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Specification of leaf dorsiventrality via a prepatterned binary readout of a uniform auxin input


Developmental boundaries play an important role in coordinating the growth and patterning of lateral organs. In plants, specification of dorsiventrality is critical to leaf morphogenesis. Despite its central importance, the mechanism by which leaf primordia acquire adaxial versus abaxial cell fates to establish dorsiventrality remains a topic of much debate. Here, by combining time-lapse confocal imaging, cell lineage tracing and molecular genetic analyses, we demonstrate that a stable boundary between adaxial and abaxial cell fates is specified several plastochrons before primordium emergence when high auxin levels accumulate on a meristem prepattern formed by the AS2 and KAN1 transcription factors. This occurrence triggers a transient induction of ARF3 and an auxin transcriptional response in AS2-marked progenitors that distinguishes adaxial from abaxial identity. As the primordium emerges, dynamic shifts in auxin distribution and auxin-related gene expression gradually resolve this initial polarity into the stable regulatory network known to maintain adaxial–abaxial polarity within the developing organ. Our data show that spatial information from an AS2–KAN1 meristem prepattern governs the conversion of a uniform auxin input into an ARF-dependent binary auxin response output to specify adaxial–abaxial polarity. Auxin thus serves as a single morphogenic signal that orchestrates distinct, spatially separated responses to coordinate the positioning and emergence of a new organ with its patterning.

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Fig. 1: Adaxial–abaxial polarity is specified before primordium emergence.
Fig. 2: Conversion of a uniform auxin input into a binary auxin response output during the specification of adaxial–abaxial polarity.
Fig. 3: PIN1 convergence patterns orient towards the adaxial side of incipient leaf primordia.
Fig. 4: Concentric rings of AS2 and KAN1 expression prepattern adaxial–abaxial polarity at the shoot apical meristem.
Fig. 5: AS2 and KAN1 prepattern adaxial–abaxial polarity at the shoot apical meristem.
Fig. 6: ARF3 expression distinguishes adaxial from abaxial founder cells.
Fig. 7: Antagonistic interactions between KAN1, AS2 and ARF3 positions the adaxial auxin response.
Fig. 8: Spatial information from the AS2–KAN1 meristem prepattern triggers conversion of a uniform auxin input into an ARF-dependent binary nuclear auxin response output to specify adaxial–abaxial polarity.

Data availability

Data for the current study are available from the paper and its Supplementary Information or from the corresponding author on reasonable request.


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We thank our colleagues in Tübingen and Katowice who have contributed ideas and thoughtful comments to the manuscript. We also thank C. Kuhlemeier for constructive discussions, D. Vranjkovic and A. Feller for technical assistance, R. Smith and A.-L. Routier-Kierzkowska for their help with MorphoGraphX, C. Brancato and K. Berendzen for protoplast production, G. Jürgens for sharing the pLucTraP and pJIT60 plasmids and C. Kuhlemeier, L. Ostergaard, Y. Jiao, J. Long, B. Möller and D. Weijers for sharing materials. A.B. and M.R-S are supported by a research grant SONATA BIS 6 (2016/22/E/NZ3/00342) from the National Science Centre, Poland. Work on leaf polarity in M.T.’s laboratory is supported through an Alexander von Humboldt Professorship.

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Authors and Affiliations



A.B., G.P. and M.T. designed the project and experiments. K.N. performed the ChIP analyses and S.M. performed the transient expression assays. All other experiments and data analyses were performed by A.B. and G.P. with help from M.R.-S. The manuscript was written by A.B., G.P. and M.T.

Corresponding author

Correspondence to Marja C. P. Timmermans.

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Nature Plants thanks Lars Østergaard, Dolf Weijers, José Luis Micol and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 The spatial sequence of leaf primordia at the shoot apex reflects primordium development in time.

a-b, A representative sequence of leaf primordium stages (b) at the shoot apex (a). Leaf primordia are numbered relative to the first bulging primordium (p1), numbers <1 indicate incipient primordia. Arrow, the first bulging primordium. c-d, Time-lapse series of the shoot apex (c) showing a temporal sequence of leaf development. Development of p-2 and p-1 primordia over 3 days (d) mirrors consecutive leaf primordia at the meristem. The p-2 and p-1 incipient primordia after 24 h attain developmental stages corresponding to p1 and p2, respectively (b), indicating a plastochron under the growth conditions used is approximately 8 h. Primordium stage numbers are maintained from the first time point. (a, c) Top views of the meristems. (b, d) Optical longitudinal sections through leaf primordia. Cell walls were stained by PI. Scale bars, 50 µm. Representative images from n = 6 apices (a, c) and n = 6 primordia (b, d) are shown.

Extended Data Fig. 2 The adaxial–abaxial boundary is specified prior to primordium emergence.

a, Optical longitudinal section of a p5 leaf primordium illustrating positions of the adaxial (yellow) and abaxial (blue) domains relative to the first forming trichome (arrow). Domains were discerned based on patterns of reporter gene expression (Fig. 1a). b, Backward tracing of cell lineages from the adaxial (yellow), middle, and abaxial (blue) domains of a p5 primordium (72 h) to their progenitors at p-2 (0 h) shows the adaxial–abaxial boundary is specified prior to primordium emergence at p-2, while the middle domain forms several plastochrons later from cells at the adaxial–abaxial boundary. c, Backward tracing of cell lineages from the adaxial (yellow), middle, and abaxial (blue) domains of p5 (top), p6 (middle) (96 h), and p6 (bottom) (72 h) primordia to their progenitors at p-4, p-3, and p-2 (0 h), respectively, reveals that the adaxial–abaxial boundary is not defined at p-4 or p-3 when most progenitors along the future boundary contribute both adaxial and abaxial daughter cells (for quantification, see Fig. 1b). However, at p-2, the adaxial–abaxial boundary for nearly all primordia analysed is stably defined and undetermined cells giving rise to both adaxial and abaxial daughters are rarely observed. d, The number (mean ± SD) of cells corresponding to the adaxial and abaxial domain, as well as along the width of the (future) adaxial–abaxial boundary, at p-3 to p1; n, the number of primordia analysed. Yellow, adaxial; blue, abaxial; red dotted line (v), midvein; arrow or hash, the first forming trichome. Cell walls were stained by propidium iodide (PI). Scale bars, 20 µm (a,b,c left panels) and 50 µm (c right panel). Representative examples from n = 20 primordia (a-b), or n is as indicated in panel d (c), are shown.

Extended Data Fig. 3 The middle domain emerges gradually from the adaxial–abaxial boundary after primordium emergence.

a-b, Cell lineage tracing of adaxial (circle) and abaxial (triangle) leaf founder cells from p-1 to p6 (a) and the resulting cell lineage graph (b) show that the middle domain derives from both adaxial and abaxial cells at the boundary that are respecified to middle domain identity (red) around p4 (48 h). The number of cells at the middle domain has increased at 72 h. White, adaxial or abaxial; red, middle identity; circle, adaxial and triangle, abaxial founder cells and their respective derivatives. c-d, The pattern of pWOX1:GFP expression at the shoot apex shows WOX1 transcription marks the emerging middle domain. (c) Top view of the meristem. (d) Front view of select leaf primordia. Cell walls were stained by PI. Scale bars, 20 µm (a), 50 µm (c-d). Representative examples from n = 24 primordia (a, b), n = 5 apices (c), and n = 5 primordia per stage (d), are shown.

Extended Data Fig. 4 Auxin input patterns change dynamically during primordium emergence.

a-e, Expression patterns for DII-Venus (a), mDII-tdTomato (b), or both (c) at the shoot apex show auxin levels are uniformly high at p-2 incipient primordia but are gradually depleted (increasing DII relatively to mDII signal intensities) first from the adaxial and subsequently from the abaxial side following primordium emergence. (a–c) Top views of the meristem. (d, e) Optical longitudinal sections through leaf primordia. Yellow, adaxial; blue, abaxial; asterisks, meristem centre. The middle domain (not outlined) gradually emerges from the adaxial–abaxial boundary. f-g, Signal intensities of DII-Venus (green) and mDII-tdTomato (dark grey) (f) or the DII/mDII signal intensity ratio (g) measured along the apex radius at respective leaf primordium stages (c) illustrate the gradual quantitative depletion of auxin during primordium development. The distance 0 µm corresponds with the meristem centre. Positions of adaxial and abaxial identity discerned from backward lineage tracing are indicated by yellow and blue segments, respectively. Scale bars, 50 µm (a–c), 20 µm (d, e). Representative data from n = 12 apices (a–c), n = 6 primordia per developmental stage (d-e), and n = 5 primordia per developmental stage (f,g) is shown.

Extended Data Fig. 5 An asymmetric auxin response output is specific for dorsiventral organs.

a–f, The patterns of pDR5v2:GFP expression at the shoot apex during the transition to flowering (a, b) and at the reproductive phase (c–f) show dorsiventral organs (green) derive from meristem sites marked by a polarized transcriptional auxin response output. Early primordia of cauline leaves (b, p1, p2), bracts (d, p1, p2), and sepals (f). Subsequently, the auxin response output shifts to the middle domain and marks the primordium tip and underlying future midvein (b, p3, p4; d, p4, p5). In contrast, axillary (b) and floral (d) meristems (white), originate from sites marked by a uniform nuclear auxin response. (a, c, e) Meristem top views. (b, d, f) Optical longitudinal sections through primordia. Insets show the position of the optical section through the flower meristem (f). Green, dorsiventral leaf-like primordium; white, axillary or flower meristem; asterisks, shoot apical or flower meristem centre. Cell walls were stained by PI. Scale bars, 20 µm. Representative images from n = 5 (a) or n = 6 (c, e) meristems, and n = 5 (b) or n = 6 (d, f) primordia per developmental stage are shown.

Extended Data Fig. 6 Strong PIN1 expression is associated with sites of auxin biosynthesis.

a-b, Quantitative projection of pPIN1:PIN1-GFP signal at the meristem L1 (a) and patterns of pTAA1:TAA-GFP, pYUC1:GFP, and pYUC4:GFP expression (b) at the shoot apex show PIN1 upregulation coincident with predicted sites of auxin biosynthesis (arrowheads). Top views of meristems (a, b, upper panels) or leaf primordium (a, lower panel), and optical transverse sections through select leaf primordia (b, lower panels). Arrowheads, flanks of primordium-meristem boundaries where local PIN1 upregulation coincides with sites of auxin biosynthesis deduced from co-expression of TAA1, YUC1, and YUC4. Cell walls were stained by PI. Asterisks, meristem centre; p, leaf primordium. Scale bars, 20 µm. Representative images from n = 6 apices (a), and n = 10 apices per line (b) are shown.

Extended Data Fig. 7 PIN1-mediated polar auxin transport constrains auxin-induced gene expression laterally.

a–d, Time-lapse series of representative p-1 leaf primordia without or with application of 100 µM NPA at 0 h (a, c) shows perturbation of PIN1-mediated polar auxin transport does not block the shift in auxin-induced gene expression (DR5v2) from the adaxial side at p-1 (0 h) to the middle domain 72 h later. Instead, in comparison to mock-treated primordia (b), NPA-treated primordia are wider and show a lateral expansion of the DR5v2 expression domain (arrows) (d). (a, c) Optical longitudinal sections and (b, d) top views of select leaf primordia. e-f, Wild type (e) and pin1 mutant (f) shoot apices showing pin1 leaf primordia are comparatively wider and occasionally laterally fused (arrow). Note also the irregular phyllotaxy of pin1 apices (f). Cell walls were stained by PI. Asterisks, meristem centre; p, leaf primordium. Scale bars, 20 µm (a–d), 50 µm (e-f). Representative data from n = 6 primordia per treatment (a–d), and n = 12 apices per line (e-f) is shown.

Extended Data Fig. 8 REV expression before p0 is limited to inner tissues.

The pattern of pREV:REV-YPet expression at the shoot apex showing weak internal REV accumulation just apical to p-1. Top view of the meristem (a) and optical longitudinal section at p-1 (b). Progenitors for the adaxial (yellow), abaxial (blue), and middle (not present at this developmental stage) domains were discerned by backward lineage tracing. Asterisks, meristem centre. Cell walls were stained by PI. Scale bars, 50 µm. Representative data from n = 7 apices (a), and n = 5 primordia (b) is shown.

Extended Data Fig. 9 Incipient leaf primordia up to p-2 show a near uniform level of MP expression.

a–c, Representative pattern of pMP:GFP-MP expression at the shoot apex showing a mostly uniform accumulation of MP at p-4 and p-3 incipient primordia. (a, b) Top view of the meristem (a) and leaf primordia (b). (c) Optical longitudinal sections at p-4 and p-3 leaf primordia. White, incipient primordium; asterisks, meristem centre. Cell walls were stained by PI. Scale bars, 50 µm (a), 20 µm (b, c). d, GFP-MP signal intensities measured along the apex radius at indicated primordium stages illustrate the near uniform level of MP accumulation. The distance 0 µm corresponds with the meristem centre. Positions of incipient primordia discerned by lineage tracing are marked in grey. Representative data from n = 10 apices (a) and n = 5 primordia per developmental stage (b-d) is shown.

Extended Data Fig. 10 Expression of KAN1, AS2, and ARF3 is anticorrelated during early primordium development.

a, Dual pARF3:ARF3-Turquoise and pKAN1:KAN1-YPet reporter lines show a complementary pattern of KAN1 and ARF3 expression at the meristem from p-2 until primordium emergence at p1. Subsequently, ARF3 expression gradually shifts from the adaxial side of the primordium onto the emerging middle domain and then the abaxial side, where its expression then overlaps with that of KAN1. b, Dual pARF3:ARF3-Turquoise and pAS2:AS2-VENUS reporter lines show expression of AS2 and ARF3 is anticorrelated. Induction of ARF3 at p-2 correlates with a local decrease in AS2 expression, and ARF3 levels adaxially increase further as growth at the meristem displaces the primordium basipetal to the AS2 prepattern (p-1, p0). Subsequently, as the primordium emerges (p1) and ARF3 expression shifts towards the middle domain and abaxial side, AS2 levels again increase adaxially. Asterisks, meristem centre. Cell walls were stained by PI. Scale bars, 50 µm. Representative images from n = 5 (a) and n = 2 (b) independent apices are shown.

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Burian, A., Paszkiewicz, G., Nguyen, K.T. et al. Specification of leaf dorsiventrality via a prepatterned binary readout of a uniform auxin input. Nat. Plants 8, 269–280 (2022).

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