Stratified seeds of null mutants of G-protein complex show increased sensitivity to ABA-induced inhibition of seed germination. Seeds from matched seed lots were surface sterilized and pretreated with water (A–D) or 100 μmol fluridone (E–H) at 4°C in darkness for 48 h. Seeds were washed extensively with water and plated on 0.5× MS media plates containing 1% Suc in the absence or presence of 1 μm ABA. Plates were kept at 4°C in darkness for 48 h and then transferred to growth chambers (16-h light/8-h darkness regime) at 22°C. Germination was recorded starting 12 h after transfer of the plates to growth chambers until 60 h and expressed as a percentage of total seeds. The figure shows germination of the single mutant alleles gcr1-2, gpa1-4, and agb1-2 compared to Col seeds in the absence (A) or presence (B) of ABA. Germination of the double and triple mutant seeds agb1 gpa1, gcr1 gpa1, agb1 gcr1, and agb1 gcr1 gpa1 in the absence and presence of ABA is shown in C and D, respectively. Germination percentage of fluridone pretreated seeds of the single mutants gcr1-2, gpa1-4, and agb1-2 is shown in absence of or in the presence of ABA compared to Col seeds in E and F, respectively, and of the double and triple mutant seeds agb1 gpa1, gcr1 gpa1, agb1 gcr1, and agb1 gcr1 gpa1 in the absence and presence of ABA is shown in G and H, respectively. The experiment was repeated three times and data were averaged, n = 60 for each experiment. The error bars represent sd. P < 0.05 for gcr1-2, <0.001 for gpa1-4 and gcr1 gpa1, and <0.0001 for all the genotypes with agb1 mutation as determined by t test in comparison to Col control at 24 h time point for water pretreated seeds and at 36 h time point for fluridone pretreated seeds.

Null mutants of G-protein complex show increased sensitivity to Glc-induced inhibition of seed germination. Seeds from matched seed lots were surface sterilized and pretreated with water or 100 μm fluridone at 4°C in darkness for 48 h. Seeds were washed extensively with water and plated on 0.5× MS media plates containing 6% Glc. Germination was recorded at 60 h after transfer to growth chambers at 22°C. The experiment was repeated three times, n = 60 for each experiment. The error bars represent sd. P < 0.005 for water pretreated seeds of genotypes lacking GCR1, GPA1, or both, and <0.001 for all genotypes lacking AGB1 compared to Col as determined by t test. For fluridone pretreated seeds, P < 0.001 for all the genotypes (except agb1-1) compared to Col, as determined by t test. For agb1-1 genotype P < 0.0001 relative to Col. Single asterisks (*) represent significant (P < 0.005) and double asterisks (**) represent highly significant (P < 0.001) values.

Inhibition of lateral root growth in G-protein complex null mutants by ABA. Seeds germinated on 0.5× MS plates were transferred after 60 h to control plates (no ABA) or plates containing 2 μm ABA and seedlings were allowed to grow vertically for 12 d under 16-h light/8-h dark condition. Number of lateral roots was counted. Figure shows number of lateral roots in different genotypes in presence or absence of ABA. Inset shows representative Col and agb1-2 mutant seedlings without or with 2 μm ABA treatment. Each experiment represents the average value from 40 seedlings. The experiment was repeated twice independently and data were averaged. Error bars represent sd. For difference in number of lateral roots without and with ABA, P > 0.5 for gcr1, gpa1, and gcr1 gpa1 and <0.0005 for all the genotypes with agb1 mutation relative to Col, as determined by t test.

Expression of stress-induced genes in G-protein complex null mutants by ABA. Five-day-old seedlings were treated with ethanol (control) or 100 μm ABA for 1 h and cDNA was synthesized. cDNA was used for real-time PCR in the presence of SYBR-green dye. Data are expressed as fold change in response to ABA based on 2^{−ΔΔ Ct} values (ΔΔ Ct = Δ Ct specific gene expression − Δ Ct Actin gene expression). The experiment was repeated three times and data were averaged. Error bars represent sd. Single asterisks (*) represent significant (<0.005) and double asterisks (**) represent highly significant (<0.001) values for change in ABA-induced gene expression compared to Col as determined by t test.

Expression of G-protein complex component genes during germination. A, Expression levels of GCR1, GPA1, and AGB1 genes. Expression was determined by real-time quantitative PCR using SYBR green dye. Expression of the Actin gene under identical conditions served as an internal control. Expression was determined starting 6 h postimbibition until 3 d after transfer of seeds to growth chambers (16-h light/8-h darkness regime) at 22°C. Asterisk indicates the time point when seeds were transferred to 22°C. Inset shows relative expression of Actin and GPA1 genes and expression level of GPA1 protein in nonstratified seeds. The experiment was repeated three times and data were averaged. The error bars represent sd. B, Western blotting with GPA1 antibodies. Steady-state level of GPA1 protein was determined in the microsomal fraction prepared from seeds and seedlings during germination and postgermination growth. Faint GPA1-specific signal appeared 1 d after transfer of seeds to 22°C and showed a large increase at day 2 (2 d), consistent with the expression profile obtained by real-time PCR. C, Effect of ABA on expression of GCR1, GPA1, and AGB1. Expression of GCR1, GPA1, AGB1, and Rd29A (a known ABA-responsive gene, used as a positive control) genes was determined by real-time quantitative PCR with control and ABA-treated seeds at 1 and 2 d after transfer to 22°C. Values represent fold change in expression level upon ABA treatment compared to control (ethanol) treatment. The experiment was repeated three times and data were averaged. The error bars represent sd. Inset shows the level of GPA1 protein at day two (2 d) after transfer of seeds to 22°C, before and after ABA treatment. Top, Western blot with GPA1 antibody; bottom, Coomassie Blue-stained gel.

Nonstratified seeds of null mutants of G-protein complex show increased sensitivity to ABA-induced inhibition of seed germination. Seeds from matched seed lots were surface sterilized and plated on 0.5× MS media plates containing 1% Suc in the absence or presence of 1 μm ABA. Plates were transferred to growth chambers (16-h light/8-h darkness regime) at 22°C. Germination was recorded starting 12 h after transfer of the plates to growth chambers until 92 h and expressed as a percentage of total seeds. The figure shows germination of the single mutant alleles gcr1-2, gpa1-4, and agb1-2 compared to Col seeds in the absence (A) or presence (B) of ABA. Germination of the double and triple mutant seeds agb1 gpa1, gcr1 gpa1, agb1 gcr1, and agb1 gcr1 gpa1 in the absence and presence of ABA is shown in C and D, respectively. The experiment was repeated three times and data were averaged, n = 60 for each experiment. The error bars represent sd. For B and D, P < 0.05 for gcr1-2, <0.001 for gpa1-4 and gcr1 gpa1, and <0.0001 for all the genotypes harboring an agb1 mutation as determined by t test in comparison to Col (ABA) at 72 h time point.

Effect of ABA on postgermination growth of G-protein complex mutants. Seeds germinated on 0.5× MS media were transferred after 24 h to control plates (no ABA) or 1 μm ABA-containing media plates under similar growth conditions and growth was recorded after 5 d (A). Representative seedlings from control and ABA-containing plates are shown. B, Percentage reduction in length of primary roots by ABA in different mutant genotypes. Each value represents average length of 20 seedlings. The experiment was repeated twice independently. Data from one experiment are presented. Error bars represent sd. For the difference in root length without and with ABA, P < 0.001 for gcr1, gpa1, and gcr1 gpa1 and <0.0005 for all the genotypes with agb1 mutation relative to Col, as determined by t test. Single asterisks (*) represent significant (<0.005) and double asterisks (**) represent highly significant (<0.001) values.

Effect of ABA in the presence of auxin on lateral root formation in G-protein complex mutants. Seeds germinated on 0.5× MS plates were transferred after 60 h to control plates (no ABA) or 0.5× MS plates containing 2 μm ABA, 100 nm NAA, or both. Seedlings were allowed to grow vertically for 12 d in 16-h light/8-h dark condition and number of lateral roots (A) as well as length of primary root (B) was measured. Each experiment represents the average value from 40 seedlings. The experiment was repeated twice independently and data were averaged. Error bars represent sd. For number of lateral roots in the presence of NAA, P > 0.5 for gcr1, gpa1, and gcr1 gpa1 and <0.0001 for all the genotypes with agb1 mutation, relative to Col. For number of lateral roots in the presence of ABA alone or both NAA + ABA, P < 0.0001 for all the genotypes compared to in the presence of NAA alone and P > 0.5 for all genotypes relative to Col as determined by t test. For inhibition of primary root length in the presence of NAA or ABA, relative to control media P < 0.001 for all the genotypes. In the presence of both NAA and ABA, P > 0.5 for gcr1, gpa1, and gcr1 gpa1 and P < 0.001 for all the genotypes lacking agb1 subunit, compared to Col as determined by t test. Single asterisks (*) represent significant (<0.005) and double asterisks (**) represent highly significant (<0.001) values.