An essential role for cullin 3 ubiquitin ligases and phototropin 2 turnover in seedling chloroplast development (de-etiolation)

Transgenic Arabidopsis seedlings RV86-5 (contains a dexamethasone-inducible ubiquitin variant that contains Arg instead of Lys at position 48; ̈Schlögelhofer et al., 2005) were surface-sterilized and sown on Uhelon 120T (Silk & Progress, Czech Republic) mesh placed on 1% (w/v) agar containing a half-strength Murashige and Skoog medium (pH 5.7), stratified at 4 °C for 3 d, and cultivated at 21 °C/19 °C day/night temperatures, with a 16 h photoperiod (90 μmol m−2 s−1 light intensity) for 14 d. On the fourteenth day (after the first 2 h of the day period), the Uhelon mesh with the seedlings was transferred onto a new solid medium supplemented with (i) 5×10−4% (v/v) DMSO (mock); (ii) 0.7 or 7.0 μM dexamethasone (DEX) and rinsed with DMSO or DEX-supplemented water for mock and DEX-treated seedlings, respectively, and incubated for 24 hours.