Figure 2.

Expression and localization of CnAIP2 in yellow cedar seeds. A, Steady-state levels of CnAIP2 mRNA and proteins in mature soaked dormant seeds, during a dormancy-breaking treatment, and during germination and early postgerminative growth. ES, Embryos and young seedlings; MG, megagametophytes; 3d-Soak, mature seeds subjected to a 3-d water soak (25°C); 4ww, mature seeds subjected to a 3-d soak and 4 weeks of warm, moist conditions (25°C); 4ww+4wc and 4ww+8wc, mature seeds subjected to a 3-d soak and 4 weeks of warm, moist conditions, followed by 4 weeks or 8 weeks of moist chilling (4°C), respectively; G2d, dormancy-terminated seeds placed in germination conditions for 2 d; RE, seeds at radicle emergence; R2-4mm, young seedlings with radicles of 2 to 4 mm. Ten micrograms of total RNA (for northern blots) and 100 µg of total proteins (for western blots) were loaded in each lane. B, CnAIP2 protein in embryos of mature seeds subjected to the full 3-month dormancy-breaking treatment and seedling roots (approximately 10 mm) after incubation overnight with 0 and 20 µm 2,4-D in MS medium. Fifty micrograms of total proteins was loaded on each lane. C, Immunohistochemistry to localize CnAIP2 in yellow cedar seeds. Sections of mature seeds previously subjected to the 3-month dormancy-breaking treatment (including embryo [E] and megagametophyte [M]) were incubated with either anti-CnAIP2 antibodies (top panel) or preimmune serum (taken from the same mouse) as a control (bottom panel). Brown color indicates positive labeling with anti-CnAIP2 antibodies. Sections were counterstained with hematoxylin; lithium carbonate was used to stain the nucleic acids blue and to show nuclei (N). Bars = 1,000, 10, and 25 µm for the left, middle, and right panels, respectively. D, CnABI3 protein in the same tissues and stages as shown in A for the CnAIP2 protein.