Auxin depletion from leaf primordia contributes to organ patterning

Abstract

Stem cells are responsible for organogenesis, but it is largely unknown whether and how information from stem cells acts to direct organ patterning after organ primordia are formed. It has long been proposed that the stem cells at the plant shoot apex produce a signal, which promotes leaf adaxial-abaxial (dorsoventral) patterning. Here we show the existence of a transient low auxin zone in the adaxial domain of early leaf primordia. We also demonstrate that this adaxial low auxin domain contributes to leaf adaxial-abaxial patterning. The auxin signal is mediated by the auxin-responsive transcription factor MONOPTEROS (MP), whose constitutive activation in the adaxial domain promotes abaxial cell fate. Furthermore, we show that auxin flow from emerging leaf primordia to the shoot apical meristem establishes the low auxin zone, and that this auxin flow contributes to leaf polarity. Our results provide an explanation for the hypothetical meristem-derived leaf polarity signal. Opposite to the original proposal, instead of a signal derived from the meristem, we show that a signaling molecule is departing from the primordium to the meristem to promote robustness in leaf patterning.

Keywords: auxin; leaf polarity; meristem; stem cell.

Fig. 1.

Transient adaxial low auxin domain is important for leaf polarity patterning. (A and B) Longitudinal and transverse sections through Arabidopsis SAM and leaf primordia region. DII-Venus signals are shown in green in A and B, and chlorophyll autofluorescence is in red (B). DII-Venus signal is enriched in the adaxial domain from P₂ to P₉. The abaxial domain in A has pFIL::DsRed-N7 (red) expression. Stronger DII-Venus signals in the boundary and adjacent adaxial domain indicated weaker auxin signaling input. Images in Fig. S1 show DII-Venus signals in leaf primordia of additional stages. (C–F) Control tomato leaf primordia, showing schematic diagram of the meristem surface auxin flux direction (C), early primordium 4–5 d after emergence (D), 7 d after emergence (E), and a transverse section through the midrib and adjacent laminal regions with close-up insertion of vascular strand (F). Note that phloem cells (p) surround the xylem (x) elements. (G–J), Tomato leaf primordia after adaxial IAA microapplication, showing site of microapplication (G), early leaf (H), and more mature (I) primordia with strong defects in adaxial-abaxial polarity, and a transverse section through the midrib and adjacent laminal regions (J). More images are shown in Figs. S5 and S6. (Scale bars: A and B, 20 μm; C–J, 200 μm; F and J Inset, 50 μm.)

Fig. 2.

Ectopic adaxial MP activity induces abaxialized leaves. (A) A transverse section of meristem and primordia, stained by propidium iodide (PI, red), showing pMP::MP-GFP (green) signals in entire young leaf primordia with adaxial enrichment. (B and C) Transgenic pMP::MPΔ (B) and pAS2::MPΔ (C) plants showing disrupted leaf adaxial-abaxial polarity (arrowheads), including lacking lamina expansion and trumpet-shaped leaves. Transverse sections through the petiole regions (D and F) and blade regions (E and G) of wild-type leaves (D and E), and trumpet-like leaves from pAS2::MPΔ (F and G) indicate abaxialization in transgenic plants. (Scale bars: A, 20 μm; B and C, 1 mm; D–G, 100 μm.)

Fig. 3.

Spatial distribution of PIN1 expression and polarity patterns, and effects of NPA and IAA on auxin gradient and PIN1 localization in early leaf primordia. (A) View of vegetative tomato meristem stained by PI (red) from above showing a 3D volume rendering of pAtPIN1::AtPIN1-GFP expression and localization (green). The earliest primordium is marked as P₁ and the oldest incipient primordium is labeled as I₁. Regions in colored dotted line boxes were imaged at enhanced resolution and are shown in corresponding boxes after denoising and contrast enhancement to highlight polar localization of AtPIN1-GFP in each cell (images without denoising and contrast enhancement are shown in Fig. S10 A and B). Cells adaxial to I₁ show AtPIN1-GFP polarity (arrows) toward the primordium, and cells adaxial to P₁ show reversed AtPIN1-GFP polarity (arrows) toward the meristem center. (B and C) Signal of DII-Venus in the first pair of true leaves of Arabidopsis seedlings after mock NPA treatment (B) and after NPA treatment (C) for 5 h. Maximum intensity projections from the YFP channel (green) were overlaid with maximum intensity projections from the Nomarski channel. Quantifications of YFP channel intensities along blue lines in B and C are shown below corresponding images. (D and E) show maximum intensity projections of tomato pAtPIN1::AtPIN1-GFP (green) expressing meristems stained by PI (red) view from above. Regions in yellow dotted line boxes were imaged at enhanced resolution (and a slightly different angle) and are shown in yellow boxes within each image after denoising and contrast enhancement to highlight polar localization of AtPIN1-GFP (images without denoising and contrast enhancement are shown in Fig. S10 C and D. Tomato meristems were treated with NPA-containing lanolin paste outside I₁ (D), or with lanolin paste containing IAA (E), with treated area highlighted by blue dotted line circles. Images (D and E) were acquired 24 h and 12 h, respectively, after treatment. (Scale bars: 20 μm.)

Fig. 4.

Polar auxin transport inhibitors induced abaxialized radially symmetric leaves. Tomato leaf primordia after local NPA microapplication, showing site of microapplication (A), early leaf (B), and more mature (C) primordia with strong defects in adaxial-abaxial polarity, and a transverse section through the midrib and adjacent laminal regions (D). More images are shown in Figs. S6 and S12. (Scale bars: 200 μm, except D Inset, 50 μm.)

Fig. 5.

Adaxial-abaxial polarity phenotypes associated with polar auxin transport defects. (A) A radialized trumpet-shaped leaf found in a pin1-1 plant. (B) Radialized trumpet-shaped and rod-shaped leaves found in a pid-7.1.2.6 rev-1 plant. Arrowheads indicate trumpet-shaped leaves, and arrows indicate rod-shaped leaves. Transverse section through the petiole regions of a wild-type leaf (C) and a trumpet-like leaf from pin1-1 (D) indicate abaxialization in pin1 mutants. Transverse section through a wild-type leaf (E) and a pin1-1 leaf of regular shape (F) showing the four mesophyll layers. (G) Conceptual model of the influence on polarity of polar auxin transport from the leaf axil to the SAM. p, phloem cells; x, xylem cells. (Scale bars: A and B, 500 μm; C–F, 100 μm.)