Figure 1.
Comparison of the shape and structure of hulled grain in the fst mutant and WT.
(A–C) The WT plants produced seeds with a normal embryo (EM) and endosperm (EN) 20 DAP. SH, shoot; RO, root. (D–F) The fst mutant plants produced pseudo seeds with an abnormal seed coat but no embryo (EM) and endosperm (EN) 20 DAP. Longitudinal sections of mature seeds (B, C, E & F). Starch of endosperm stained with 1% I2-KI (C & F). Scale bars, 100 µm.
Figure 2.
Characterization of ovule and female gametophyte in the fst mutant.
(A-I) Comparison of ovule development in FST (A, E &G) and fst (B–D, F, H and I). Embryo sac development completion at the stage just BF (A–F), antipodal cells (ANC) of fst were unable to mature as antipodal tissues (ANT) and were located at the correct site within the embryo sac (B), while the ANC of FST were fully developed to ANT (A). Polar nuclei (PN) and egg apparatus (EA) in both FST and fst were developed and appeared normal in shape. At the stage just BF (A–D), abnormalities in mature ovaries showed defects in the development of integuments (INT) and chalaza (CHA) in the fst mutant (B–D) compared to the FST plants (A). (A–D) was stained with toluidine blue. (E–I) Shape and structure of an FST (E & G) and fst (F, H & I) mature ovule. Structure of mature ovules observed under SEM (E–I). Arrows: normal structures. OIN, outer integument; PAR, pericarp (seed coat). Red arrows show abnormalities specific to fst with defects in antipodal tissues (ANT), chalaza (CHA), integuments (INT) and micropyle (MI). NU, nucellus. Scale bars, 100 µm.
Figure 3.
Embryogenesis and endosperm development in the fst mutant.
(A&B) Proembryo (PEM) and free endosperm tissue (ENT) development of FST (W) and fst (M) 2 (A) and 3 DAP (B).(C) Growth of the initial shoot apex (ISA) and seminal root primordium differentiation 5 DAP.(D) First leaf primordium differentiation 7 DAP.(E) Developmental changes illustrating embryo length. Arrows: EM, embryo; EN: endosperm; ENT, endosperm nuclei tissues; INT, integument; PAR, pericarp (seed coat); ROA, root apex; TENT, trace of endosperm nuclei tissues; YEL, young embryonic leaves; n, number of embryos measured. Scale bars, 100 µm.
Figure 4.
Functional analysis and complementation test of FST.
(A) Effect of FST RNAi (35S::RNAi-FST) repression on seed development, showing incomplete grain filling in T2 plants. W, WT. (B&C) Comparison of grain filling of the endosperm (EN) (B), starch stained with 1% I2-KI (C) in the FST (W) and RNAi transgenic seeds at the T2 generation. (D) Comparison of pollen fertility, seed set, degree of grain filling in the FST (W), fst (M) and transgenic plants derived by a complementation test (C) and RNAi silencing (R) of FST gene. n, number of individual plants measured. (E) Seed germination frequencies of FST (W) and transgenic plants derived by a complementation test (C) and RNAi silencing (R) of FST gene. (F) Comparison of flowering time of WT (FST), mutant (fst) and transgenic plants derived by repression (RNAi) and over-expression of the FST gene. Arrows: EM, embryo; EN, endosperm; n, number of individual plants measured. Scale bars, 1 mm. (G) Comaprision of total protein content of WT (FST) and transgenic plants derived by repression (RNAi) of the FST gene. (H) Comaprision of fat content of WT (FST) and transgenic plants derived by repression (RNAi) of the FST gene.
Figure 5.
Expression pattern and biological function of FST.
(A–O) Expression of FST during ovule and seed development by GUS and in situ hybridization. (A–D) FST expression in glumes (GL), ovule primordium (OUP), the ovule and the base of stigma (BST) during floral development at the flower primordium stage (A), ovule primordium differentiation stage (B), stigma differentiation stage (C) and stage just BF (D). LE, lemma; OWP, ovary wall primordium; PA, palea; SP, stamen primordia. (E–G) FST expression in the nucellus (NU), inner integument (IIN), the tip of nucellus near to egg apparatus (EA) and micropyle (MI) during ovule (OV) and embryo sac (EMS) development at the inner-outer integument differentiation stage (E&F) and stage just BF (G). IINP, inner integument primordium; INP, integument primordium; OIN, outer integument; OINP, outer integument primordium. (H–L) FST expression in the apical-basal regions of the proembryo (PEM), endosperm nuclei tissues (ENT), shoot apex (SHA) coleorhiza (COR) and shoot/root primordium (SHP/ROP) of an embryo, and pericarp (PAR) during embryogenesis and endosperm development 3 (H&I), 4 (J), 5 (K) and 7 DAP (L). SC, seed coat. (M–O) FST expression at the shoot/root apex (SHA/RTA) of the embryo (EM), endosperm (EN), seed coat (SC) and pericarp (PAR) during seed development 5 (M) and 10 DAP (N&O). (P–R) Models for the expression pattern and biological function of FST. Red squares (▪) or arrows () indicate the site of FST expression. Blue arrows (
) represent the path of the signal factor (SF). During the development of ovules, embryo sacs and seeds, the SFs induced by prominent FST gene products at NUP (P1), EMS (P2&P3), IIN (P3), PEM( Q1&Q2), ISA/COR (Q3&Q4) and ENT (R) could convey positional information for chalaza formation, integument morphogenesis (P2&P3), embryonic patterning (Q1–Q4) and endosperm proliferation (R), respectively. NUP, nucellus primordium. Scale bars, 100 µm.
Table 1.
Genes involved in female organ development with expression changes of at least 4-fold (fst/ WT) in fst floral organs just BF.
Table 2.
Genes involved in seed development with expression changes of at least 2-fold (fst/ WT) in fst seeds and ovaries 5 DAP.