Figure 1.
Germination defects of HRS1 knockout mutant on different media.
The seeds of wild type (WT) control, HRS1 knockout mutant (hrs1-1) and complementation (CP) line (CP6-13) were stratified for 48 h at 4°C, followed by transfer to 23°C to allow germination to start on 1/2 MS medium or those supplemented with the indicated concentrations of abscisic acid (ABA) or NaCl. (A–C) The germination time courses of WT, hrs1-1 and CP genotypes on 1/2 MS medium or those containing 1 µM ABA or 100 mM NaCl. The percentages of radicle emergence (means ± SD) at the indicated time points were each determined using the results from triplicate samples. The data shown are all typical of five separate germination experiments, and the two additional CP lines (CP19-1 and CP23-3) behaved similarly as CP6-13 during the experiments. HOG, hours of germination at 23°C.
Figure 2.
Effects of different concentrations of exogenous abscisic acid and NaCl on hrs1-1 germination.
(A, B) The more severe delay of hrs1-1 germination, as compared to that of WT control and two CP lines (CP6-13 and CP23-3), conferred by increasing concentrations of abscisic acid (ABA) or NaCl added to the media. The data shown were recorded after 4 days of germination at 23°C, and are representative of five separate assays. (C, D) Comparisons of the germination rates among WT control, hrs1-1 and CP line (CP6-13) on the media with the indicated concentrations of ABA or NaCl. Each germination rate value (TI, mean ± SD) was calculated using the measurements from triplicate samples after 6 days of germination at 23°C. The higher the value, the more rapid the germination proceeds. The data presented are all representative of three independent experiments, with the additional CP lines (CP19-1 and CP23-3) performing similarly as CP6-13 during the experiments. The means are labeled by different letters or letter combinations according to multiple statistical comparisons, and those labeled by one or more identical letters do not differ significantly from each other (P≤0.05).
Figure 3.
Genetic analysis of hrs1-1 germination.
Germination rate (TI, mean ± SD, each calculated using the measurements from triplicate samples) was used to compare the germination behavior of different genotypes. The datasets presented are each representative of at least three independent experiments. Based on multiple statistical comparisons, the means labeled by one or more identical letters do not differ significantly from each other (P≤0.05). (A) Comparisons of the germination rates of WT control, four single mutants (hrs1-1, abi3-8, abi4-1 and abi5-7), and three double mutants (hrs1-1abi3-8, hrs1-1abi4-1 and hrs1-1abi5-7) on 1/2 MS medium or those with two different concentrations of ABA. (B) Comparisons of the germination rates of WT control, two single mutants (hrs1-1 and abi1-2), two double mutants (hrs1-1abi1-2 and hab1-1abi1-2), and one triple mutant (hrs1-1hab1-1abi1-2) on 1/2 MS medium or those with two different concentrations of ABA. (C) The germination rates of WT control, four single mutants (hrs1-1, abi3-8, abi4-1 and abi5-7), and three double mutants (hrs1-1abi3-8, hrs1-1abi4-1 and hrs1-1abi5-7) on 1/2 MS medium or those containing two concentrations of NaCl. (D) The germination rates of WT control, two single mutants (hrs1-1 and abi1-2), two double mutants (hrs1-1abi1-2 and hab1-1abi1-2), and one triple mutant (hrs1-1hab1-1abi1-2) on 1/2 MS medium or those supplemented with two concentrations of NaCl.
Figure 4.
Analysis of ABI3, ABI4 and ABI5 expression during the germination of wild type control and hrs1-1 seeds.
Quantitative PCR was conducted with gene specific oligonucleotide primers. The amplification of ACT8 (At1g49240, encoding Arabidopsis ACTIN8) served as the internal control. The normalized expression levels (means ± SD) were each calculated using the results from three technical repeats, and are representative of three independent experiments. (A–C) Comparisons of ABI3, ABI4 and ABI5 expression levels between wild type (WT) control and hrs1-1 samples collected at the indicated time points during germination on 1/2 MS medium or the media supplemented with 1 µM ABA or 100 mM NaCl. Asterisk indicates statistical significance from WT control at P≤0.05.
Figure 5.
Temporal and spatial patterns of HRS1 expression during seed germination.
(A) Comparisons of HRS1 expression levels at 0, 24, 36 and 48 HOG on 1/2 MS medium or those supplemented with 1 µM ABA or 100 mM NaCl by quantitative PCR. The amplification of ACT8 (At1g49240) served as the internal control. The normalized expression levels (means ± SD) were each determined using the data from three technical repeats, and are typical of four independent experiments. (B–D) The spatial patterns of HRS1 expression during Arabidopsis seed germination on 1/2 MS medium or those supplemented with 1 µM ABA or 100 mM NaCl investigated using the HRS1 promoter::GUS reporter line RL3-11. HRS1 expression, detected at the designated time points, is indicated by the blue signals generated by histochemical staining of GUS activity. The arrowhead and arrow indicate the zone of strong HRS1 expression detected at 36 HOG on 1/2 MS medium or 48 HOG on ABA containing medium. The three sets of data shown are each representative of four independent GUS staining experiments. Similar results were obtained with the two additional reporter lines RL4-9 and RL5-1. Bars = 0.4 mm.
Figure 6.
Elongation growth of the cells in lower hypocotyl and transition zone, and the occurrence of strong HRS1 expression in lower hypocotyl cells, during Arabidopsis seed germination.
Lower hypocotyl (LH) cells are marked by asterisks, whereas the cells in transition zone (TZ) are labeled by filled dots. (A) Comparison of LH and TZ cells in the radicle samples collected at 24 or 48 HOG. Both LH and TZ cells were elongated from 24 to 48 HOG, with the magnitude of the elongation being substantially larger for LH cells. At 48 HOG (immediately post germination), an elongation zone (EZ), differentiated mainly from elongated LH cells, was found in the young seedling root. In addition, four newly developed TZ cells (indicated by diamonds) were observed on top of the root meristematic region. (B) The occurrence of strong HRS1 expression in the LH cells (of the promoter::GUS reporter line RL3-11) as indicated by the blue signals produced by histochemical staining of GUS activity at 36 HOG. The expression pattern shown was typical of four separate staining experiments, and was found for all three independent promoter::GUS reporter lines of HRS1. EZ, elongation zone; H, hypocotyl; LH, lower hypocotyl; RM, root meristem; TZ, transition zone. Bars = 50 μm.
Figure 7.
Examination of cell elongation growth in germinating embryo axis.
The seeds of WT control, hrs1-1 and CP line (CP6-13) were cold stratified for 48 h, and then allowed to germinate on 1/2 MS medium at 23°C. The measurements were made at 24 and 36 HOG, respectively. (A) The average cell length values (means ± SD, n = 30∼35 samples measured per data point) in transition zone (TZ) and lower hypocotyl (LH) in WT control, hrs1-1 and CP6-13. The data displayed are representative of three separate experiments. (B) The average length values (means ± SD, n = 33∼37 samples measured per data point) of TZ, LH, and TZ plus LH in WT control, hrs1-1 and CP6-13. The data shown are typical of three independent experiments. The additional CP lines (CP19-1 and CP23-3) behaved similarly as CP6-13 during these experiments. Asterisk indicates statistical significance from WT control and CP line at P≤0.05.
Figure 8.
The effects of inhibitor treatment on the germination of wild type control and hrs1-1.
The seeds of wild type (WT) control and hrs1-1 were cold stratified for 48 h, followed by germination on the media without, or with the indicated concentrations of, mannitol, Na3VO4 or DES. The germination rate (TI, mean ± SD, each calculated with the results of triplicate samples) was recorded after six days of germination at 23°C. The data sets displayed are each typical of four independent experiments. (A) The germination rates of WT control and hrs1-1 on the media without (0), or with three concentrations (100, 200 or 400 mM) of, mannitol. (B) The germination rates of WT control and hrs1-1 on 1/2 MS medium or the media with two different concentrations of Na3VO4 or DES. Single, double and triple asterisks indicate statistical significance from WT control at P≤0.05, 0.01, and 0.001, respectively.
Figure 9.
Analysis of plasmalemma H+-ATPase activity during the germination of wild type control, hrs1-1 and complementation line.
The seeds of wild type (WT) control, hrs1-1 and complementation line CP6-13 were cold stratified for 48 h, followed by germination on 1/2 MS medium at 23°C. The levels of H+-ATPase activity (means ± SD, each calculated with the results of triplicate samples) were determined using plasma membrane enrich fractions prepared from the seed samples taken at the indicated time points. The data shown are typical of three independent experiments. The results obtained with the additional CP lines (CP19-1 and CP23-3) were highly similar to those determined for CP6-13 during the experiments. Single and double asterisks indicate statistical significance from WT control at P≤0.05 and 0.01, respectively.
Figure 10.
The effects of fusicoccin treatment on the germination of wild type control and hrs1-1.
The seeds of wild type (WT) control and hrs1-1 (KO) were cold stratified for 48 h, followed by germination on 1/2 MS medium or that supplemented with 1 µM fusicoccin (FC). The percentages of radicle emergence (means ± SD) at the indicated time points were each calculated using the results from triplicate samples. The data shown are typical of three separate germination experiments.