Rice GERMIN-LIKE PROTEIN 2-1 Functions in Seed Dormancy under the Control of Abscisic Acid and Gibberellic Acid Signaling Pathways

Abstract

Seed dormancy is a natural phenomenon in plants. It ensures that seeds complete the grain-filling stage before germination and prevents germination in unsuitable ecological conditions. In this study, we determined the previously unknown function of the rice (Oryza sativa) gene GERMIN-LIKE PROTEIN 2-1 (OsGLP2-1) in seed dormancy. Using artificial microRNA and CRISPR/CAS9 approaches, suppression of OsGLP2-1 expression in rice resulted in the release of dormancy in immature seeds. Conversely, overexpression of OsGLP2-1 driven by the OsGLP2-1 native promoter led to greater seed dormancy. Seed scutellum-specific expression of OsGLP2-1 was increased by exogenous abscisic acid, but decreased with gibberellic acid treatment. We provide evidence that OsGLP2-1 is antagonistically controlled at the transcriptional level by ABA INSENSITIVE5 and GAMYB transcription factors. We conclude that OsGLP2-1 acts as a buffer, maintaining appropriate equilibrium for the regulation of primary dormancy during seed development in rice.

Figure 1.

Expression pattern of P_GLP2-1:GUS in transgenic rice plants at different growth stages. A, Specific expression in minor veins of the leaf in the vegetative phase. Leaves from 14-d-old seedlings: left, negative control (empty vector transformant [EV]) lacking signal; right, P_GLP2-1:GUS leaf with strong blue GUS signal in minor veins. Leaves from 64-d-old plant: left, negative control; center, P_GLP2-1:GUS leaf showing staining in the minor veins; right, CaMV 35S promoter (P_35S) driving constitutive expression of GUS in the leaf. B, GUS staining in organs from mature plant, including leaf, spikelet, stem, internode, leaf sheath, and seed. Among these organs of P_GLP2-1:GUS plants, light signal was observed in the leaf sheath and strong blue signal displayed specifically in the scutellum. C, GUS staining of developing seeds of P_GLP2-1:GUS transformant. Seeds were sampled at different growth stages (17 and 35 DAF). Scutellum-specific expression pattern was exhibited by all developing seeds. EV seed was used as negative control, and P_35S showing constitutive GUS expression was used as positive control.

Figure 2.

Dormancy-breaking phenotype exhibited on the rice CRISPR-osglp2-1-seeds. A, Schematic diagram of CRISPR/CAS9-mediated targeted mutagenesis in OsGLP2-1. The ORF consists of two exons indicated as black boxes. The mutated sites (323 and 513) in 20-bp single guide RNA targeting sequences and protospacer adjacent motif are shown in bold italic letter and in red letter, respectively. B, and C, Comparisons of wild type and CRISPR-osglp2-1 developing seeds dormancy state. B, Dormancy-breaking phenotype exhibited on the CRISPR-osglp2-1-seeds (7 d after sowing). C, Compared with the low germination rate of wild-type seeds at 3, 5, 7, and 9 d after sowing, two lines of T-DNA–free homozygous CRISPR-osglp2-1-seeds, cr-glp-323 and cr-glp-513, displayed significantly higher germination percent, respectively. The surface sterilized 20 DAF-dehulling seeds were sown in MS medium, 15 seeds for each sample, and 6 replicates were performed. Error bars = SE. Asterisks indicate significant difference at *P = 0.05 and **P = 0.01 in SPSS one-way ANOVA analysis with post hoc test.

Figure 3.

Response of the expression of OsGLP2-1 to ABA and GA. A and B, Expression of GUS driven by P_GLP2-1 was stimulated by exogenous ABA and suppressed by GA. C and D, Altered expression of OsGLP2-1 in response to ABA and GA in wild-type seeds. The induction/suppression of OsGLP2-1 expression was detected after ABA/GA treated within 3 h, respectively. The P_GLP2-1:GUS or wild-type rice embryo including the scutellum was freshly cut from dehusked 30 DAF seeds, and immediately treated with exogenous 50 μm ABA (A and C), 1 or 10 μm GA (B and D) for different time points in a growth chamber (25°C, 12-h light/12-h dark). The samples were ground in liquid nitrogen for further monitoring of GUS or OsGLP2-1 expression by RT-qPCR. Collection of 15 embryos for each sample and three replicates were performed. Error bars = SE (n = 3). Asterisks indicate significant difference at *P = 0.05 and **P = 0.01 in SPSS independent-sample t test (A, C, and D) and one-way ANOVA analysis with post hoc test (B), respectively.

Figure 4.

Effect of ABI5 and GAMYB on activity of GUS driven by P_GLP2-1. Effector construct: ABI5 (P_35S:ABI5) and GAMYB (P_35S:GAMYB); Reporter construct (P_GLP2-1:GUS); Internal control (P_NOS:RiLuc). The means of GUS/RiLuc ratios were obtained from three replicates of five pooled N. benthamiana leaves. Error bars = SE (n = 3). Mock, leaf sample infiltrated with MMA solution. RiLUC was used as an internal control in the agroinfiltration system. Asterisks indicates sgnificant difference at **P = 0.01 in SPSS independent-sample t test. ABI5: LOC_Os01g64000; GAMYB: LOC_Os01g59660.

Figure 5.

Binding activity of ABI5 and GAMYB to P_GLP2-1 detected by yeast one-hybrid assay. A, Schematic diagram of the potential cis-elements in P_GLP2-1 and promoter fragments. F1: −932 to −637 (295 bp); F2: −507 to −237 (270 bp); F3: −932 to −237 (696 bp); F4: −1419 to −1 (1419 bp); P_GLP2-1: −2086 to −1 (2086 bp). B, Compared with the negative controls (empty vector GAD with pLacZi; F1::LacZ, or F2::LacZ, respectively, and GAD-ABI5 with pLacZi), ABI5 directly bound to the P_GLP2-1 promoter F1 fragment and activated expression of the LacZ reporter gene. GAMYB directly bound to the F2 fragment and induced LacZ expression; moreover, the signal increased progressively with 2 or 3 GARE tandemly repeated in 2×F2::LacZ and 3×F2::LacZ, respectively. The combinations of GAD with pLacZi, F2::LacZ, 2×F2::LacZ, or 3×F2::LacZ; and GAD-GAMYB with pLacZi were used as negative controls. These binding interactions of P_GLP2-1 with GAMYB and ABI5 were confirmed in at least three repeated experiments.

Figure 6.

Physical interaction of ABI5 and GAMYB to the ABRE and GARE in P_GLP2-1. A to C, EMSA results displayed that the transcription factors (ABI5 and GAMYB) could directly recognize and bind to P_GLP2-1 fragments containing the ABRE or GARE, respectively. A, The probes used in EMSA were designed based on the predicted binding sequence of ABRE (red letters) and GARE (blue letters) in the F1 and F2 fragments, respectively, together with their flanking nucleotides (underlined), two overlapped binding sites were synthesized. B and C, Binding activity of ABI5 and GAMYB with their corresponding probes. The fusion proteins MBP-ABI5 and Trx-His-GAMYB specifically bound to the corresponding probes and resulted in the retardant band, whereas no binding activity was observed with the corresponding negative controls of MBP and Trx-His. The unlabeled cold probes (10, 50, 100×) gradually competed for the corresponding binding activity, whereas the mutated biotin-labeled probes (10, 50, 100×) completely abolished the protein-DNA interaction, and the mutated cold-probes showed no competitive activities. D to G, MST analysis showing that the specially binding activity of Trx-His-ABI5 with OsGLP2-1 promoter fragments of A5-1, A5-2 (E), and Trx-His-GAMYB with Ga-1 (G), respectively. The negative control, Trx-His, did not interact with these fragments (D and F). D and E, MST measurements of the interaction between ABI5 and A5-1, A5-2 fragments. F and G, MST assay testing the interaction between GAMYB and Ga-1 fragment. D to G data are average ± sd, n = 3. The data were analyzed with the software of MO. Affinity Analysis v2.2.4.

Figure 7.

Subcellular localization of OsGLP2-1-eGFP in transgenic rice seedling. In 7-d-old rice seedling, the control P_35S:eGFP leaves showed green fluorescence throughout the cytoplasm and nucleus (C); in contrast, green fluorescence from P_35S: HA-GLP_2-1-eGFP leaves exhibited moving needle-shaped signals in the cytosol, and no signal was observed in the nucleus (A). Moreover, the mobile round green fluorescent signal was also observed in P_35S:HA-GLP_2-1-eGFP seedling root (B). These dynamic green fluorescent signals of OsGLP2-1-eGFP in rice were confirmed in at least three independent experiments. Bars = 6 μm.