Lifting della repression of Arabidopsis seed germination by nonproteolytic gibberellin signaling

Abstract

DELLA repression of Arabidopsis (Arabidopsis thaliana) seed germination can be lifted either through DELLA proteolysis by the ubiquitin-proteasome pathway or through proteolysis-independent gibberellin (GA) hormone signaling. GA binding to the GIBBERELLIN-INSENSITIVE DWARF1 (GID1) GA receptors stimulates GID1-GA-DELLA complex formation, which in turn triggers DELLA protein ubiquitination and proteolysis via the SCF(SLY1) E3 ubiquitin ligase and 26S proteasome. Although DELLA cannot be destroyed in the sleepy1-2 (sly1-2) F-box mutant, long dry after-ripening and GID1 overexpression can relieve the strong sly1-2 seed dormancy phenotype. It appears that sly1-2 seed dormancy results from abscisic acid (ABA) signaling downstream of DELLA, since dormant sly1-2 seeds accumulate high levels of ABA hormone and loss of ABA sensitivity rescues sly1-2 seed germination. DELLA positively regulates the expression of XERICO, an inducer of ABA biosynthesis. GID1b overexpression rescues sly1-2 germination through proteolysis-independent DELLA down-regulation associated with increased expression of GA-inducible genes and decreased ABA accumulation, apparently as a result of decreased XERICO messenger RNA levels. Higher levels of GID1 overexpression are associated with more efficient sly1 germination and increased GID1-GA-DELLA complex formation, suggesting that GID1 down-regulates DELLA through protein binding. After-ripening results in increased GA accumulation and GID1a-dependent GA signaling, suggesting that after-ripening triggers GA-stimulated GID1-GA-DELLA protein complex formation, which in turn blocks DELLA transcriptional activation of the XERICO inhibitor of seed germination.

Figure 1.

Rescue of sly1 germination by GID1 overexpression is not associated with decreased RGL2 protein levels. A, Germination of 0.5-month-old wild-type (WT), sly1-2, sly1-2 abi1-1, sly1-2 GID1a-OE, sly1-2 GID1b-OE, and sly1-2 GID1c-OE seeds imbibed on 0.5× MS-moistened filter paper at 22°C. Percentage germination was scored daily. B, Accumulation of RGL2 protein during the time course of seed imbibition. Seeds were imbibed on filter paper at 22°C and harvested daily over 4 d for protein-blot analysis of RGL2 accumulation. Corresponding germination rates are shown.

Figure 2.

Overexpression of GID1b in the gai1-1 background does not reduce the ABA sensitivity of gai-1. An ABA dose-response curve was performed using wild-type Ler, Ler GID1b-OE, gai-1, and gai-1 GID1b-OE seeds. Seeds were incubated on MS-agar containing the indicated ABA concentrations at 4°C for 3 d followed by 96 h at 22°C. Error bars indicate se for three replicates (n = 30). [See online article for color version of this figure.]

Figure 3.

Hormone measurement of imbibing seeds. Hormone measurements were conducted using seeds after-ripened for 2 weeks except for sly1-2(AR), which was after-ripened for 28 months. All seeds were imbibed on MS-agar at 4°C for 60 h followed by 22°C for 12 h. ABA and GA₄ levels are averages of six to 10 samples. Error bars show se. DW, Dry weight.

Figure 4.

GID1a is required for after-ripening and controls RGL2 protein accumulation in the sly1 mutant background. A, GID1a is needed for after-ripening of sly1-2. Germination is shown for seeds after-ripened for 1 and 20 months incubated on MS-agar plates for 3 d at 4°C followed by 7 d at 22°C. B and C, Seeds after-ripened for 20 months were incubated on MS-moistened filter paper for 3 d at 4°C followed by 48 h at 22°C and then used to examine RGL2 protein (B) and RGL2 mRNA (C) accumulation by protein-blot and RT-PCR analyses, respectively. Total protein (60 µg) was loaded, and Ponceau staining was used as a loading control.

Figure 5.

Additive effects of gid1 and sly1-2 mutations on plant height, fertility, and associated DELLA protein levels. A, Sixty-five-day-old wild-type (WT) and homozygous mutant plants with and without 100 µm GA₄ treatment (every 3 d). B, Vegetative DELLA RGA protein accumulation appeared to depend on the gid1 genotype in protein-blot analysis of 40 µg of total protein extracted from 28-d-old whole plants. Two exposures of the same blot are shown with Ponceau staining as a loading control. Normalized RGA mRNA accumulation based on the mean values from three independent qRT-PCR experiments is plotted on the histogram. Error bars show se. C, Flower morphology at anthesis. D, DELLA RGA and RGL2 protein accumulation in these flowers depended on the presence of GID1 genes. [See online article for color version of this figure.]

Figure 6.

Higher germination potential correlates with higher accumulation of FLAG-GID1b and formation of the GID1b-DELLA complex. A, Germination rates of sly1-10 (black squares) and three independent sly1-10 FLAG-GID1b seed lines (A, B, and C) sown on MS-moistened filter paper and incubated 5 d at 22°C. Under these conditions, sly1-10 does not germinate. B, A co-IP experiment performed using protein extracted from the indicated sly1-10 FLAG-GID1b seeds imbibed for 48 h at 22°C. Total protein was incubated with anti-FLAG matrix agarose. Immunoprecipitated FLAG-GID1b was detected by immunoblot with anti-FLAG, and DELLA RGA in complex with GID1b was detected with anti-RGA. Total protein (60 mg) was loaded (input). A Ponceau-stained membrane provided a loading control.

Figure 7.

HA-GID1b protein levels and HA-GID1b-RGA complex formation correlate with the suppression of sly1-2 dwarfism. A, sly1-2 plants expressing higher (line 386) or lower (line 388) levels of the HA-GID1b fusion protein during stem elongation. B, HA-GID1b was immunoprecipitated from protein extracted from 12-d-old sly1-2 plants expressing either high (line 386) or low (line 388) levels of HA-GID1b protein. Coimmunoprecipitated RGA was detected by protein-blot analysis. Protein extract was incubated with HA matrix agarose in the presence of 0.1% ethanol (mock), 1 µm GA₃, and 100 µm GA₃ and loaded on an SDS-PAGE gel. Protein-blot analysis was performed using anti-RGA and anti-HA. Protein extract (40 µg) was loaded (input). [See online article for color version of this figure.]

Figure 8.

Effect of GID1 overexpression on gene expression in seeds. The expression of the ABA biosynthesis gene, XERICO, GA20ox2, and XTH31 was examined by qRT-PCR. For each experiment, total was RNA extracted from seeds imbibed on MS-agar for 60 h at 4°C followed by 24 h at 22°C and then incubated for a further 12 h after the addition of a mock treatment or 10 mm GA₃. All seeds were dry after-ripened for 2 weeks, including dormant sly1-2 (D), except for sly1-2 after-ripened (AR) for 28 months For each condition, there were three biological replicates, and each replicate was subsampled three times. Error bars represent sd. Statistical significance was determined with an ANOVA using Tukey’s adjustment. P values are given in Supplemental Figure S8.

Figure 9.

Model for the rescue of sly1 seed germination by GID1-GA-DELLA complex formation. A, After-ripening of sly1-2 stimulates seed germination by increasing endogenous GA levels that promote GID1-GA-DELLA complex formation. GID1 overexpression also increases GID1-GA-DELLA formation, thereby inactivating DELLA repression of GA-responsive (GA-R) gene expression and responses. B, GID1 can prevent DELLA induction of XERICO transcription either via SLY1-directed DELLA degradation or directly through GID1-GA-DELLA complex formation. Reduced XERICO expression results in decreased ABA accumulation in after-ripened sly1 and sly1 GID1 overexpression lines.