A postgermination developmental arrest checkpoint is mediated by abscisic acid and requires the ABI5 transcription factor in Arabidopsis

ABA stabilizes ABI5 protein. (A) Eight-day-old Ws/35S∷HA-ABI5 seedlings (T3 generation) transferred to MS medium with or without 50 μM ABA. Proteins were extracted at the time points indicated and ABI5 protein levels were monitored by Western blot analysis using antibodies to HA. Each lane contained 5 μg protein. (B) Eight-day-old Ws/35S∷HA-ABI5 seedlings were incubated in liquid MS medium supplemented with 100 μM cycloheximide for 15 h. After the treatment, 50 μM ABA was added to the medium and proteins were extracted at the times indicated. Equal amounts (5 μg) of protein were loaded on a 10% SDS/polyacrylamide (NOVEX) gel and analyzed by Western blot using an anti-HA antibody. (C) Extracts prepared from 8-day-old Ws/35S∷HA-ABI5 seedlings were treated with a mixture of anti-protease (complete Mini from Amersham Pharmacia) supplemented with 1 mM PMSF with or without proteasome inhibitors ALLN, MG115, MG132, and PS1 (10 μM each, Calbiochem), as described in Materials and Methods. An equal volume of SDS-loading buffer was added to stop the reactions. Equal amounts of sample were then analyzed by Western blots using rabbit antibody to HA. (D) Eight-day-old Ws/35S∷HA-ABI5 transgenic seedlings were treated in liquid MS medium with [³²P]orthophosphoric acid (100 μCi) for 1 h 30 min. Seedlings were further treated with or without 50 μM ABA for 30 min. HA-ABI5 was immunoprecipitated by using rabbit antibody to HA coupled to beads. Beads were further treated with or without γ phosphatase. 90% of the sample was then analyzed by PhosphorImager (Bio-Rad); 10% of the sample was analyzed by Western blot using mouse monoclonal antibody to HA (Santa Cruz Biotechnology) as the first antibody.

ABA regulates ABI5 accumulation within a narrow developmental window. (A) WT Ws seeds were kept in darkness at 4°C for 3 days without ABA and then transferred to 5 μM ABA plates at the indicated times poststratification. ABI5 levels were monitored by Western blot analysis. Photographs depict representative seedlings 5 days after the different treatments. (Scale bar, 0.5 mm.) (B) ABI5 protein level rapidly decreased on ABA removal. WT Ws seeds treated with ABA (5 μM) as described in Fig. 1B were transferred to plates without ABA, 2 days poststratification. ABI5 levels were monitored by Western blot analyses as described in Fig. 1. (C) WT Ws seeds eventually germinated in the presence of ABA (5 and 10 μM). (Scale bar, 0.5 mm.) (D) WT Ws seedlings 4 and 30 days poststratification in the presence or absence of ABA (5 μM). (Scale bar, 0.5 mm.)

ABI5 protein accumulation is induced by salt and drought stress. (A) WT Ws seeds were kept in darkness at 4°C for 3 days with or without NaCl (200 mM) and then transferred to constant light at 22°C for 1, 2, 3, 4, and 5 days (0 indicates the time immediately following transfer). Western blot analyses using antibodies to ABI5 were performed as described in Fig. 1. (B) One-day-old seeds were transferred to absorbing paper for 1, 2, 3, and 4 h. Western blot analyses using antibodies to ABI5 were further performed as described in Fig. 1.