Seed germination of GA-insensitive sleepy1 mutants does not require RGL2 protein disappearance in Arabidopsis

Abstract

We explore the roles of gibberellin (GA) signaling genes SLEEPY1 (SLY1) and RGA-LIKE2 (RGL2) in regulation of seed germination in Arabidopsis thaliana, a plant in which the hormone GA is required for seed germination. Seed germination failure in the GA biosynthesis mutant ga1-3 is rescued by GA and by mutations in the DELLA gene RGL2, suggesting that RGL2 represses seed germination. RGL2 protein disappears before wild-type seed germination, consistent with the model that GA stimulates germination by causing the SCF(SLY1) E3 ubiquitin ligase complex to trigger ubiquitination and destruction of RGL2. Unlike ga1-3, the GA-insensitive sly1 mutants show variable seed dormancy. Seed lots with high seed dormancy after-ripened slowly, with stronger alleles requiring more time. We expected that if RGL2 negatively controls seed germination, sly1 mutant seeds that germinate well should accumulate lower RGL2 levels than those failing to germinate. Surprisingly, RGL2 accumulated at high levels even in after-ripened sly1 mutant seeds with 100% germination, suggesting that RGL2 disappearance is not a prerequisite for seed germination in the sly1 background. Without GA, several GA-induced genes show increased accumulation in sly1 seeds compared with ga1-3. It is possible that the RGL2 repressor of seed germination is inactivated by after-ripening of sly1 mutant seeds.

Figure 1.

Structure of the SLY1 Gene and the SLY1 mRNA Expression Detected by RT-PCR in Three Mutant Alleles. (A) The mutation sites are shown in three alleles. The sly1-10 mutant has an 8-kb insertion at +429 nucleotides from translational start. The sly1-2 mutant has a 2-bp deletion at the +333-nucleotide position. The T-DNA is inserted at the +219-nucleotide position in sly1-t2. (B) The top panel shows the SLY1 expression in three mutant alleles and the wild type using the sly1-2f and sly1-2r primers (pair A) that flank the insertion in sly1-t2 giving no product. The middle panel shows the expression using the 2-63f and sly1-r primers (pair B) that flank the sly1-10 rearrangement giving no product. The bottom panel shows the ACTIN2 control.

Figure 2.

Germination Rates of sly1 Mutant Seeds Varied but Were Dependent on Allele and Age of Seeds. Seeds plated on MS-agar were incubated 3 d at 4°C followed by 7 d at 22°C. Percentage of germination was calculated based on cotyledon emergence for independent seed lots of: sly1-10 (A), sly1-2 (B), and sly1-t2 (C). Duration of dry storage or age of seed is indicated above the bars (months). N.G., none of the seeds germinated. Error bars show se (n = 3). Seed lot numbers (#) are indicated on the x axis.

Figure 3.

Determination of Seed Viability by Tetrazolium Staining and Germination Rescue by Cutting the Seed Coat. (A) Seeds of the wild type (0.5, 26, and 63 months old), after-ripened sly1-2 (26 months old, seed lot #21), and dormant sly1-2 (0.5 and 63 months old, seed lots #35 and #7, respectively) were stained with 0.5% tetrazolium for 10 h. Fully stained seeds were counted as viable. (B) To examine seed viability, dormant sly1-2 (seed lot #35, 0.5 months old) and sly1-t2 (seed lot #12, 0.5 months old) seed coats were nicked with forceps to stimulate seed germination. ga1-3 (0.5 months old) seeds were used as a positive control for seed germination rescue. Age of each seed stock is shown below the graph (months). Germination rates (G) are shown below the figure (%). Error bars show se (n = 30).

Figure 4.

sly Mutant Seeds Germinate More Slowly Than the Wild Type, and Germination Is Rescued by a Mutation in RGL2. Seeds were plated on MS-agar and the number of germinated seeds scored daily after transfer to 22°C. The wild type (28 months old), sly1-10 (27 months old, #3), after-ripened (A) sly1-2 (26 months old, #21), dormant (D) sly1-2 (0.5 months old, #35), 1-month-old wild type (Col), sly1-t2 rgl2-13 (1 month old), and sly1-t2 (1 month old, crossed to wild-type Col) were used. Error bars show se (n = 3). (A) Percentage of radicle emergence based on radicle protrusion from seed coat. (B) Percentage of cotyledon emergence based on appearance of green cotyledons outside the seed coat. (C) Rescue of sly1-t2 seed germination by the rgl2-13 mutation based on percentage of radicle emergence.

Figure 5.

Accumulation of RGL2 Protein during a Time Course of Seed Imbibition. RGL2 protein was detected by protein gel blot analysis. (A) Ler seeds were incubated for 6 d at 4°C and then time points were at indicated intervals after transfer to 22°C (blot exposure >30 min). The correlation between RGL2 protein accumulation and radicle emergence (%) was determined. RGL2 protein disappeared within 12 h, before radicle emergence. (B) Time-course analysis of RGL2 protein in wild-type and mutant backgrounds. Protein gel blot analysis (exposure time 5 to 10 min) of protein isolated from seeds imbibed at 4°C for 1, 3, 5, and 6 d and from seeds first imbibed for 3 d at 4°C then transferred to 22°C for indicated number of days. Protein from dry seeds was also examined. Seed samples used were Ler (28 months old), ga1-3 (25 months old), sly1-10 (27 months old, #3), after-ripened (A) sly1-2 (26 months old, #21), dormant (D) sly1-2 (0.5 months old, #35), Ws (0.5 months old), and sly1-t2 (0.5 months old, #12). Asterisks indicate nonspecific bands.

Figure 6.

RGL2 mRNA Accumulation during a Time Course of Seed Imbibition for Wild-Type, ga1-3, and sly1 Mutant Seeds. RT-PCR analysis of RGL2 mRNA expression in seeds imbibing using the same time courses in Figure 6 of stratified (4°C for 1, 3, 5, and 6 d) seeds and seeds imbibing at 22°C after 3 d of stratification at 4°C. Seed samples used were identical to Figure 6 and include Ler (28 months old), ga1-3 (25 months old), sly1-10 (27 months old, #3), after-ripened (A) sly1-2 (26 months old, #21), dormant (D) sly1-2 (0.5 months old, #35), Ws (0.5 months old), and sly1-t2 (0.5 months old, #12).

Figure 7.

GA Partially Rescued sly1 Germination without Reducing RGL2 Protein Levels. (A) Percentage of germination on indicated GA₃ concentration in ga1-3 biosynthesis mutant (25 months old), sly1-10 (5 months old, #13), after-ripened (A) sly1-2 (26 months old, #21), dormant (D) sly1-2 (0.5 months old, #35), and sly1-t2 (0.5 months old, #12; 18 months old, #2). Germination was calculated based on cotyledon appearance. Age of the stock is shown below the line (months). Error bars show se (n = 30 to 60). (B) Percentage of radicle emergence determined during time-course experiment examining RGL2 protein accumulation in the presence of 10 μM GA₃ using the same seed lots used in (A) except for 0.5-month-old sly1-t2. Radicle emergence was determined at 60, 72, and 84 h at 22°C following 3 d of 4°C. (C) RGL2 protein expression in the absence and presence of 10 μM GA₃. Protein was extracted from imbibing seeds at indicated time points at 22°C following 3 d at 4°C. (D) RGL2 mRNA accumulation in the absence and presence of GA₃ during the same time-course experiment in (C). Left panel, RGL2 mRNA; right panel, ACTIN2 mRNA.

Figure 8.

Effect of sly1 Mutations on Accumulation of GA-Regulated Transcripts in Imbibing Seeds. mRNA accumulation of seven GA-induced genes was determined by RT-PCR. mRNA was isolated from ungerminated imbibing seeds.

Figure 9.

Model for Control of Seed Germination by SLY1 and RGL2. (A) There is an alternative pathway controlling seed germination that is stimulated by GA. This pathway may bypass the sly1 mutation either by inhibiting RGL2 activity without causing decreased RGL2 protein levels possibly by posttranslational modification or by activating seed germination independently of SLY1/RGL2. (B) In the ga1-3 mutant (no GA), RGL2 does not interact with the GA receptor GID1. In this case, RGL2 functions purely as a negative regulator. Addition of GA rescues ga1-3 seed germination by causing RGL2 to be destroyed following interaction with GA-GID1 and SCF^SLY1. In the sly1 mutant (GA present), RGL2 cannot be destroyed because SLY1 is defective. In this case, RGL2 acts as a repressor of GA responses when not bound to GID1, and GA-GID1-RGL2 may be inactive as a repressor or act as a positive regulator of seed germination.