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Combined SHOOT MERISTEMLESS and WUSCHEL trigger ectopic organogenesis in Arabidopsis

Jean-Luc Gallois^1,*, Claire Woodward^1,*, G. Venugopala Reddy² and Robert Sablowski^1,

¹ Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK
² California Institute of Technology, Division of Biology, Pasadena, CA91125, USA
^* These authors contributed equally

View larger version (116K): [in a new window]   Fig. 1. Activated STM-GR rescued the meristem in stm1 mutants and inhibited leaf development. (A) 35S::STM-GR seedlings grown on medium without dexamethasone. One of the seedlings is homozygous for stm1 (arrow). Bar: 5 mm. (B) 35S::STM-GR seedlings (including one-quarter of stm1 homozygotes), grown on medium with 1 µM dexamethasone. Bar: 5 mm. (C) Scanning electron micrograph of the apex of a 35S::STM-GR seedling two weeks after germination on medium with 1 µM dexamethasone. The meristem (m) is surrounded by leaf primordia (p) in the normal spiral phyllotaxis. The arrows indicate stipules, whose identity was confirmed using a stipule-specific reporter gene (not shown). Bar: 100 µm. (D) Cryo-scanning electron micrograph of a leaf from a 2-weeks old 35S::STM-GR seedling grown on medium with 1 µM dexamethasone. Arrows indicate ectopic stipules between leaf lobes. Bar: 100 µm. (E) Tip of a leaf from a 35S::STM-GR seedling grown for 2 weeks on medium with 1 µM dexamethasone. The arrow indicates a pair of guard cells. Bar: 100 µm. (F,G) 35S::STM-GR plants grown for 2 weeks on soil, sprayed once a day for 4 days with 0.015% v/v Silwet L-77 (F) or 0.015% v/v Silwet L-77 with dexamethasone 0.1 µM (G) and photographed 20 days after germination (moved to agar medium only for photography). The area around in the centre or the rosette in G is contains arrested leaf primordia similar to those seen in B, and early floral buds. Seedlings similar to the one shown in G were seen after spraying with 0.1-10 µM dexamethasone, although the degree of growth arrest was more severe at higher steroid concentrations. Bar: 5 mm.

View larger version (132K): [in a new window]   Fig. 2. STM-GR activated ectopic expression of KNAT2::uidA and cycD3::uidA, but not cycB::uidA. 35S::STM-GR seedlings were grown for 9 days on medium without dexamethasone (A,C,E) or with 1 µM dexamethasone (B,D,F). The seedlings also contained the reporters KNAT2::uidA (A,B), cycD3::uidA (C,D) or cycB::uidA (E,F).

View larger version (34K): [in a new window]   Fig. 3. Cre-loxP-based system for mosaic expression. (A) The system was tested in plants containing heat-shock-inducible Cre recombinase (hsp18.2::Cre) and a reporter construct consisting of loxP-flanked uidA inserted between the 35S promoter and GFP (35S::lox-uidA-lox-GFP). After a brief heat shock, transient induction of Cre caused excision of uidA and GFP activation in random cells and their descendants. (B,C) Optical sections of GFP-expressing sectors (arrows) on the cotyledon epidermis (B) and in hypocotyls (C) were recorded 7 days after heat shock.

View larger version (117K): [in a new window]   Fig. 4. Mosaic WUS expression combined with STM-GR activation caused ectopic organogenesis. (A) Control seedling (35S::lox-uidA-lox-GFP; hsp18.2::Cre) with no morphological effects 7 days after heat shock induction of GFP sectors (see Fig. 3). Inset: top view of comparable seedling. (B) Phenotype caused by mosaic WUS expression in 35S::lox-uidA-lox-WUS; hsp18.2::Cre seedlings, 7 days after heat shock. Inset: top view of similar seedling. (C,D) Phenotype after induction of mosaic WUS expression and STM-GR activation, in 35S::lox-uidA-lox-WUS; hsp18.2::Cre; 35S::STM-GR seedlings grown on medium with 1 µM dexamethasone 10 days after heat shock. The arrows indicate ectopic organs on the hypocotyl (C) and cotyledons (D). (E-H) Cryo-scanning electron micrographs of hypocotyls. (E) Hypocotyl from a control seedling as in A; (F) hypocotyl after WUS induction as in B; (G) example of outgrowth (arrow) observed on the hypocotyl at low frequency 2 weeks after WUS induction as in B; (H) hypocotyl after activation of STM-GR but not WUS (35S::STM-GR seedling grown for 10 days on medium with 1 µM dexamethasone). Bar: 100 µm. (I-K) Cryo-scanning electron micrographs of ectopic organs induced by combined WUS and STM-GR activation (as in C,D). (I) Outgrowth on cotyledon (as in D); (J,K) outgrowths on hypocotyls (as in C). Arrows in K indicate outgrowths at early stages. Bar: 100 µm.

View larger version (117K): [in a new window]   Fig. 5. Ectopic organogenesis was a non cell-autonomous response to WUS. (A,B) Whole seedlings stained for GUS activity after induction of ectopic organogenesis. 35S::lox-uidA-lox-WUS; hsp18.2::Cre; 35S::STM-GR seedlings were grown on medium with 1 µM dexamethasone for 10 days (A) or 7 days (B) after heat shock. Arrows indicate GUS-expressing outgrowths. (C) Transverse section through the hypocotyl of a seedling treated as described in B, showing an outgrowth with GUS-expressing cells. Bar: 100 µm. (D-G) Detection of WUS mRNA by in situ hybridisation. (D) Control showing endogenous WUS expression (arrow) below the centre of the meristem, in a transverse section through the apical meristem (m) and leaf primordia (p) of a 35S::STM-GR seedling (equivalent to the seedlings in Figs 1B and 2B). (E-G) Patches of ectopic WUS mRNA in sections through a leaf primordium (p) and cotyledons (c) in seedlings with mosaic WUS expression combined with STM-GR activation (equivalent to the seedlings shown in Fig. 4C,D). The arrow indicates outgrowths on cotyledons, consisting of cells that did not express WUS. Bar: 100 µm.

View larger version (73K): [in a new window]   Fig. 6. The CLV1 promoter was transiently activated during ectopic organogenesis. (A,B) Optical sections through the apex of 3-day old control seedlings. (A) Seedling lacking CLV1::GFP. The red chlorophyll fluorescence marks the base of the cotyledon petioles; the space in the centre is occupied by the meristem and the first two leaf primordia. (B) CLV1::GFP seedling. GFP signal is visible in the meristem (arrow); the dark areas adjacent to the meristem is occupied by leaf primordia. (C-F) Longitudinal optical sections through the hypocotyls of seedlings with ectopic organs (35S::lox-uidA-lox-WUS; hsp18.2::Cre; 35S::STM-GR, heat shocked and plated on medium with 1 µM dexamethasone; equivalent to the seedlings shown in Figs 4C,D and 5B). (C-E) CLV1::GFP seedlings, (C) 2 days, (D) 7 days and (E) 10 days after heat shock. The arrows in C indicate the division of epidermal cells seen in early stages of outgrowth formation. (F) Control seedling lacking CLV1::GFP, 10 days after heat shock. (G,H) Longitudinal optical sections through the hypocotyls of control seedlings (35S::lox-uidA-lox-WUS; hsp18.2::Cre; 35S::STM-GR, CLV1::GFP) with activation of STM-GR or WUS alone. (G) seedling that was not heat shocked, after growth for 7 days in medium with 1 µM dexamethasone (STM-GR activation alone); (H) seedling that was heat shocked and grown for 7 days on medium without dexamethasone (WUS activation alone). The green/yellow signal seen along part of the walls of the large cells in H is an artefact of light refraction, also seen in seedlings that lacked CLV1::GFP. Bar: 100 µm.