a,b and the left panel in c) and 100 µm (insets in c).
Extended Data Fig. 8 CIN-TCPs activate KNOX-II genes.
a, Heat map of differentially expressed transcripts in the microarray experiment of 9-day old jaw-D;pTCP4::mTCP4:GR seedlings treated with either 40 µM cycloheximide (CHX) or a combination of 40 µM cycloheximide and 20 µM dexamethasone (CHX & Dex) for 4 hours (see Fig. 5c). b, Relative level of KNAT3 and KNAT4 transcripts analyzed by RT-qPCR in 9 DAS p35S::mTCP4:GR seedlings treated with 40 µM cycloheximide (CHX) or a combination of 20 µM dexamethasone and 40 µM CHX (CHX & Dex) for 4 h. Transcript levels were normalized to internal control PP2A. N = 3, Averages of three biological replicates are shown. Error bars indicate mean value SD. P, probability values of unpaired Student’s t-test. c, EMSA gel blots with radio-labeled (Probe) or unlabeled (Competitor) oligonucleotides corresponding to BS1 and BS3 (shown in Fig. 6a) and recombinant MBP-TCP4D protein. d, EMSA with radio-labeled (Probe) or unlabeled (Competitor) oligonucleotides corresponding to BS2 and BS3 (shown in Fig. 6b) and recombinant His-TCP4 protein. 25–150 fold excess competitor was used. e, Average size of abaxial pavement cells of the 1st leaves in 29 DAS seedlings. Increased cell size in the jaw-D;GR x p35S::KNAT3 leaves compared to jaw-D;GR x Col-0 leaves suggests that KNAT3 overexpression partly rescues the small-cell phenotype of jaw-D. Sample number, N = 120–150 cells per leaf were measured and averages of 3–5 leaves are shown. Unpaired Student’s t-test was used. Error bars indicate mean value SD. P, probability values of unpaired Student’s t-test. f, Relative transcript level analyzed by RT-qPCR in 9 DAS seedlings. Reduced levels of TCP2, 3, 10 & 24 in jaw-D;GR x p35S::KNAT3 suggests that the rescue of jaw-D leaf phenotype by KNAT3 overexpression is not due to the co-suppression of the jaw-D locus. Transcript levels were normalized to internal control PP2A. N = 3, Averages of three biological replicates are shown. Error bars indicate mean value SD. P, probability values of unpaired Student’s t-test.
Extended Data Fig. 9 Reproductive organ development in jk-Mock plants.
a,b, Flowers (a) and inflorescence (b) of 42 DAS mock-grown plants. White arrows indicate infertile flowers. Scale bar, 1 mm (a) or 5 mm (b).
Source Data Fig. 1
a, Complete gene list of differentially regulated genes in jaw-D;GR × knat3;4;5-amiR (jk-mock) only and not in jaw-D;GR and knat3;4;5-amiR compared to Col-0. b, Full list of division- and age-specific marker genes. c, List of division- and age-specific marker genes differentially regulated in jaw-D;GR × knat3;4;5-amiR (jk-mock) only and not in jaw-D;GR and knat3;4;5-amiR.
Source Data Fig. 2
Common DEGs obtained by comparing the microarray done using 9 DAS jaw-D;pTCP4::mTCP4:GR seedlings treated with 40 µM cycloheximide (CHX) or a combination of 40 µM CHX and 20 µM dexamethasone (CHX DEX) for 4 h with the previously published microarray28.
Source Data Fig. 3
List of primers and oligonucleotides used in this study.
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Challa, K.R., Rath, M., Sharma, A.N. et al. Active suppression of leaflet emergence as a mechanism of simple leaf development. Nat. Plants 7, 1264–1275 (2021). https://doi.org/10.1038/s41477-021-00965-3
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