Figure 1.
Phylogeny and expression patterns of Bsister genes.
Bayesian phylogeny of class D, class B and Bsister genes in seed plants. Posterior probabilities are indicated on the nodes. The expression patterns of Bsister and class D genes are depicted as far as they are known. Colors indicate expression intensity: white, no expression; light grey, weak expression; dark grey, strong expression; asterisk, expression was detected particularly in endothelial cells of inner integument; n, no expression data available; cross, tissue or organ is not present in respective species.
Figure 2.
Expression analyses of OsMADS29.
(A). RT-PCR analyses of OsMADS29 expression at different developmental stages of wild type plants. DAP, days after pollination. ACTIN1 was used as control. (B). Relative expression levels of OsMADS29 at different developmental stages. Amounts of transcripts are shown as relative values to those of UBQ. Error bars show SD (n = 3). (C). RT-PCR analyses of OsMADS29 expression in various floral organs of wild type plants at heading date stage. APT1 was used as a control [47]. (D–L). In situ hybridization analyses of OsMADS29 in ovules and developing seeds. (D). Longitudinal section of the ovule. (E–G). Longitudinal sections of seed at 1 DAP (E), and embryo of seed at 3 DAP (F) and 5 DAP (G). (H–I). Transverse sections of the mid-region of the ovary at a few hours before anthesis (H) and seed at 3 DAP (I). The arrows head in (H) and (I) indicate the inner layer cells of the pericarp. (J). Negative control with sense probes of OsMADS29. (K–L). A magnification of the transverse section of a seed at 7 DAP. (K). The ovular vascular trace in the seed at 7 DAP. (L). Part of pericarp of seed at 7 DAP. CC, cross-cells; EM, embryo; EN, endosperm; IN, integuments; II, inner layer of the inner integument; NE, nucellar epidermis; NP, nucellar projection; NU, nucellus; OV, ovular vascular trace; TC, tube-cells. Bars = 100 µm in (D) to (J), and 50 µm in (K) and (L).
Figure 3.
Phenotypes of OsMADS29 RNAi lines and molecular detections.
(A–I). Profiles of seeds at 7 DAP, 10 DAP and mature stages, respectively. (A–C), n-seeds of control plants. (D–F), m-seeds with deficient starch accumulation in the endosperm. (G–I), s-seeds that are aborted before 7 DAP. Bars = 1 mm. (J). Relative expression levels of OsMADS29 in control plants and three typical OsMADS29 RNAi lines (L5, L8 and L9). Total RNA was pooled from stage Ov10 [48]. The ACTIN1 was used as the internal control. Error bars show SD (n = 3). (K). RT-PCR analyses of OsMADS29 and the other two related Bsister genes of rice, OsMADS30 and OsMADS31, in control plants and RNAi plants. Total RNA was pooled from stage Ov10. The ACTIN1 was used as the internal control. (L). Percentage of different kinds of seeds in one panicle in control plants and different OsMADS29 RNAi lines. The error bar indicates the SE (n = 3).
Table 1.
Agronomic traits of seeds in control plants and OsMADS29 RNAi transgenic lines.
Figure 4.
Histological analyses of control and transgenic seeds.
(A). Starch granules were seen in control seeds at 7 DAP. (B, C). m- and s-seeds at 7 DAP without starch granules. (D–F). A higher magnification of the OV at 7 DAP in the control seeds (D), m-seeds (E) and s-seeds (F), respectively. The double arrow between the two transverse lines indicates the cell layers of the OV. (G, H). Semi-thin transverse sections from the mid-region of mature control seeds (G) and m-seeds (H) at 30 DAP. The red rectangle in (G) and (H) indicates the position of ovular vascular trace. The arrowhead in (H) indicates the remained pericarp. (I, J). Higher magnification for part of the pericarp in mature control seeds (I) and m-seeds (J) at 30 DAP. It shows that the pericarp cuticular layer in (I) and persisted pericarp cells in (J). The red dots indicate the cuticular seed coat in control seeds (I) and the cells of integuments and nucellar epidermis in m-seeds are not degenerated (J). (K, L). Higher magnification of the OV in control seeds (K) and m-seeds (L), which showed in the red rectangle in (G) and (H). The red circle in (L) indicates the large OV in m-seeds. AL, aleurone; NP, nucellar projecton; OV, ovular vascular trace; P, pericarp; PH, phloem; PS, the pigment strand; XY, xylem; EN, endosperm. Bars = 200 µm in (A–C), 50 µm in (D–F), 500 µm in (G) and (H), 100µm in (I) and (J), and 50 µm in (K) and (L).
Figure 5.
Histological structure of embryo and germination of mature seeds.
(A). Longitudinal sections of control embryo at 7DAP. (B, C) Longitudinal sections of embryo of s-seeds (B) and m-seeds (C) at 7 DAP, respectively. (D). The normally germinated seed of control plant. (E). Longitudinal sections of the mature embryo in control seed. The red line referred to the position of vascular bundle. (F). Magnifying observation of cross sections from the red line marked parts in (E). The arrow showed the clear vascular bundle. (G). The m-seeds failed to germinate. (H). Longitudinal sections of the mature embryo in m-seeds. (I). Magnifying observation of cross sections from the red line marked parts in (H). No obvious vascular bundles were observed at the arrow referred place. VB, vascular bundles. Bars = 20 µm in (A–C), 5 mm in (D) and (G), 0.2 mm in (E) and (H), 20 µm in (F) and (I).
Figure 6.
Detections of tissue degeneration.
(A–D). Evan’s Blue staining of control seeds. (A-C) Transverse sections at 15 DAP, 20 DAP, and 26 DAP, respectively. (D) The longitudinal section at 26 DAP. The blue tissues indicate dead endosperm cells. (E–H). Evan’s Blue staining of m-seeds. (E–G) Transverse sections at 15 DAP, 20 DAP, and 26 DAP respectively. H Longitudinal sections at 26 DAP. Bar = 500 µm in (A) to (H). EM, embryo. (I). Quantitative real-time PCR analyses of PCD-related genes in 7 DAP seeds of control and transgenic plants. ACTIN1 was used as an internal control. Error bar indicated the SD (n = 3). ** indicates an extremely significant difference (P<0.01) between control and transgenic lines.