Murashige and skoog

The rice cultivar Dongjin (Oryza sativa ssp. japonica cv. Dongjin) was used in this study. Husked rice seeds were sterilized with 70% ethanol (30 s), then with 2% NaClO (40 min), and finally were washed five times with distilled water. The sterilized seeds were grown on a 1/2-strength MS (Murashige and Skoog; Duchefa, Netherland) medium containing 0.4% phytagel (adjusted to pH 5.8 with 0.5 g/L MES and KOH) in long-day conditions (16 h light and 8 h dark) at 28 °C.
The seeds of Brassica rapa and Arabidopsis were sterilized with washing of 70% Ethanol (30 s) and 2% of NaClO (30 min). The Brassicas were grown on a 1/2-strength MS (Murashige and Skoog; Duchefa, Netherland) medium containing 0.4% phytagel (adjusted to pH 5.8 with 0.5 g/L MES and KOH) in long-day conditions (16 h light and 8 h dark) at 23 °C. The chemicals used in the screen were obtained from the Korea Chemical Bank, and were dissolved in DMSO to make stock solutions.

Luminometric experiments were performed using previously developed Arabidopsis thaliana ecotypes Col-0 and C24 expressing APOAEQUORIN under control of the CaMV-35S promoter [15 (link),28 (link)]. Seeds were surface sterilised with 70% (v/v) ethanol for 5 min, washed with sterile water five times and sown onto petri dishes containing ½ strength Murashige and Skoog (Duchefa, Harlem, Netherlands) supplemented with 0.8% (w/v) Bacto agar (Becton, Dickson and Company, Sparks, MD, USA) (adjusted to pH 5.7 with KOH). The plates were incubated horizontally in a growth chamber under 12/12 h light/dark regime, at 20°C and 80 μM m^-2 s^-1 light intensity.

The Oryza sativa japonica cultivar ‘Dongjin’ (parental line), and the oswrky5-D and oswrky5 mutants were grown in a growth chamber under LD conditions (14 h light at 28 °C/10 h dark at 25 °C) and in a rice paddy field under NLD conditions (≥14 h sunlight/day, 37° N latitude) in Seoul, Republic of Korea. The T-DNA insertion activation-tagged oswrky5-D and knockdown oswrky5 mutants were obtained from the Crop Biotech Institute at Kyung Hee University, Republic of Korea [54 (link),55 (link)].
For dark treatment, detached leaves of rice plants grown in the growth chamber for 3 weeks were incubated in 3 mM 2-(N-morpholino)ethanesulfonic (MES) buffer (pH 5.8) with the abaxial side up at 28 °C in complete darkness. To detect OsWRKY5 transcript levels under various hormone treatments, WT seeds were sterilized with 70% ethanol and 2% NaClO, and then germinated and grown on half-strength Murashige and Skoog (0.5X MS, Duchefa, The Netherlands) solid medium under LD conditions for 10 days in a growth chamber. Ten-day-old plants were transferred to 0.5X MS liquid medium containing 50 μM epibrassinolide (BR), 50 μM GA, 50 μM IAA, 50 μM 6-BA, 100 μM SA, 50 μM MeJA, 50 μM ABA, or 50 μM ACC. Total RNA was extracted from leaves harvested at 0 and 6 h of treatment.