PMC

Genetic Interaction between ABI5 and FyPPs.
(A) Germination and growth of seeds of , abi5, and self-pollinated f1^−/+ f3 and f1^−/+ f3 abi5 plants incubated on plates with 0 or 0.5 µM for 5 d in white light. Seeds from self-pollinated f1^−/+f3 plants showed increased sensitivity to , while abi5-1 seeds were insensitive to compared with controls. Seeds from self-pollinated f1^−/+ f3 abi5-1 plants were insensitive to , similar to abi5-1 seeds.
(B) Loss of FyPP1 and FyPP3 activity enhances the -related phenotypes of ABI5OE seeds. Top panel shows seedlings grown on plates without . The ungerminated seeds (25.2%) from self-pollinated f1^−/+ f3 ABI5OE plants were further phenotyped and genotyped and confirmed to be f1 f3 ABI5OE homozygotes. Bottom panel shows the seedlings grown on plates containing 0.5 µM . The germination and greening percentages of each line are shown at the bottom of each panel, and the number of seeds used for the calculation is shown in parentheses.
(C) Growth of , F3OE, ABI5OE, and F3OE ABI5OE seeds on plates with 0 or 0.5 µM . Overexpression of FyPP3 rescued the hypersensitivity phenotype of ABI5OE seeds. The germination and greening percentages of each line are shown at the bottom of each panel, and the number of seeds used for the calculation is shown in parentheses.

Antagonistic Relationship between OST1 (SnRK2.6) and FyPPs.
(A) Germination and growth of , ost1, F3DN, and ost1 F3DN seeds and seeds from self-pollinated f1^−/+ f3 and f1^−/+ f3 ost1 plants incubated on plates with or without . Seeds were incubated under white light for 5 d.
(B) Germination of , ost1, F3DN, and ost1 F3DN seeds and seeds from self-pollinated f1^−/+ f3 and f1^−/+ f3 ost1 plants. Seeds were grown under white light for 5 d on plates with the indicated concentrations of . Germination was determined with an average of >100 seeds from three independent experiments. Values are means ± sd.
(C) Greening of , ost1, F3DN, and ost1 F3DN seeds and seeds from self-pollinated f1^−/+ f3 and f1^−/+ f3 ost1 plants incubated on plates with (1 μM) or without treatment for 5 d. Greening was determined with an average of >100 seeds from three independent experiments. Values are means ± sd. Asterisks indicate the levels of statistical significance as determined by Student’s t test: *P < 0.01 versus .
(D) Immunoblot analysis of ABI5 accumulation in kinase- and phosphatase-deficient mutants. In the absence of , there was no detectable ABI5 protein in , ost1, f1^−/+ f3, F3DN, f1^−/+ f3 ost1, and F3DN ost1 seedlings. After treatment (1 μM, 8 d), ABI5 (indicated by the arrows) hyperaccumulated in seeds of self-pollinated f1^−/+ f3 or F3DN lines, in contrast with the reduced accumulation of ABI5 in seeds of self-pollinated f1^−/+ f3 ost1 plants and F3DN ost1 lines. There was no detectable ABI5 protein in and ost1 seedlings at this stage. The arrowhead indicates the nonspecific band recognized by the ABI5 antibody. Arrows indicate the ABI5 bands. RPT5 was used as a loading control. A total of 100 μg protein was loaded for each lane.

Abscisic acid (ABA)-insensitivity of rsr1-1 is Glc-dependent. Seeds of rsr1-1 (upper lanes) and Pat(B33)-Gus (lower lanes) were plated on MS-medium supplemented either with (A) 3 μM ABA or (B) 3 μM ABA + 30 mM Glc. In the presence of both Glc and ABA, rsr1-1 seedlings had developed chlorophyll and primary leaves 4 weeks after plating, whereas 3 μM ABA alone delayed germination and completely inhibited development in both rsr1-1 and Pat(B33)-Gus.

AtGASA6-OE seeds are hyposensitive, but RNAi and gasa6-1 mutant seeds are hypersensitive to Glc, ABA, and . A, The diagram of the insertion position in gasa6-1 mutant. B, Relative transcript levels of AtGASA6 in 2-week-old gasa6-1 mutant seedlings detected by -PCR; Actin1 was used to standardize gene expression. C, Relative transcript levels of AtGASA6 in germinating seeds of AtGASA6-OE and RNAi seeds. The transcript levels all were normalized to Actin1 gene expression. D, Photographs of germination phenotypes of wild-type (WT), gasa6-1, AtGASA6-OE (OE#2-1, OE#5-5, and OE#6-1), and RNAi (R#3-5, R#4-3, and R#6-7) seeds treated with 0.5 μm , 1 μm ABA, 6% (w/v) Glc, or 6% (w/v) mannitol; minimal medium was used as control. Seeds were photographed at 24, 64, 52, 64, or 48 h, respectively. At least 40 seeds were used. E, Seed germination rates of wild-type (), gasa6-1, AtGASA6-OE (OE#2-1, OE#5-5, and OE#6-1), and RNAi (R#3-5, R#4-3, and R#6-7) seeds untreated or treated with 0.5 μm , 1 μm ABA, 6% (w/v) Glc, or 6% (w/v) mannitol; minimal medium was used as control. Three technical replicates were performed for each of three biological replicates. *, Significant differences compared with control seeds (one-way ANOVA was used to analyze the significant difference: P < 0.01).