Positive and negative regulation of seed germination by the Arabidopsis GA hormone receptors, GID1a, b, and c

Abstract

Epistasis analysis of gid1 single and double mutants revealed that GID1c is a key positive regulator of seed germination, whereas the GID1b receptor can negatively regulate germination in dormant seeds and in the dark. The GID1 GA receptors were expected to positively regulate germination because the plant hormone gibberellin (GA) is required for seed germination in Arabidopsis thaliana. The three GA hormone receptors, GID1a, GID1b, and GID1c, positively regulate GA responses via GA/GID1-stimulated destruction of DELLA (Asp-Glu-Leu-Leu-Ala) repressors of GA responses. The fact that the gid1abc triple mutant but not gid1 double mutants fail to germinate indicates that all three GA receptors can positively regulate non-dormant seed germination in the light. It was known that the gid1abc triple mutant fails to lose dormancy through the dormancy breaking treatments of cold stratification (moist chilling of seeds) and dry after-ripening (a period of dry storage). Previous work suggested that there may be some specialization of GID1 gene function during germination because GID1b mRNA expression was more highly induced by after-ripening, whereas GID1a and GID1c mRNA levels were more highly induced by cold stratification. In light-germinated dormant seeds, the gid1b mutation can partly rescue the germination efficiency of gid1a but not of gid1c seeds. Thus, GID1b can function as an upstream negative regulator GID1c, a positive regulator of dormant seed germination. Further experiments showed that GID1b can negatively regulate dark germination. Wild-type Arabidopsis seeds do not germinate well in the dark. The gid1b and gid1ab double mutants germinated much more efficiently than wild type, gid1c, or gid1ac mutants in the dark. The observation that the gid1ab double mutant also shows increased dark germination suggests that GID1b, and to some extent GID1a, can act as upstream negative regulators of GID1c. Since the gid1abc triple mutant failed to germinate in the dark, it appears that GID1c is a key downstream positive regulator of dark germination. This genetic analysis indicates that the three GID1 receptors have partially specialized functions in GA signaling.

Keywords: GID1; epistasis; germination; gibberellin; seed dormancy.

Figure 1

After‐ripening time course of gid1 germination. An after‐ripening time course compared gid1 single (a,c,e,g) and double (b,d,f,h) mutant seed germination. Percentage germination was scored daily over 7 days imbibition on 0.5× MS‐agar at 22°C, over four dry after‐ripening time points (1 days, 1 week, 2 weeks and 4 weeks). Mean germination is shown with error = SD, n = 3

Figure 2

Epistasis analysis of dormant gid1 seed germination in the light. Compared are: (a) gid1a and gid1c to gid1ac, (b) gid1a and gid1b to gid1ab, and (c) gid1a and gid1c to gid1ac. Seeds were germinated at 2 days AR (as in Figure 1a,b). Error = SD, n = 3

Figure 3

Epistasis analysis of gid1 mutant GA dose–response. Compared are light germination of: (a) gid1a and gid1c to gid1ac, (b) gid1a and gid1b to gid1ab, and (c) gid1a and gid1c to gid1ac. Seeds were incubated in 10 μM PAC for 48 hr at 4°C in dark, washed, and then incubated in the light for 5 days at 22°C on MS‐agar containing 0, 0.01, 0.1 and 0.5 μM GA ₃. Germination was scored daily. The x‐axis is log₁₀ scale. Error = SD (n = 4) is shown

Figure 4

Epistasis analysis of the gid1 dark germination phenotype. Compared in order are dark germination of: (a) gid1a and gid1c to gid1ac, (b) gid1a and gid1b to gid1ab, and (c) gid1a and gid1c to gid1ac. Seeds were plated at 4 weeks AR. The x‐axis labels “Bright”, “Dim”, “Dimmer” indicate the fluorescent light intensity or quality during plating prior to incubation for 14 days in the dark at 22°C. Shown is the mean ±SE (n = 3)

Figure 5

Light and dark germination of the gid1abc segregating line. Seeds of Col WT, gid1ab, gid1ac, gid1bc, and of the gid1 b/b c/c a/+ line segregating for gid1a, were plated at 4 weeks‐AR under fluorescent lights (17 μmol m⁻² s⁻¹), then germinated either in the dark or under lights at 22°C. Error bars indicate SD (n = 3)

Figure 6

Effect of gid1 genotype on seed size. Comparison of: (a) Col WT and (b) gid1ac seed morphology showing that gid1ac seeds appear shriveled. Seed size was evaluated in two independent sets of seeds, (c) Set 1 and (d) Set 2. Student's t test was used to examine the statistical significance of seed size differences from WT, *p < 0.05, **p < 0.001. The bar indicates 0.5 mm

Figure 7

Model for GID1 regulation of seed germination. (a) In the light, GID1c and GID1a act in parallel as positive regulators of seed germination. GID1b acts as a positive regulator of seed germination in nondormant (ND) seeds, but as a negative regulator of GID1c and seed germination in dormant (D) seeds. (b) In the dark, GID1c is the stronger and GID1a the weaker positive regulator of germination. Both GID1b and GID1a can negatively regulate germination via negative regulation of GID1c