Plant Physiol. 2007 February; 143(2): 745–758

Figure 3. Effect of ABA on primary root elongation. A, Wild type, aba2, and overexpression lines 4-3, 4-4, and 5-1 were grown on 1% Suc agar plates for 5 d, then transferred to the same medium with or without ABA treatment and grown vertically for another 6 d. Arrowheads indicate the position of primary root tips. B, Primary root length of wild type, aba2, and overexpression line seedlings grown on 1% Suc for 5 d, then transferred to the same medium with or without ABA and grown vertically for another 6 d. The values are the means ± sd of 10 to 12 seedlings of each genotype. The experiment was repeated twice with consistent results. C, ABA content of wild type, aba2, and overexpression lines. Seedlings were grown on 1% Suc agar plates for 11 d and then subjected to ABA immunoassay. The values are the means ± sd of three independent experiments using different seed batches, each performed in duplicate. WT, Wild type. D, Primary root length of wild type (Ws) and gin1-1. Seedlings had the same growth conditions as in B, except with vertical growth for 7 d. The values are the means ± sd of 12 seedlings of each genotype. The experiment was repeated twice with consistent results. The seeds in A to D were subjected to cold pretreatment at 4°C for 3 d before planting on agar plates. [See online article for color version of this figure.] Plant Physiol. 2007 February; 143(2): 745–758.

Plant Physiol. 2007 February; 143(2): 745–758

Figure 4. Effect of osmoticum on seedling growth. A, Seeds with cold pretreatment were grown on 1% Suc agar plates for 5 d, then transferred to the same fresh medium with 40% PEG-infused treatment for another 4 weeks. WT, Wild type. B, Quantification of bleached cotyledons derived from A. The values are the means ± sd of three independent experiments, each genotype with 100 seedlings per experiment. [See online article for color version of this figure.] Plant Physiol. 2007 February; 143(2): 745–758.

Plant Physiol. 2007 February; 143(2): 745–758

Figure 5. Effect of salinity on germination and plant growth. A and B, Germination of wild type, aba2, and overexpression lines on 1% Suc agar plates supplemented with 125 (A) or 250 mm (B) NaCl. Germination rates were the means ± sd of three independent experiments using different seed batches, with consistent results, each with 150 to 200 seeds. WT, Wild type. C, Phenotypic comparison of wild type, aba2, and overexpression lines grown on 1% Suc agar plates plus 250 mm NaCl with or without 1 μm fluridone for 14 d. D and E, ABA levels of wild type, aba2, and transgenic lines. Seedlings were grown on 1% Suc agar plates for 5 d, then transferred to the same fresh medium supplemented with 125 mm NaCl for another 7 d (D) or 250 mm NaCl for another 2 d (E). The values are the means ± sd of three independent experiments using different seed batches, each performed in duplicate. F, Phenotypic comparison of wild type, aba2, and transgenic line 4-4. Seedlings were grown on 1% Suc plates supplemented with 200 mm NaCl for 28 d. G, Quantification of bleached plants of wild type, aba2, and transgenic line 4-4 in F. The values are the means ± sd of three independent experiments using different seed batches and with consistent results, each with 200 to 250 seeds. H, Phenotypic comparison of wild type, aba2, and overexpression line 4-4 in response to salinity. A total of 75 plants from each genotype, grown in soil for 21 d, were tested by watering with four increasing concentrations of NaCl (50, 100, 150, and 200 mm), each for 4 d for a total of 16 d. Seeds tested in this study were cold pretreated before planting on agar plates or in soil. Plant Physiol. 2007 February; 143(2): 745–758.

Plant Physiol. 2007 February; 143(2): 745–758

Figure 6. ABA2 promoter activity in response to diverse stress. A, Determination of GUS activity under drought condition. Aerial parts of tissues were removed from 21-d-old plants grown in soil and immediately placed on an electronic dry box with 40% relative humidity for 1.5 and 3 h. B, Determination of GUS activity under diverse stress. Plants were grown in soil for 21 d and subsequently treated with cold (4°C; 3 d), drought (4 d), flooding (1 d), and salt stresses (125 and 250 mm NaCl, each for 1 d), respectively. Control samples were harvested at 21 dap, at the beginning of stress treatments. C, Determination of GUS activity under Suc and NaCl treatments. Seedlings were grown on 1% Suc agar plates with or without 125 or 250 mm NaCl for 14 d. Results in A, B, and C are the means ± sd of three independent experiments, each experiment with a duplicate. Transgenic plants harboring the ABA2GUS transgene were under wild-type (lines 1-11 and 4-12) and aba2 mutant (lines 3-6 and 3-12) backgrounds. Plant Physiol. 2007 February; 143(2): 745–758.

Plant Physiol. 2007 February; 143(2): 745–758

Figure 7. Expression of ABA- and sugar-related genes in response to drought. Plants were grown in soil for 3 weeks, and then subjected to withholding water for 4 d (drought). Control samples were harvested at the same time as drought-treated samples. Three independent experiments were performed, except for control with two independent experiments, each with a duplicate and with consistent results. WT, Wild type; HXK, hexose kinase; Susy, Suc synthase; UBQ, ubiquitin. Plant Physiol. 2007 February; 143(2): 745–758.

Plant Physiol. 2007 February; 143(2): 745–758

Germination and Root Elongation Tests For germination tests, seeds harvested from the same batches were cold pretreated and then grown on modified Murashige and Skoog medium supplemented with Glc, Suc, NaCl, ABA, glufosinate ammonium, or fluridone at various concentrations listed in the “Results.” The medium was autoclaved and cooled to 50°C to 60°C prior to the addition of filter-sterilized ABA, glufosinate ammonium, or fluridone. For root elongation experiments, cold-pretreated seeds from different genotypes were first grown on agar plates with 1% Suc for 4 or 5 d, then uniform seedlings of similar size and primary root length were transferred to appropriate fresh medium and grown vertically for another 6 or 7 d.

Plant Physiol. 2007 February; 143(2): 745–758

Preparation of Low-Water-Potential Medium Plates One-half-strength modified Murashige and Skoog medium (pH 5.7) with 0.7% Phyto agar (Duchefa Biochemie B.V.) was autoclaved. The sterilized medium was cooled to 50°C to 60°C and then aliquoted into petri dishes (100- × 20-mm depth), 20 mL each, for solidification. PEG-infused plates were made by dissolving PEG-8000 (Sigma) powder into one-half-strength Murashige and Skoog solution (pH 5.7) with the above-mentioned components, except phyto agar, and then filter sterilized; this PEG solution was then overlaid on agar-solidified medium at a ratio of 3:2 (v/v) and equilibrated overnight (≥12 h). The excess PEG solution was then removed. The procedure essentially followed the protocol of Verslues and Bray (2004).

Plant Physiol. 2007 February; 143(2): 745–758

NaCl and Dehydration TreatmentsWild type, aba2, and ABA2 overexpression seeds with cold pretreatment were grown on agar plates with various NaCl concentrations for 18 or 28 d. The ratios of bleached to total plants were counted to define tolerance to salinity. Seeds with cold pretreatment were also grown in soil for 3 weeks; then plants were subjected to watering with solutions containing four gradually increased concentrations of NaCl (50, 100, 150, and 200 mm), each for 4 d for a total of 16 d (Shi et al., 2003). For dehydration, seeds with cold pretreatment were grown in soil for 21 d and then plants were excised. The aerial parts of tissues were placed on plastic weighing boats and kept in an electronic dry box (model-DX-76; Taiwan Dry Tech Corp.) with approximately 40% relative humidity. The fresh weights of tissues were measured at 0-, 1.5-, and 3-h time points.

Plant Physiol. 2007 February; 143(2): 745–758

GUS Activity AssayCold-pretreated transgenic seeds harboring the ABA2GUS transgene were grown on agar plates or in soil for different time periods, depending on experiments, and then subjected to various stresses (cold, drought, or salinity). The treated plants were harvested and ground with extraction buffer (50 mm NaHPO4 [pH 7.0], 10 mm β-mercaptoethanol, 10 mm Na2EDTA, 0.1% sodium lauryl sarcosine, 0.1% Triton X-100). After centrifugation at 13K rpm for 10 min at 4°C, the supernatants were removed for further GUS assay, essentially according to the protocol of Jefferson et al. (1987), using a DyNA Quant 200 fluorometer (Amersham-Pharmacia Biotech).

Plant Physiol. 2007 February; 143(2): 745–758

RT-PCRTotal RNA was extracted from wild type, aba2, and ABA2 overexpression lines using TRIzol reagent (Invitrogen). Six micrograms of total RNA of each genotype with 1 μg oligo(dT) primer (Invitrogen) were heated at 70°C for 5 min and then chilled on ice immediately. RNA was then subjected to RT with reverse-transcriptase Avian myeloblastosis virus (Roche) at 42°C for 1 h according to the manufacturer's protocol. Synthesized cDNA was used as a template for PCR.