Rice transcriptional repressor OsTIE1 controls anther dehiscence and male sterility by regulating JA biosynthesis

Abstract

Proper anther dehiscence is essential for successful pollination and reproduction in angiosperms, and jasmonic acid (JA) is crucial for the process. However, the mechanisms underlying the tight regulation of JA biosynthesis during anther development remain largely unknown. Here, we demonstrate that the rice (Oryza sativa L.) ethylene-response factor-associated amphiphilic repression (EAR) motif-containing protein TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORS (TCP) INTERACTOR CONTAINING EAR MOTIF PROTEIN1 (OsTIE1) tightly regulates JA biosynthesis by repressing TCP transcription factor OsTCP1/PCF5 during anther development. The loss of OsTIE1 function in Ostie1 mutants causes male sterility. The Ostie1 mutants display inviable pollen, early stamen filament elongation, and precocious anther dehiscence. In addition, JA biosynthesis is activated earlier and JA abundance is precociously increased in Ostie1 anthers. OsTIE1 is expressed during anther development, and OsTIE1 is localized in nuclei and has transcriptional repression activity. OsTIE1 directly interacts with OsTCP1, and overexpression of OsTCP1 caused early anther dehiscence resembling that of Ostie1. JA biosynthesis genes including rice LIPOXYGENASE are regulated by the OsTIE1-OsTCP1 complex. Our findings reveal that the OsTIE1-OsTCP1 module plays a critical role in anther development by finely tuning JA biosynthesis and provide a foundation for the generation of male sterile plants for hybrid seed production.

Figure 1.

OsTIE1 is a key factor in maintaining rice male fertility. A) Conserved sequence alignment between OsTIE1 and TIE1. The same amino acid residues are marked with a star sign, while amino acid residues of the same type are marked with a colon. B) Heterologous expression of OsTIE1 in Arabidopsis. Scale bar: 5 cm. C) WT, Ostie1-1, and Ostie1-2 plants at the pustulation period. The mutant plants produced few seeds. Scale bar: 10 cm. D) Statistical analysis of the seed-setting rate of the WT, Ostie1-1, and Ostie1-2. Data are expressed as mean (±Sd) (n ≥ 4). The chi-square test was used to compare the seed-setting rate of different phenotypes. ***P < 0.001. E) Reciprocal cross between WT and mutant alleles. Scale bar: 2 cm. F) Statistical analysis of crossing combination. WT♀×WT♂ was used as the control. The seed-setting rate exhibited no significant difference from that of the control when the mutant was used as the female, while plants were completely sterile when the mutant was used as the pollen donor. Data are expressed as mean (±Sd) (n ≥ 3). The chi-square test was used to compare the seed-setting rate of different cross groups. ***P < 0.001.

Figure 2.

The Ostie1-1 and Ostie1-2 mutants displayed premature anther dehiscence. A to D) Florets were observed at Stage 12 with a stereomicroscope. Ostie1-1 and Ostie1-2 showed premature anther dehiscence compared to the WT. Scale bar: 1 mm. E, F) SEM observation of the anthers of the WT and Ostie1-1 at Stage 12. Scale bar: 100 μm. G, H) SEM observation of the pistils of the WT and Ostie1-1 at Stage 12. Pollen grains marked with arrows were prematurely released and adhered to pistils at Stage 12 in Ostie1-1. Scale bar: 200 μm. I) Statistical analysis of the anther dehiscence rate at Stage 12 and Stage 13. Data are expressed as mean (±Se) (n ≥ 5). Two-tailed t-test was applied for comparisons between groups. ***P < 0.001. J) Comparison of WT, Ostie1-1, and Ostie1-2 anthers at Stage 12. Scale bar: 1 mm. K) Statistical analysis of the length of filaments and anthers in the WT, Ostie1-1, and Ostie1-2 at Stage 12. Data are expressed as the mean (±Sd) (n ≥ 100). Fisher's protected least significant difference was applied for multiple comparisons. Means were judged significantly different at P < 0.05. L, M) Cross-section of anthers at Stage 12. Anthers were prematurely dehisced in Ostie1-1. Scale bar: 100 μm. N, O) I₂-KI staining of pollen grains at Stage 12. Pollen grains were inviable in Ostie1-1. Scale bar: 100 μm.

Figure 3.

OsTIE1 is a nucleus-localized transcriptional repressor mainly expressed in anthers after Stage 9. A) RT-qPCR analysis of OsTIE1 expression during anther development. The ordinate represents the ratio of the gene expression level to that of the internal control OsUBQ. Data are expressed as the mean (±Sd) of 3 biological replicates. B to G) Tissue-specific staining with GUS driven by the OsTIE1 promoter. The signal was consistent with the results of the real-time assays. Scale bar: 1 mm. H, I) OsTIE1 was colocalized with FIB2, which was used as a nuclear marker in N. benthamiana and rice protoplasts. Scale bar: 50 μm. J) OsTIE1 is a transcriptional repressor. REN LUC was used as the internal control. The ratio of the luminescent signals from firefly LUC and REN LUC was calculated. OsTIE1△EAR indicates OsTIE1 without the EAR-motif. Data are expressed as the mean (±Sd) of 3 biological replicates. Two-tailed t-test was used for comparisons between groups. **P < 0.01.

Figure 4.

OsTIE1 regulates the JA synthesis pathway. A) Bar plot of DEGs at Stage 10 and Stage 12. The criteria for DEGs were log2 fold change of ≥1.5 or ≤−1.5 and q ≤ 0.05. B, C) GO enrichment analysis of DEGs between Ostie1-1 and WT at Stage 10 and Stage 12, respectively. Terms under the biological process category were enriched. Several terms related to JA pathway or cell wall biogenesis appeared among DEGs. D) Heat map of JA synthesis genes with log2 fold change revealed by RNA-seq. E, F)OsLOX6 and OsLOX11 were upregulated in Ostie1-1. Data are expressed as the mean (±Sd) of 3 biological replicates. Two-tailed t-test was used for comparisons between groups. *P < 0.05. ***P < 0.001. G) The concentration of JA in anthers at Stage 10 and Stage 12. Data are expressed as the mean (±Sd) of 3 biological replicates. Two-tailed t-test was used for comparisons between groups. ***P < 0.001.

Figure 5.

Abnormal JA accumulation in Ostie1-1 is the main reason for premature anther dehiscence. A, B) DIECA treatment of Ostie1-1 rescued the phenotype of anther dehiscence. Dehisced anthers are marked with red arrows. Scale bar: 1 mm. C, I) Statistical analysis of the anther dehiscence rate of Ostie1-1 and WT plants treated with the mock treatment, 1 mm DIECA or 100 μm JA. A 1‰ ethanol solution was used as the mock treatment. Data are expressed as the mean (±Se) (n ≥ 5). Fisher’s protected least significant difference was used for multiple comparisons. Means were judged significantly different at P < 0.05. D, H) Effects of DIECA or JA treatment on WT and Ostie1-1 anthers at Stage 12. Scale bar: 1 mm. E, J) Statistical analysis of the length of anthers and filaments under mock, JA, or DIECA treatment. Data are expressed as the mean (±Sd) (n ≥ 100). Fisher’s protected least significant difference was used for multiple comparisons. Means were judged significantly different at P < 0.05. F, G) I₂-KI staining of Ostie1-1 pollen grains under mock or DIECA treatment at Stage 12. Scale bar: 100 μm.

Figure 6.

OsTIE1 functions by interacting with OsTCP1. A) The interactions between OsTIE1 and CIN-like TCPs in rice were tested using yeast 2-hybrid assays. B) The LUC signal was detected only when nLUC-OsTIE1 and cLUC-OsTCP1 were coexpressed in N. benthamiana leaves. DBD indicates the DBD of yeast GAL4, which was used as the control. C) OsTCP1 fused with His was detected when OsTIE1-MYC was enriched. D) Statistical analysis of the seed-setting rate of WT and OsTIE1pro:OsTCP1 transgenic plants. Data are expressed as the mean (±Sd) (n ≥ 5). The chi-square test was used to compare the seed-setting rate. ***P < 0.001. E to G) Comparison of WT and OsTIE1pro:OsTCP1 transgenic plants at Stage 12. Premature dehisced anthers are marked with red arrows. Scale bar: 1 mm. H) Statistical analysis of the anther dehiscence rate. Data are expressed as the mean (±Se) (n ≥ 5). Two-tailed t-test was used for comparisons between groups. ***P < 0.001. I)OsLOX11 was upregulated in OsTIE1pro:OsTCP1 anther. Data are expressed as the mean (±Sd) of 3 biological replicates. Two-tailed t-test was used for comparisons between groups. ***P < 0.001. J) The dual-LUC assay was employed to test the transcriptional regulation activity of the OsTIE1-OsTCP1 module. Firefly LUC was driven by the promoter of LOX11, which was 2,100-bp upstream of LOX11 coding region. The expression level of firefly LUC was increased when OsTCP1 was transformed and declined when OsTIE1 and OsTCP1 were cotransformed. Data are expressed as the mean (±Sd) of 3 biological replicates. Two-tailed t-test was used for comparisons between groups. **P < 0.01. K) EMSA revealed that OsTCP1 directly bound to the promoter region of OsLOX11.

Figure 7.

TIE-TCP modules regulate anther dehiscence in Arabidopsis. A to C) Increased expression of OsTIE1 in Arabidopsis produced a phenotype of anther indehiscence and shortened filaments, opposite to that of Ostie1-1. D, E) The TIE1 gain-of-function mutant tie1-D displayed a phenotype similar to that of 35Spro:OsTIE1. Scale bar: 1 mm in A) and D) and 100 μm in B), C), and E). F) Statistical analysis of anther dehiscence rate at flowering stage. Data are means ± Se (n ≥ 5). Two-tailed t-test was applied for comparison between groups. ***P < 0.001. G) Statistical analysis of the length of filament or anther in WT, tie1-D, and 35S:OsTIE1 at flowering stage. Data are means ± Sd (n ≥ 100). Two-tailed t-test was applied for comparison between groups. ***P < 0.001. H to K) Phenotype observation of WT and tie1 tie2 tie3 tie4 1 d before flowering. tie1 tie2 tie3 tie4 exhibited premature dehiscence that phenocopied Ostie1. Scale bar: 1 mm in H) and I) and 200 μm in J) and K). L) Statistical analysis of anther dehiscence rate in H) to K). Data are means ± Se (n ≥ 5). Two-tailed t-test was used. ns, P > 0.05, *P < 0.05, and ***P < 0.001. M to P) GUS staining assay revealed that TIE1 protein was expressed in anthers. Scale bar: 1 mm in M) and N), 50 μm in O), and 20 μm in P). Q to U) Anther dehiscence of WT, tcpSEP, tcpOCT, and tcpUND at flowering stage. tcpOCT, tcpUND exhibited indehiscent anthers that were opposite to that of Ostie1. Scale bar: 1 mm in Q) and 200 μm in R) to U). V) Statistical analysis of anther dehiscence rate at flowering stage in Q) to U). Data are means ± Se (n ≥ 5). Two-tailed t-test was used. ns, P > 0.05, *P < 0.05, and ***P < 0.001. W to Z) GUS staining assay revealed that TCP4 protein was expressed in anthers. Scale bar: 1 mm in W), 500 μm in X), 50 μm in Y), and 20 μm in Z).

Figure 8.

Working model of OsTIE1 in the suppression of rice anther dehiscence. JA synthesis is suppressed by the OsTIE1-OsTCP module before Stage 12 in rice to ensure normal timing of anther dehiscence. OsTCP1 acts as a positive regulator of JA synthesis by activating the expression of JA synthesis genes, including LOXs, while OsTIE1 is a transcriptional repressor that functions by interacting with OsTCP1 and suppressing the expression of JA synthesis genes. In Ostie1-1, hyperactive JA synthesis causes premature accumulation of JA and anther dehiscence, while JA synthesis is suppressed until the normal dehiscence time in WT plants expressing OsTIE1.