Arabidopsis glutamate receptor homolog3.5 modulates cytosolic Ca2+ level to counteract effect of abscisic acid in seed germination

Abstract

Seed germination is a critical step in a plant's life cycle that allows successful propagation and is therefore strictly controlled by endogenous and environmental signals. However, the molecular mechanisms underlying germination control remain elusive. Here, we report that the Arabidopsis (Arabidopsis thaliana) glutamate receptor homolog3.5 (AtGLR3.5) is predominantly expressed in germinating seeds and increases cytosolic Ca2+ concentration that counteracts the effect of abscisic acid (ABA) to promote germination. Repression of AtGLR3.5 impairs cytosolic Ca2+ concentration elevation, significantly delays germination, and enhances ABA sensitivity in seeds, whereas overexpression of AtGLR3.5 results in earlier germination and reduced seed sensitivity to ABA. Furthermore, we show that Ca2+ suppresses the expression of ABSCISIC ACID INSENSITIVE4 (ABI4), a key transcription factor involved in ABA response in seeds, and that ABI4 plays a fundamental role in modulation of Ca2+-dependent germination. Taken together, our results provide molecular genetic evidence that AtGLR3.5-mediated Ca2+ influx stimulates seed germination by antagonizing the inhibitory effects of ABA through suppression of ABI4. These findings establish, to our knowledge, a new and pivotal role of the plant glutamate receptor homolog and Ca2+ signaling in germination control and uncover the orchestrated modulation of the AtGLR3.5-mediated Ca2+ signal and ABA signaling via ABI4 to fine-tune the crucial developmental process, germination, in Arabidopsis.

Figure 1.

Seed germination is enhanced by external calcium. A, Effect of calcium on germination. Arabidopsis wild-type seeds sown on modified MS medium containing 0 or 5 mm CaCl₂ or 0 mm CaCl₂ supplemented with 10 mm EGTA were incubated at 4°C (in the dark) for 3 d and transferred to the growth chamber (22°C) for 36 (top) or 42 h (bottom) prior to analysis. Three biological replicates were performed, and representative images are shown. B, Quantification of germination percentage at various concentrations of external Ca²⁺. Seeds were sown on modified MS medium containing indicated amounts of CaCl₂ and scored for germination 36 h after incubation under the germination conditions. C, Effect of EGTA on germination. Seeds were sown on modified MS medium containing no CaCl₂ supplemented with 5 or 10 mm EGTA and scored for germination 42 h after incubation under the germination conditions. D, Time course quantification of germination at various amounts of external Ca²⁺. Seeds were incubated on the same medium as in A at 22°C for 24 to 60 h and scored for germination. Asterisk indicates the germination percentage at 36 h after incubation of the seeds under the germination conditions. E, Effect of Ca²⁺ channel blocker LaCl₃ on germination. Seeds were incubated on MS medium supplemented with indicated concentrations of LaCl₃ and scored for germination 42 h after incubation under the germination conditions. For the germination analysis in B to E, data from three independent replicates are shown; error bars indicate se of the mean. Bar = 0.5 mm.

Figure 2.

AtGLR3.5 is required for seed germination. A, Time course analysis of AtGLR3.5 expression during germination by qRT-PCR. Wild-type (WT) seeds incubated in water were stratified and collected at the indicated time points after incubation under the germination conditions. Data shown indicate means ± se of the mean (n = 3). B, AtGLR3.5p::GUS reporter analysis in seed embryo during germination. Seeds were stratified and collected for staining at the indicated time points after incubation under the germination conditions. Images were taken after removal of the seed coat. C, Germination phenotype of wild-type, AtGLR3.5 RNAi, and AtGLR3.5 OE seeds on MS medium 40 h after incubation under the germination conditions. Three biological replicates were performed, and a representative image is shown. D, Germination analyses of wild-type, AtGLR3.5 RNAi, and AtGLR3.5 OE seeds 36 h after incubation under germination conditions. Bars = 100 µm (B) and 2 mm (D).

Figure 3.

AtGLR3.5 modulates [Ca²⁺]_cyt fluctuation. A, Germination analysis of AtGLR3.5 RNAi seeds in the absence and presence of LaCl₃. Seeds sown on MS medium supplemented with indicated concentrations of LaCl₃ were scored for germination 36 h after incubation under the germination conditions. B, Germination analysis of AtGLR3.5 RNAi seeds at various concentrations of CaCl₂. Seeds sown on modified MS medium containing indicated amounts of CaCl₂ were scored for germination 36 h after incubation under the germination conditions. C, Ca²⁺-sensitive photoprotein aequorin imaging analyses of [Ca²⁺]_cyt in 8-d-old wild-type (WT) and AtGLR3.5 RNAi seedlings. D, Aequorin luminescence-based [Ca²⁺]_cyt quantification in 8-d-old seedlings before and after 10 mm CaCl₂ treatment. Data shown are means ± se of the mean (n = 30 seedlings). E, [Ca²⁺]_cyt-dependent FRET efficiency changes in wild-type and AtGLR3.5 RNAi seedlings in response to CaCl₂ (10 mm). The primary roots of 4-d-old Arabidopsis plants expressing YC3.60 were used for the analysis. Arrows indicate the time point of CaCl₂ addition. F, Quantification of [Ca²⁺]_cyt changes in the wild type and AtGLR3.5 RNAi mutant 300 s after CaCl₂ (10 mm) application, based on FRET efficiency changes as in E. Data shown are means ± se of the mean (n = 10 seedlings). G, Effects of CaCl₂ (10 mm) and LaCl₃ (10 mm) on FRET efficiency changes in wild-type plants. Data shown are means ± se of the mean (n = 16 seedlings). In A and B, data shown indicate means ± se of the mean (n = 3).

Figure 4.

AtGLR3.5 and calcium affect ABA sensitivity in seed germination. A, Calcium alleviates the inhibitory effect of ABA on germination. B, Phenotypes of 15-d-old wild-type (WT) and AtGLR3.5 RNAi seedlings treated with or without ABA (1 µm). C, Germination analysis of wild-type and AtGLR3.5 RNAi seeds treated with or without ABA (1 µm). D, Phenotypes of 9-d-old wild-type and AtGLR3.5 OE seedlings in the absence and presence of ABA (1 µm). E, Germination analysis of wild-type and AtGLR3.5 OE seeds in the presence and absence of ABA (1 µm). For the germination analysis in A, C, and E, seeds were scored for germination 36 h after incubation under the germination conditions, and data shown are means ± se of the mean (n = 3). In B and D, three independent experiments were performed and representative images are shown.

Figure 5.

AtGLR3.5 influences the expression of key ABA regulators in seed germination. Expression analyses of genes involved in ABA signaling (A–C) and metabolic pathways (d–F) in wild-type (WT) and AtGLR3.5 RNAi seeds at indicated time points after incubation under germination conditions. Seeds were stratified at 4°C for 3 d, transferred to a growth chamber for indicated time periods, and collected for analysis. Data shown are means ± se of the mean (n = 3).

Figure 6.

Interplay between calcium and ABA converges at ABI4. A, Relative expression of ABI4 in response to CaCl₂ (1 mm) or EGTA (5 mm) in wild-type (WT) seeds. B, Histochemical staining of the GUS reporter driven by the ABI4 promoter (ABI4p::GUS) in response to CaCl₂ (1 mm) or EGTA (5 mm) treatment. Images were taken after removal of the seed coat. C, Expression analysis of ABI4 in wild-type or AtGLR3.5 RNAi lines in response to ABA (20 µm). D, Germination analysis of wild-type and Dex-inducible ABI4 OE seeds at various concentrations of CaCl₂ in the absence or presence of Dex (2 µm). E, Germination analysis of abi4-1 mutant at various concentrations of CaCl₂. In A to C, seeds were stratified in indicated solution and transferred to the growth chamber for 24 h before analysis. In D and E, seeds were scored for germination 36 h after incubation under the germination conditions. In A and C, data shown are means ± se of the mean (n = 3). Bar = 100 µm.

Figure 7.

AtGLR3.5 affects the expression of cellular Ca²⁺-sensing molecules. Expression analyses of CaM1, CaM7, and CPK3 in wild-type (WT) and AtGLR3.5 RNAi seeds at indicated time points after incubation under the germination conditions. Seeds were stratified and collected at 0 or 24 h after transfer to a growth chamber. Data shown are means ± se of the mean (n = 3).

Figure 8.

A working model for seed germination regulated by the AtGLR3.5-cytosolic Ca²⁺-ABI4 network and Ca²⁺-independent ABA signaling pathway. Arrows and t bars indicate activation and repression, respectively. Black lines represent direct effects, and dotted lines represent effects that have not yet been known to occur directly. PP2Cs, Type 2C protein phosphatases; SnRK2s, SNF1-related protein kinases.