DEV01320 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. Markers under the control of the SHOOT MERISTEMLESS (STM) promoter are expressed in the boundary domain around organ primordial. Expression of the GUS reporter (1,2) or endoplasmic reticulum targeted GFP (3-6) was characterised following overnight ethanol-induction of STM::ALCR-alcA::GUS or STM::ALCR-alcA::erGFP lines, respectively, and compared with a CUC1 enhancer trap line (line M0223, 7-9). (1) Longitudinal section of a GUS-stained 12-day-old STM::ALCR-alcA::GUS seedling. (2) Dissected apex of a GUS-stained 12-day-old STM::ALCR-alcA::GUS seedling. (3) Apical view of a 12-day-old STM::ALCR-alcA::erGFP seedling. (4) Apical view of a stage 2 STM::ALCR-alcA::erGFP flower. (5) Apical view of a stage 4 STM::ALCR-alcA::erGFP flower. (6) Apical view of a stage 6-7 STM::ALCR-alcA::erGFP flower. (7) Apical view of a stage 2 CUC1 enhancer trap line flower. (8) Apical view of a stage 4 CUC1 enhancer trap line flower. (9) Apical view of a stage 6-7 CUC1 enhancer trap line flower. Scale bars: 100 mm. In 12-day-old seedlings (1-3), the GUS or erGFP marker under the control of STM regulatory sequences is expressed in the boundary domain two to four cells wide between the leaf primordium (lp) and the meristem (m). The maker is also weakly expressed on the abaxial base of the leaf primordium (1) and (3). During flower development, expression of erGFP under the control of STM regulatory sequences marks the boundaries of the incipient (s, 4) or growing sepal primordia (s, 5). GFP expression is maintained in the adaxial side of the floral meristem that corresponds to the ancient boundary between floral and apical meristems (arrow in 4). The boundary around the petals (arrow, p) and stamens (st) express GFP in a stage 6-7 flower (6). Similar expression patterns can be observed in the CUC1 enhancer trap line (7-9), that reflects CUC1 mRNA accumulation pattern (Takada et al., 2001). The mRNA accumulation patterns of CUC1 and CUC2 largely overlap during flower development (Ishida et al., 2000; Takada et al., 2001). In conclusion, STM regulatory sequences are active in a boundary domain largely overlapping with CUC1 and CUC2 mRNA accumulation domain.

• Supplemental Figure 2 -

  Fig. S2. Activation of the ethanol-inducible system does not interfere with plant growth or cell cycle progression. (A) The effects of the activation of the ethanol-inducible system on plant growth were investigated. For this, LFY::ALCR-alcA::GUS plants were ethanol induced with ethanol vapour for 1 hour or 24 hours, or not induced. Inflorescence length and flower number were determined 12 days (white), 15 days (blue), 20 days (red) or 23 days (black) after the beginning of the treatment (data represent mean values and standard deviations of 10 plants). This showed that no growth perturbation resulted from the activation of the ethanol-inducible system. (B) The effects of the activation of the ethanol-inducible system on cell cycle progression were investigated. For this, we used a LFY::ALCR-alcA::GUS alcA::H4GFP line expressing a translational fusion of Histone H4 with GFP that allows the recognition of mitotic cells (1) in a similar way to propidium iodide staining of the DNA (2). This allowed the calculation of the mitotic index (MI) in stage 2-4 flower primordia in the LFY::ALCR-alcA::GUS alcA::H4GFP line following overnight ethanol-induction with vapour produced either from 30%, 75% or 95% ethanol (3). Similarly, the mitotic index of propidium iodide-stained wild-type plants that were either ethanol-induced with 95% ethanol or not was determined. No significant differences were observed between the MI of wild-type plants that were ethanol induced or not, showing that at the used concentration, the ethanol had no effect on cell cycle progression. The MI calculated from the LFY::ALCR-alcA::GUS alcA::H4GFP line were not significantly different from those of wild-type plants showing that ethanol-activation of the ALCR factor does not interfere with cell cycle progression and that ethanol-induced expression of H4GFP allows the determination of the MI.