Figure 1.
Decreased primary seed dormancy and vivipary phenotype of abi4.
(A)–(F) Germination of WT and abi4 seeds on 1/2 MS medium with or without stratification treatment. Seeds were stored for 1, 2 weeks or 6 months after harvest and subjected to analysis. Quantitative analysis of germination rates are shown in the right panels (n≥45). One representative image per genotype (1.5 days after sowing) is shown (left panels). Bar = 0.25 mm. Percentages are the average of three repeats ± standard error. (G) Representative images of vivipary phenotype of abi4 on 1/2 MS medium (a, c) or on soil (b, d) are shown. Immature long-green siliques were collected from plants with various genotypes plants grown under the identical growth conditions.
Figure 2.
ABA quantification in abi4 and WT dry and imbibed seeds.
ABA contents were determined in dry and imbibed WT and abi4 mutant seeds. Two-week stored seeds were used for analysis. The * stands for significant level of 0.05.
Figure 3.
GA biogenesis is impaired in seeds of abi4 mutant.
(A) Two independent homozygous OE-ABI4 lines were identified through qRT-PCR. (B) Western blot confirmed the two OE-ABI4 transgenic lines. (C) ABI5 expression analysis in abi4, WT and two OE-ABI4 transgenic lines. (D)–(F) Germination analysis of WT, abi4, OE-1 and OE-2 seeds on 1/2 MS medium (D) 1/2 MS medium supplemented with 15 µM PAC. (E) 1/2 MS medium supplemented with 0.5 µM GA (F). Quantitative analysis of germination rates are shown in the right panels (n≥45). One representative image (time points indicated in figures) per genotype is shown (left panels). Bar = 0.25 mm. Percentages are average of three repeats ± standard error. (G) Endogenous GA4 levels in abi4 and WT seeds were determined. Two-week stored seeds were used for analysis. Percentages are average of three repeats ± standard error.
Figure 4.
Gene expression analysis in dry and imbibed seeds.
Gene expression was investigated by qRT-PCR during the course of the imbibition process. Two-week stored seeds were used for mRNA extraction, and three replications were performed. Primers used in the qRT-PCR assay are listed in Table S1. (A) GA biosynthesis genes. (B) GA catabolism genes. (C) RGL3, a negative regulator of GA signaling. (D) ABA biosynthesis genes. (E) ABA catabolism genes.
Figure 5.
ABI4 represses CYP707A1 and CYP707A2 expression by directly binding to these promoters.
(A)–(C) Promoters of CYP707A1 (A), CYP707A2 (B) and CYP707A3 (C) were analyzed. Fragments of 2,000–3,000 bp upstream of ATG were chosen as the promoter regions. (D) ChIP-qPCR was performed using specific primers corresponding to different promoter regions. TUB4 was employed as an internal control, and the promoter of ABI5 was used as a positive control. Primers used in the ChIP-qPCR assay are indicated by arrows and listed in Table S1. (E)–(H) Transient expression assay showed that ABI4 inhibits CYP707A1 and CYP707A2 transcription. Representative images of N. benthamiana leaves are shown in (E) and (G). Quantitative analyses of luminescence intensity are shown in (F) and (H). The experiments were performed three biological repeats and obtained the similar trend.
Figure 6.
The abi4 rescues the non-germination phenotype of ga1-t and restores the delayed germination of cyp707a1 and cyp707a2.
Quantitative analysis of germination rates are shown in the right panels (n≥45). One representative image (time points indicated in figures) per genotype is shown (left panels). Two-week stored seeds were used for analysis. Bar = 0.25 mm. Percentages are the average of three repeats ± standard error. (A) Seed germination of abi4, ga1-t and abi4/ga1-t in the absence of exogenous GA. (B) Seed germination of abi4, ga1-t and abi4/ga1-t in the presence of exogenous GA. (C) Seed germination of abi4, cyp707a1 and abi4/cyp707a1 mutants on 1/2 MS medium. (D) Seed germination of abi4, cyp707a2 and abi4/cyp707a2 mutants on 1/2 MS medium.