ADH | Alcohol dehydrogenase (hypoxia marker)

HRA1 contributes to plant submergence survival.(A) Effect of HRA1 misexpression on rosette growth in air, or after recovery from 72 h submergence in darkness. Scale bar, 2 cm. (B) Percentage of plants surviving flooding-induced hypoxia (n?=?5), dry weight of rosette plants kept under control growth conditions (n?=?6), and dry weight of rosettes after postsubmergence recovery (n?=?6). Data are mean ± s.d.; *p<0.05, significant differences from the wild type after one-way ANOVA. (C) HRA1 regulates target gene transcripts in an age-dependent manner in leaves of plants treated with complete submergence. Transcripts were measured before submergence (“control conditions”), after 4 h submergence in darkness (“submergence”), and after 1 h de-submergence in the light (“reoxygenation”). Relative transcript values were calculated using old leaves of the wild type under control conditions as the reference sample. Data are mean ± s.d. (n?=?3); letters indicate statistically significant differences between genotypes after one-way ANOVA (p<0.05) performed independently on each leaf type. (D) Western blot analysis of ADH and PDC protein accumulation in leaves at different developmental stages from control and submerged (4 h) plants. The full-size images of the hybridized membranes can be found in Figure S10. (E) Stability of the translational fusion RAP2.12:RrLuc protein (RrLuc, Renilla reniformis luciferase) in Arabidopsis mesophyll protoplasts upon transfection with increasing amounts of 35S:HRA1. RAP2.12:RrLuc abundance was evaluated from the RrLuc relative activity, measured through a dual luciferase assay. Data are mean ± s.d. (n?=?4), and the asterisks indicate statistically significant differences (p<0.05) from protoplasts expressing RAP2.12:RrLuc alone, after one-way ANOVA.

Assessment of ethanol fermentation in ADH and PDC mutants. (A) ADH activity in 6-day-old Arabidopsis seedlings (Col-0 wild type, and adh1, adh1adh2, and pdc1pdc2 mutants) after incubation under normoxic (21% O2 v/v) or hypoxic (1% O2 v/v) conditions for 12 h. The box plots are built on the bases of four replicates. (B) ADH1 expression in the wild type (Col-0), and adh1 and pdc1pdc2 mutants. The box plots are built on the bases of four replicates. (C) Immunodetection of ADH1 protein in aerobic wild-type (Col-0), adh1, and pdc1pdc2 seedlings, grown in liquid MS medium for 4 d, in the presence of 1% sucrose (w/v). Three biological replicates for each genotype are shown. Gel staining with Coomassie G-250 is shown as a protein loading control (D) Ethanol production in the same genotypes used for the ADH activity quantification shown in (A). The box plots are built on the bases of four replicates.

FLM intron 1 retention prevents nuclear export of FLM messenger RNA. (A) Expression analysis and sub-cellular localization of different FLM isoforms. Top panels, FLM-?, FLM-?, and FLMi1, in fractionated cell extracts (T-total, C-cytoplasmic, N-nuclear) by RT-PCR. FLM-? (high contrast) show nuclear localization for this isoform. Middle panel, The SEF factor splice variants in fractionated cell extracts. The SEF factor was used as a fractionation control as only the mature form (FS) is exported to the cytoplasm. FS, fully spliced; ir1, intron 1 retention; ir1+2, retention of introns 1 and 2. Lower panels, polyacrylamide gel electrophoresis of protein extracts from the same cell fractionation. The cytoplasmic fraction is free of the nuclear protein histone 3B (H3B), and the nuclear fraction is free of the cytoplasmic alcohol dehydrogenase (ADH).