FIGURE 5.

Defective female gametophyte development in ABAP1^OE. (A) Schematic representation of the events taking place during maturation of the female gametophyte. The final embryo sac is a polarized structure with two main distinct regions: the micropyla and the chalaza. Red arrows indicate gene markers of embryo sac developmental stages. Red arrowhead represents the timing of EDA24-like expression. (B–E) Micrographs of wild-type and ABAP1^OE ovules. Embryo sacs and embryo sac cells were artificially outlined for better visualization. DIC microscopy (B) and DAPI staining (C) of wild-type ovules indicate the central cell nucleus (Ccn and white arrowhead) formed by fusion of the two polar nuclei. DIC microscopy (D) and DAPI staining (E) of ABAP1^OE ovules show two unfused polar nuclei (PN and red arrowheads). EC, egg cell; Ccn, central cell nucleus; PN, polar nuclei. Scale bars at (B–E) represent 20 μm. (F) Percentage of unfused polar nuclei quantified in emasculated flowers at stage 13 (FG6-FG7) from wild-type Col, ABAP1^OE and eda24-like^KD plants. For WT, 6 ovaries were analyzed, with a minimum of 25 and a maximum of 39 ovules analyzed per ovary. For ABAP1^OE, 14 ovaries were analyzed, with a minimum of 25 and a maximum of 36 ovules analyzed per ovary. For eda24-like^KD, 8 ovaries were analyzed, with a minimum of 23 and a maximum of 34 ovules analyzed per ovary. A statistical analysis was performed by t-test (p-value < 0.05). Asterisks (*) indicate significant changes. (G) Relative mRNA levels of ABAP1, Agamous 80, CCG, Agamous 61 and EDA24-like were determined by qRT-PCR in wild-type and ABAP1^OE flower buds. Data were normalized with UBI14 as reference gene and were compared with wild-type. Each biological replicate was performed with a pool of 15 flower buds. Bars indicate mean ± standard error of biological replicates. A statistical analysis was performed by t-test (p-value < 0.05). Asterisks (*) indicate significant changes.