Fig. 1. Western blot under reducing and non-reducing conditions, and the model of ApoA-I dimer by Gogonea et al (2013). A, Western blot under reducing conditions (with β-mercaptoethanol); C+, ApoA-I recombinant; C-, Untransformed rice; ID 15, 20, 23, and 24 rice plants were positive in the screening PCR. All plants showed a band at 28 kDa, similar to the positive control, which was not present in the wild type (WT, untransformed rice) seeds. B, Relative expression of ApoA-IMilano gene in rice plants. RNA was extracted from rice seeds of different plants, and beta-tubulin was used as housekeeping gene. ID 15, 20, 23, and 24 rice plants were positive in the screening PCR. Results are presented as Mean ± SE from three biological replicates. Different lowercase letters above the bars indicate statistically significant differences at the level of P ≤ 0.05.

Fig. 2. Temporal and spatial expression of ApoA-IM gene. A, Western blot under reducing conditions (with β-mercaptoethanol). Rice seeds from the same panicle were collected at different days after flowering. Lines 1, 2, 3, 4, 5, 6, and 7 represent 4, 8, 12, 16, 20, and 25 days after flowering, and maturity stage, respectively. B, qRT-PCR analysis of ApoA-IM gene expression levels in seeds at different ripening stages. Results are presented as Mean ± SE from three biological replicates. Different lowercase letters above the bars indicate statistically significant differences at the level of P ≤ 0.05. C, Western blot under reducing conditions was performed on different plant tissues of the same transformed rice plant. The presence of the signal corresponding to positive control confirmed the seed-specificity of 13 kDa promoter. D, RT-PCR carried out on different plant tissues. Beta-tubulin was used as the internal control. C+, Recombinant hApoA-I; L, Proteins extracted from the leaf; S, Proteins extracted from transformed rice seeds; Cu, Proteins extracted from culm; R, Proteins extracted from roots; C-, Rice seeds from the untransformed plant.

Fig. 3. Immunofluorescence and bright field images of caryopses I (A-F) and II (G-N) of ApoA-IM transformed plants. A-F, Immunofluorescence (A, C, and E) and bright field images (B, D, and F) of caryopsis I of ApoA-IM transformed plant. For A and C, a high fluorescence was observed only in the amyloplasts of endosperm cells particularly in the stroma, surrounding starch granules in C with arrows. E and F, Negative controls showed no cross-reaction with seed tissues. G-N, Immunofluorescence (G, I, K, and M) and bright field images (H, J, L, and N) of caryopsis II of ApoA-IM transformed plant. Pericarp and nucellus appeared thinner in G, H, I, J, M, and N. ApoA-IM was localized in organelles in endosperm cells, seed coats, and aleurone cells in I and K with arrows. G and I, High fluorescence was observed in the seed coat cells. M and N, Only autofluorescence was observed in negative control. a, Aleurons cell; n, Nucellus; Pr, Pericarp; sc, Seed coat; se, Seed endosperm cell.

Fig. 4. Immunogold analysis on caryopsis II of ApoA-IM transformed plants. A and B, ApoA-IM was localized into plasmids in the chloroplast grana in the seed coat (B is a magnification of box in A). C, Dark spots were observed in the primary cell wall and not in middle lamellae (with arrows). D, In aleuron cells, ApoA-IM was localized in storage vacuoles (with arrows). E, ApoA-IM was localized into plasmids in amyloplasts of the endosperm cells (with arrows). F, In aleuron cells, ApoA-IM was localized in vesicles associated with dictyiosomes (with arrowhead). cl, Chloroplast; cw, Cell wall; ml, Middle lamellae; a, Amyloplast; sv, Storage vacuole; g, Golgi body.