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. 2009 May;150(1):463–481. doi: 10.1104/pp.109.135269

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Copyright © 2009, American Society of Plant Biologists

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Figure 4. — Genetic relationship between RHA2a and ABI5. A to C, Overexpression of RHA2a in abi5-7 leads to ABA hypersensitivity. A, Quantification of cotyledon greening. Cotyledon-greening percentage of Col-0, abi5-7, and RHA2a overexpression plants in the genetic background of Col-0 (35S:RHA2a 7#) and two lines of RHA2a overexpression plants in the genetic background of abi5-7 (abi5-7/35S:RHA2a 1# and abi5-7/35S:RHA2a 2#) grown on medium containing different concentrations of ABA was recorded at 5 d after the end of stratification. Data show the mean ± sd of three replicates. At least 100 seeds per genotype were measured in each replicate. B, Quantification of chlorophyll content. Seedlings described in A were collected for chlorophyll a/b extraction and measurement. Values represent the mean ± sd of three replicates. FW, Fresh weight of whole seedlings. C, Root growth measurements. Seedling root length of the indicated genotypes grown on medium containing different concentrations of ABA was measured at 5 d after the end of stratification (see “Materials and Methods”). Relative root growth compared with that on ABA-free medium is indicated. Data show the mean ± sd of three replicates. At least 30 seedlings per genotype were measured in each replicate. Asterisks indicate the significance of the difference from the corresponding wild-type values determined by Student's t test (* 0.01 ≤ P < 0.05, ** P < 0.01). At least three independent experiments were conducted and similar results were obtained. D to F, Overexpression of ABI5 in rha2a leads to ABA hypersensitivity. D, Quantification of cotyledon greening. Cotyledon-greening percentage of Col-0, rha2a, and ABI5 overexpression plants in the genetic background of Col-0 (Col-0/35S:ABI5) and two lines of RHA2a overexpression plants in the genetic background of rha2a (rha2a/35S:ABI5 1# and rha2a/35S:ABI5 2#) grown on medium containing different concentrations of ABA was recorded at 5 d after the end of stratification. Data show the mean ± sd of three replicates. At least 100 seeds per genotype were measured in each replicate. E, Quantification of chlorophyll content. Seedlings described in D were collected for chlorophyll a/b extraction and measurement. Values represent the mean ± sd of three replicates. F, Root growth measurements. Seedling root length of the indicated genotypes grown on medium containing different concentrations of ABA was measured at 5 d after the end of stratification (see “Materials and Methods”). Relative root growth compared with that on ABA-free medium is indicated. Data show the mean ± sd of three replicates. At least 30 seedlings per genotype were measured in each replicate. Asterisks indicate the significance of the difference from the corresponding wild-type values determined by Student's t test (* 0.01 ≤ P < 0.05, ** P < 0.01). At least three independent experiments were conducted and similar results were obtained. G to J, Double mutant analysis between rha2a and abi5-7. G, Quantification of cotyledon greening. Cotyledon-greening percentage of the indicated genotypes grown on medium containing different concentrations of ABA was recorded at 5 d after the end of stratification. Data show the mean ± sd of three replicates. At least 100 seeds per genotype were measured in each replicate. H, Quantification of chlorophyll content. Seedlings described in G were collected for chlorophyll a/b extraction and measurement. Values represent the mean ± sd of three replicates. I, Root growth measurements. Seedling root length of the indicated genotypes grown on different concentrations of ABA was measured at 5 d after the end of stratification. Relative root growth compared with that on ABA-free medium is indicated. Data show the mean ± sd of three replicates. At least 30 seedlings per genotype were measured in each replicate. J, Photographs of seedlings at 5 d after the end of stratification. Twenty-four hours after stratification, germinated seeds were transferred from ABA-free medium to medium containing different concentrations of ABA. Asterisks indicate the significance of the difference from the corresponding abi5-7 values determined by Student's t test (* 0.01 ≤ P < 0.05, ** P <0.01). At least three independent experiments were conducted and similar results were obtained. [See online article for color version of this figure.]

Figure 4. — Genetic relationship between RHA2a and ABI5. A to C, Overexpression of RHA2a in abi5-7 leads to ABA hypersensitivity. A, Quantification of cotyledon greening. Cotyledon-greening percentage of Col-0, abi5-7, and RHA2a overexpression plants in the genetic background of Col-0 (35S:RHA2a 7#) and two lines of RHA2a overexpression plants in the genetic background of abi5-7 (abi5-7/35S:RHA2a 1# and abi5-7/35S:RHA2a 2#) grown on medium containing different concentrations of ABA was recorded at 5 d after the end of stratification. Data show the mean ± sd of three replicates. At least 100 seeds per genotype were measured in each replicate. B, Quantification of chlorophyll content. Seedlings described in A were collected for chlorophyll a/b extraction and measurement. Values represent the mean ± sd of three replicates. FW, Fresh weight of whole seedlings. C, Root growth measurements. Seedling root length of the indicated genotypes grown on medium containing different concentrations of ABA was measured at 5 d after the end of stratification (see “Materials and Methods”). Relative root growth compared with that on ABA-free medium is indicated. Data show the mean ± sd of three replicates. At least 30 seedlings per genotype were measured in each replicate. Asterisks indicate the significance of the difference from the corresponding wild-type values determined by Student's t test (* 0.01 ≤ P < 0.05, ** P < 0.01). At least three independent experiments were conducted and similar results were obtained. D to F, Overexpression of ABI5 in rha2a leads to ABA hypersensitivity. D, Quantification of cotyledon greening. Cotyledon-greening percentage of Col-0, rha2a, and ABI5 overexpression plants in the genetic background of Col-0 (Col-0/35S:ABI5) and two lines of RHA2a overexpression plants in the genetic background of rha2a (rha2a/35S:ABI5 1# and rha2a/35S:ABI5 2#) grown on medium containing different concentrations of ABA was recorded at 5 d after the end of stratification. Data show the mean ± sd of three replicates. At least 100 seeds per genotype were measured in each replicate. E, Quantification of chlorophyll content. Seedlings described in D were collected for chlorophyll a/b extraction and measurement. Values represent the mean ± sd of three replicates. F, Root growth measurements. Seedling root length of the indicated genotypes grown on medium containing different concentrations of ABA was measured at 5 d after the end of stratification (see “Materials and Methods”). Relative root growth compared with that on ABA-free medium is indicated. Data show the mean ± sd of three replicates. At least 30 seedlings per genotype were measured in each replicate. Asterisks indicate the significance of the difference from the corresponding wild-type values determined by Student's t test (* 0.01 ≤ P < 0.05, ** P < 0.01). At least three independent experiments were conducted and similar results were obtained. G to J, Double mutant analysis between rha2a and abi5-7. G, Quantification of cotyledon greening. Cotyledon-greening percentage of the indicated genotypes grown on medium containing different concentrations of ABA was recorded at 5 d after the end of stratification. Data show the mean ± sd of three replicates. At least 100 seeds per genotype were measured in each replicate. H, Quantification of chlorophyll content. Seedlings described in G were collected for chlorophyll a/b extraction and measurement. Values represent the mean ± sd of three replicates. I, Root growth measurements. Seedling root length of the indicated genotypes grown on different concentrations of ABA was measured at 5 d after the end of stratification. Relative root growth compared with that on ABA-free medium is indicated. Data show the mean ± sd of three replicates. At least 30 seedlings per genotype were measured in each replicate. J, Photographs of seedlings at 5 d after the end of stratification. Twenty-four hours after stratification, germinated seeds were transferred from ABA-free medium to medium containing different concentrations of ABA. Asterisks indicate the significance of the difference from the corresponding abi5-7 values determined by Student's t test (* 0.01 ≤ P < 0.05, ** P <0.01). At least three independent experiments were conducted and similar results were obtained. [See online article for color version of this figure.]