Fig. 1.

Essential role of PpSnRK2 in ABA responses and osmostress tolerance. a, b Activity of kinases from ABA-treated (a) or osmostress-treated (b) protonemata of wild-type (WT) and Ppsnrk2 disruptants. Total soluble proteins extracted from protonemata treated with 10 µM ABA or with 0.4 M mannitol for indicated periods were subjected to in-gel phosphorylation assay using histone IIIS as a substrate. The bottom panel shows Coomassie Brilliant Blue (CBB) staining of the protein samples to show equal loading. c Protonemal growth of WT and the Ppsnrk2 QKO plants with or without 100 µM ABA. d RNA gel blot analysis of ABA-inducible LEA gene expressions of WT, Ppsnrk2 TKO, and QKO. Protonemata were treated with 10 µM ABA for indicated periods, and total RNA was used for the analysis. The bottom panel shows ethidium bromide staining of rRNA to show equal loading. e, f Accumulation of e boiling soluble proteins and f a LEA (17B9) protein in protonemata of WT or QKO plants treated with ABA or mannitol. Protonemata were treated with or without the indicated concentration of ABA or mannitol for 1 d prior to protein extraction. e Extracted proteins were boiled, and the supernatant (boiling soluble proteins) was separated by SDS-PAGE and stained by CBB. f Extracted proteins were separated by SDS-PAGE, transferred onto a nylon membrane, and reacted with anti-17B9 antibody. g Desiccation tolerance of Ppsnrk2 QKO plants. The ABA-insensitive mutant ark was used as a control. Protonemata were dehydrated in an atmosphere of relative humidity of 97% for indicated periods. The dehydrated tissues were hydrated in water and grown on normal medium for two weeks. h Comparison of osmoshock tolerance among ABA mutants of P. patens. Protonemata of WT, QKO, ABA-deficient Ppaba1, and ark were treated with 0.4 M mannitol solution for 15 min, then stained by 0.5% Evans Blue. i Protonemata of WT, QKO, and ark were subjected to an acclimation assay. One-week-old protonemata were treated with 0.1 M mannitol solution or 10 µM ABA for 1 h or 12 h, then subjected to osmoshock treatment with 0.35 M mannitol for 20 min and stained with 0.5% Evans Blue. Scale bars, 0.5 cm in c, h, i