Molecular characterization of three independent RGL2 Ds-insertion mutant lines. (A) RNA gel-blot hybridizations using DIG-labeled RGL2 and GUS probes (see Materials and Methods) and total RNA from inflorescences (28 days old). The RGL2 probe identified a ∼2-kb hybridization band in RNA from the wild-type control. This band was absent in RNA from all three mutant lines. The GUS probe identified hybridization bands in RNA from all three mutant lines, but failed to hybridize to RNA from wild-type controls. Below the GUS-hybridization panel, the gel fluorescence serves as a control for equal RNA sample loading on the gel. (B) Schematic diagram showing the Ds-insertion sites in the rgl2-1, rgl2-5, and rgl2-12 mutant alleles (thin line, noncoding region; filled thick line, RGL2-coding region, inverted triangle, Ds insertion). The Ds element insertion in all three alleles orientated 3′ to 5′ as shown. Numbers represent the site of insertion in the RGL2 ORF (in nucleotides, the A of the start ATG = 1).

RGL2 plays a relatively minor role in the regulation of stem elongation and leaf expansion. (A) Comparison of stem lengths of wild-type and of rgl2-1, rgl1-1, gai-t6, and rga-t2 mutants grown on medium containing 10⁻⁷ M PAC (30 days old; n = 15–20). (B) Comparison of rosette size (a measure of leaf expansion) of two rgl2-1 lines, gai spy-7 gar2-1 (positive control) and wild-type (negative) control grown on medium containing 10⁻⁶ M PAC (30 days old). Each genotype is represented by three typical plants arranged in a column. (C) Rosette size of the rgl2-1 ga1-3 double mutant is indistinguishable from that of the ga1-3 single mutant (30 days old). Each genotype is represented by two typical plants arranged in a column. (D) rgl2-1 does not suppress the dwarf phenotype of ga1-3. All plants shown (except for the wild-type control) are homozygous for ga1-3. rgl2-1 and gai-t6 do not appreciably suppress ga1-3 phenotype, whereas rga-t2 partially, and the gai-t6 rga-24 combination fully, suppresses the dwarfing phenotype conferred by ga1-3 (adult plants, 45 days old, are shown).

RGL2 is highly expressed in the inflorescences of bolting plants. (A) RNA gel-blot analysis of RGL2, GAI, and RGA transcripts in different plant tissues (28 days old). (B) GUS staining was detected mainly in the inflorescences and siliques of bolting plants heterozygous for rgl2-5. No GUS activity was detected at the seedling stage from day 4 to 12 after sowing (data not shown). (C) GUS staining was displayed in almost all flower organs. (D) Close up of a silique showing GUS staining at the base of the young developing seeds.

Amino-acid sequence alignment comparing the predicted RGL2, GAI, RGA, RGL1, and RGL3 proteins of Arabidopsis (for nomenclature, see Dill and Sun 2001). Gaps are introduced to maximize alignment. Regions I to VII are as defined previously (Peng et al. 1999a). Regions I and II are GA-response regions, which, when deleted from GAI, Rht (wheat), d8 (maize), SLR1 (rice), or RGA, confer reduced GA responses and dwarfism (Peng et al. 1997, 1999a; Dill et al. 2001; Ikeda et al. 2001).

RGL2 is a GA-response negative regulator of seed germination. Germination begins when quiescent dry seeds are imbibed. Following this, the radicle begins to elongate. Germination becomes visible when the radicle protrudes outside of the seed coat. In these experiments, the frequency of radicle protrusion was used as a measure of germination. (A) Seed germination of RGL2 alleles on medium containing PAC. All three alleles (rgl2-1, rgl2-5, and rgl2-12) confer strong resistance to the inhibitory effects of PAC on seed germination. The gai spy-7 gar2-1 mutant line also displays PAC-resistant seed germination (Peng et al. 1999b) and was used here as a positive control. (B) A genomic DNA fragment containing RGL2 complements the rgl2-1 phenotype in five independent transgenic lines, PJR369, 376, 378, 379, and 381. Vector control, pCambia1300 (Cambia, Australia). (C) Comparison of the effects of increasing concentrations of PAC on the seed germination of wild-type and of the rgl2-1, rgl1-1, gai-t6, and rga-t2 mutants. At the highest PAC concentration, only the rgl2-1 mutant, and the gai spy-7 gar2-1-positive control seeds can germinate. (D) Suppression of the nongerminating phenotype of ga1-3 by the rgl2-1 mutation, but not by rgl1-1, gai-t6, rga-t2, or by gai-t6 rga-t2 in combination. Seeds were germinated on SM medium. All tests yielding germination rates between zero and <2% were given a value of 2% for the convenience of drawing the histogram. The seed germination experiments were repeated multiple times and the results shown are those of a single experiment and are clearly representative of what was seen in the repeat experiment.

RGL2 expression in seeds is imbibition inducible. (A) RNA gel-blot analysis of RGL2 expression in wild-type seeds at different stages of imbibition and germination. RGL2 transcripts first become detectable at 24 h after the onset of imbibition at 4°C, then rise to a peak that is maintained for as long as the seeds are kept at 4°C. When wild-type seeds are subsequently moved to 23°C, RGL2 transcript levels begin to fall rapidly, and are barely detectable 48 h after the seeds were moved into the warmth, by which time germination had been completed. (B) RGL2 expression in wild-type seeds is also induced by imbibition at 23°C. Transcript levels rise more rapidly than they did at 4°C, with high levels being achieved by 24 h. (C) RGL2 transcripts remain at high levels in ga1-3 mutant seeds. Unlike RGL2 transcript levels in wild-type seeds, RGL2 transcript levels remained high in ga1-3 seeds following transfer from 4 to 23°C. (D) RGL2 expression is down-regulated in ga1-3 seeds grown at 23°C and treated with GA (10⁻⁴ M GA₃). (E) In situ GUS expression patterns in seeds of rgl2-5 heterozygotes during seed germination. GUS staining is observed in the radicle of seeds that have been imbibed at 4°C (seed coat was removed after staining for observation). Following transfer to 23°C, GUS staining rapidly disappears, being still faintly detectable in the radicle at 24 h and barely detectable at 48 h, by which time germination is relatively advanced. In A, C, D, and E, seeds were chilled at 4°C for 7 d and then moved to a 23°C growth chamber with a 24 hr photoperiod.

Model outlining the roles of GAI/RGA-like GA-signaling components in the plant life cycle. In this study, we have shown that RGL2 links an environmental cue (moisture) with the GA-signaling pathway during the regulation of seed germination. Sequence similarity suggests that RGL3 may also regulate seed germination in response to GA. Signaling through GAI and RGA mediates GA-promoted stem elongation and leaf expansion (Peng et al 1997, 1999a; Silverstone et al. 1998; Dill and Sun 2001; King et al. 2001; this work). Signaling through GAI, RGA, and RGL2 may mediate GA-promoted flower and pollen development (Dill and Sun 2001; S.C. Lee, H. Cheng, and J. Peng, unpubl.). The activities of GAI, RGA, and RGL1 may well be modulated in response to environmental variables at stages other than seed germination, and possible variables are shown.