The regulator of G-protein signaling proteins involved in sugar and abscisic acid signaling in Arabidopsis seed germination

Abstract

The regulator of G-protein signaling (RGS) proteins, recently identified in Arabidopsis (Arabidopsis thaliana; named as AtRGS1), has a predicted seven-transmembrane structure as well as an RGS box with GTPase-accelerating activity and thus desensitizes the G-protein-mediated signaling. The roles of AtRGS1 proteins in Arabidopsis seed germination and their possible interactions with sugars and abscisic acid (ABA) were investigated in this study. Using seeds that carry a null mutation in the genes encoding RGS protein (AtRGS1) and the alpha-subunit (AtGPA1) of the G protein in Arabidopsis (named rgs1-2 and gpa1-3, respectively), our genetic evidence proved the involvement of the AtRGS1 protein in the modulation of seed germination. In contrast to wild-type Columbia-0 and gpa1-3, stratification was found not to be required and the after-ripening process had no effect on the rgs1-2 seed germination. In addition, rgs1-2 seed germination was insensitive to glucose (Glc) and sucrose. The insensitivities of rgs1-2 to Glc and sucrose were not due to a possible osmotic stress because the germination of rgs1-2 mutant seeds showed the same response as those of gpa1-3 mutants and wild type when treated with the same concentrations of mannitol and sorbitol. The gpa1-3 seed germination was hypersensitive while rgs1-2 was less sensitive to exogenous ABA. The different responses to ABA largely diminished and the inhibitory effects on seed germination by exogenous ABA and Glc were markedly alleviated when endogenous ABA biosynthesis was inhibited. Hypersensitive responses of seed germination to both Glc and ABA were also observed in the overexpressor of AtRGS1. Analysis of the active endogenous ABA levels and the expression of NCED3 and ABA2 genes showed that Glc significantly stimulated the ABA biosynthesis and increased the expression of NCED3 and ABA2 genes in germinating Columbia seeds, but not in rgs1-2 mutant seeds. These data suggest that AtRGS1 proteins are involved in the regulation of seed germination. The hyposensitivity of rgs1-2 mutant seed germination to Glc might be the result of the impairment of ABA biosynthesis during seed germination.

Figure 1.

Arabidopsis seed germination in the absence or presence of stratification conditions. A, Seeds after 1 or 2 weeks storage were sterilized and sowed on petri dishes. The germination rates were recorded after 5 d of germination. B, Germination of Col and rgs1-2 seeds after 2 weeks of storage in the absence of stratification (4°C, 48 h). C, Germination of Col and rgs1-2 seeds after 2 weeks of storage in the presence of stratification (4°C, 48 h). Values presented are the mean germination rate from three separate experiments, and the error bar indicates ±se.

Figure 2.

Effects of different kinds of sugars on seed germination. Matched seed lots of Col, rgs1-2, and gpa1-3 were sterilized, stratified, and sown on plates containing the indicated concentrations of Glc (A), Suc (B), mannitol (C), and sorbitol (D) under continuous white light at 22°C. The germination was scored after 5 d. Values presented are the mean germination rate from three separate experiments, and the error bar indicates ±se.

Figure 3.

Altered sensitivities to ABA in rgs1-2 mutant seed germination. Matched seed lots were pretreated with deionized water or 100 μm fluridone for 48 h at 4°C before being placed at 22°C under continuous white light for germination. A, Germination of wild-type Col, rgs1-2, and gpa1-3 mutant seeds in the presence of the indicated concentrations of ABA at 5 d after stratification. B, Germination of wild-type Col, rgs1-2, and gpa1-3 mutant seeds in the presence of 2 μm ABA over time (days after stratification). C, Seeds pretreated with deionized water (no fluridone treatment, No Flu) or 100 μm fluridone (Flu) were washed and sown on plates with or without 2 μm ABA. D, Seeds pretreated with deionized water or 100 μm fluridone were washed and sown on plates containing 6% Glc. Values presented are the mean germination rate from three separate experiments, and the error bar indicates ±se. Data in A and B were statistically analyzed with t test. Single asterisk indicates significance at P < 0.05 level and double asterisk indicates significance at P < 0.01 level.

Figure 4.

The seed germination responses of overexpressor of AtRGS1 to ABA and Glc. A, Matched seed lots of Col, rgs1-2, and 35S-RGS1 were sterilized, stratified, and sown on plates containing 2 μm ABA or 6% Glc. The germination was scored after 5 d. B, Seeds were sterilized, stratified, and sown on plates containing various concentrations of ABA. The germination was scored after 5 d. Values presented are the mean germination rate from three separate experiments, and the error bar indicates ±se.

Figure 5.

Effects of Glc on expression of ABA2 and NCED3 analyzed by RT-PCR. Seeds were surface disinfected and stratified for 2 d in water at 4°C, then grown on plates containing 0.5 strength of MS medium with or without 6% Glc. RT-PCR analyses were performed three times with consistent results (for details, see “Materials and Methods”).

Figure 6.

A proposed scheme of AtRGS1-mediated Glc and ABA signaling pathways in controlling seed germination. Based on the rgs1-2 mutant and 35S-RGS1 transgenic plant phenotypes, the results here suggest that high Glc inhibits seed germination through induction of ABA accumulation. AtRGS1 appears to be a positive regulator for ABA biosynthesis by stimulating the NCED3 and ABA2 expression. GPA1 negatively regulates ABA effect (Chen et al., 2004). However, although AtRGS1 interacts with AtGPA1 (Chen et al., 2003), we cannot arrive at the conclusion of whether the opposite effects of AtRGS1 and AtGPA1 on seed germination share the same mechanism.