OsELF3.1-OsCATA-Ghd7 Pathway Regulates Rice Heading

doi:10.1016/j.rsci.2025.06.001

Abstract

Abstract:

Rice, a critical global staple crop, relies heavily on heading date, a key agronomic trait marking the transition from vegetative to reproductive growth. Understanding the genetic regulation of heading date is vital for enhancing the adaptability of high-quality rice varieties across diverse geographical regions and for bolstering local food security. In this study, we uncovered a novel role for OsCATA, a catalase gene, in the regulation of photoperiodic flowering in rice. We identified a novel allele of OsELF3.1, whose mutation resulted in delayed heading. Further analyses revealed that OsELF3.1 physically interacted with OsCATA. Notably, OsCATA exhibited rhythmic expression patterns similar to OsELF3.1 and, when mutated, also delayed flowering. Expression analyses showed that the delayed heading phenotype could be attributed to elevated Ghd7 expression under both long-day and short-day conditions, with OsCATA expression positively regulated by OsELF3.1. Double mutants of OsELF3.1 and OsCATA displayed a heading delay similar to that of oself3.1 single mutants. Additionally, OsELF3.1 could interact with Ghd7 in vivo, alleviating its suppression of Ehd1. Luciferase assays confirmed that Ghd7 repressed Ehd1 expression, while OsELF3.1 mitigated this repression. Collectively, our findings reveal that OsCATA is critical in suppressing Ghd7 expression through the OsELF3.1-OsCATA-Ghd7 transcriptional pathway, thereby regulating rice heading.

Key words: rice, heading date, OsELF3.1, Ghd7, OsCATA

Wu Zhaozhong, Zhong Zhengzheng, Xu Peng, Liu Ling, Wang Beifang, Yang Qinqin, Wen Xiaoxia, Ma Guifang, Luo Mili, Zhang Yingxin, Liu Qun’en, Peng Zequn, Zhan Xiaodeng, Cao Liyong, Cheng Shihua, Wu Weixun. OsELF3.1-OsCATA-Ghd7 Pathway Regulates Rice Heading[J]. Rice Science, 2025, 32(5): 658-672.

Figures/Tables 9

Fig. 1. Role of OsELF3.1 in rice heading. A and B, Phenotypes of Nipponbare (Nip, wild type) and elh4 mutant under natural long-day (NLD) conditions in Hangzhou, Zhejiang Province, China (A), and natural short-day (NSD) conditions in Lingshui, Hainan Province, China (B). Scale bar in A and B are 20 and 30 cm, respectively. C, Days to heading comparisons for Nip and elh4 under NLD and NSD conditions. D and E, Phenotypic analysis of Nip, elh4, and elh4 complementation (elh4-com) lines under NLD conditions (D) and corresponding heading date statistics (E). Scale bar in D is 20 cm. F and G, Phenotypic identification (F) and heading date statistics (G) for oself3.1 knockout lines (oself3.1-ko1 and oself3.1-ko2) under NLD and NSD conditions. Scale bar in F is 20 cm. In C, E, and G, data are mean ± SD (n values shown in columns). *** above bars indicate statistically significant differences at the 0.001 level by Student’s t test.

Fig. 2. Interaction between OsELF3.1 and OsCATA. A, Yeast two-hybrid (Y2H) assays reveal interactions between OsELF3.1 and OsCATA. OsELF3.1 and its truncated fragments served as prey against bait constructs, with co-transformed Y2H Gold yeast cells assessed on QDO (SD-Leu/-Trp/-His/-Ade) media. AD, GAL4 activation domain; BD, GAL4 DNA-binding domain. B, Bimolecular fluorescence complementation (BiFC) assays validate the interaction. Constructs were agroinfiltrated into Nicotiana benthamiana, and fluorescence signals were detected 4 d post-infiltration using a confocal microscope (Zeiss 700). mCherry-NLS served as a nuclear marker. Scale bars, 20 µm. YFP, Yellow fluorescent protein; DIC, Differential interference contrast. C, Luminescence complementation imaging (LCI) assays confirm the interaction of OsELF3.1 with OsCATA. Constructs were transiently expressed in N. benthamiana, with nLuc and cLuc as negative controls. Luminescence was visualized using a low-light, cooled charge-coupled device (CCD) camera. cLuc, C-terminal fragment of luciferase; nLuc, N-terminal fragment of luciferase; EV, Empty vector. D, Co-immunoprecipitation (Co-IP) assays demonstrate interaction in rice protoplasts co-transfected with OsELF3.1 and OsCATA. Immunoprecipitation was performed with anti-MYC sepharose beads, and proteins were detected using anti-GFP and anti-MYC antibodies.

Fig. 3. Phenotypes of oscata knockout and oself3.1 oscata double mutants. A, CRISPR-induced mutations in oscata. Protospacer adjacent motif (PAM) sequences are shown in orange, with mutation sites in red tangerine. B and C, Phenotypes of Nipponbare (Nip, wild type) and oscata knockout mutants (oscata-ko) under natural long-day (NLD) conditions at the heading stage of Nip (B) and days to heading (C) of Nip and oscata-ko mutants under NLD and natural short-day (NSD) conditions. Scale bar in B is 20 cm. D, Phenotypes were assessed of Nip and oscata-ko mutants at 60 d post-heading of Nip under NLD conditions. Scale bar, 20 cm. E, CRISPR-induced mutations in oself3.1 oscata double mutants (oself3.1 oscata-ko1 and oself3.1 oscata-ko2). PAM sequences are shown in orange, with mutation sites in red. F and G, Phenotypes (F) and days to heading (G) of Nip and oslelf3.1 oscata double mutants (oself3.1 oscata-ko1 and oself3.1 oscata-ko2) under NLD conditions. Scal bar in F is 20 cm. H and I, Phenotypes were assessed in Nip, oslelf3.1 mutants (oslelf3.1-ko1 and oslelf3.1-ko2, H), and oslelf3.1 oscata double mutants (oself3.1 oscata-ko1 and oself3.1 oscata-ko2, I) at 60 d post-heading under NLD conditions. Scale bars, 20 cm. In C and G, data are mean ± SD (n values are shown in columns). ***, Significant differences at the 0.001 levels by Student’s t test.

Fig. 4. Diurnal expression patterns of OsCATA and OsELF3.1. A-D, qRT-PCR analysis of OsCATA and OsELF3.1 in leaves of Nipponbare (Nip) plants under controlled long-day (CLD, A and B) and controlled short-day (CSD, C and D) conditions. E-H, Expression profiles of OsCATA and OsELF3.1 in leaves of Nip and oself3.1 plants under CLD (E and F) and CSD (G and H). Leaf samples were collected at 50 d after sowing in CLD (A, B, E, and F) and 40 d after sowing in CSD (C, D, G, and H) conditions. Transcript levels were normalized to ubiquitin (Os03g0234350), with data as mean ± SD (n = 3). Zeitgeber time (ZT) defines light onset (ZT8). Light and dark periods are represented by blue and black bars, respectively.

Fig. 5. Expression levels of genes related to heading date. qRT-PCR analyses of Ehd1 (A), Ghd7 (B), Hd1 (C), Hd3a (D), and RFT1 (E) in leaves of Nipponbare (Nip), oself3.1, oscata, and oself3.1 oscata double mutants, and OsELF3.1 (F) in Nip and oscata plants at 50 d after sowing under nature long-day conditions in Hangzhou filed. Data were normalized to ubiquitin (Os03g0234350) and represent mean ± SD (n = 3). Blue and black bars indicate light and dark periods, respectively.

Fig. 6. OsCATA interacts with Ghd7. A, Yeast two-hybrid (Y2H) assays confirm interactions between OsCATA and Ghd7 using co-transformed Y2HGold yeast cells on DDO (SD-Leu/-Trp) and QDO (SD-Leu/-Trp/-His/-Ade) media. T7 and P53 serve as a positive interaction control pair; AD, GAL4 activation domain; BD, GAL4 DNA-binding domain. B, Bimolecular fluorescence complementation (BiFC) assays demonstrate OsCATA-Ghd7 interaction in rice protoplasts. Fluorescence signals were observed using a confocal microscope (Zeiss 980) 20-24 h after infiltration. mCherry-NLS was used as a nuclear marker. YFP, Yellow fluorescent protein; NLS, Nuclear localization signal; DIC, Differential interference contrast; HYNE, N-terminal half of YFP; HYCE, C-terminal half of YFP. Scale bars, 20 µm. C, Luminescence complementation imaging (LCI) assays validate the interaction between OsCATA and Ghd7 in Nicotiana benthamiana. Negative controls included nLuc and cLuc. Luminescence was captured using a Charge-Coupled Device camera at 3 d post-infiltration. cLuc, C-terminal fragment of luciferase; nLuc, N-terminal fragment of luciferase; EV, Empty vector. D, Co-immunoprecipitation (Co-IP) assays reveal interaction in protoplasts co-transfected with OsCATA-MYC and Ghd7-eGFP, detected with anti-MYC and anti-GFP antibodies. GFP, Green fluorescent protein; MYC, MYC epitope tag; IB, Immunoblot; IP, Immunoprecipitation.

Fig. 7. OsELF3.1 interacts with Ghd7. A, Yeast two-hybrid (Y2H) assays confirm OsELF3.1-Ghd7 interaction on DDO (SD-Leu/-Trp) and QDO (SD-Leu/-Trp/-His/-Ade) media. T7 and P53 serve as a positive interaction control pair; AD, GAL4 activation domain; BD, GAL4 DNA-binding domain. B, Bimolecular fluorescence complementation (BiFC) assays illustrate OsELF3.1-Ghd7 interaction in rice protoplasts, with fluorescence detected 20-24 h after infiltration using a confocal microscope (Zeiss 980). mCherry-NLS was used as a nuclear marker. cYFP, C-terminal fragment of yellow fluorescent protein; nYFP, N-terminal fragment of yellow fluorescent protein; Scale bars, 20 µm. C, Luminescence complementation imaging (LCI) assays verify the interaction of OsELF3.1 and Ghd7 in Nicotiana benthamiana. Luminescence was captured using a Charge-Coupled Device camera. cLuc, C-terminal fragment of luciferase; nLuc, N-terminal fragment of luciferase; EV, Empty vector. D, Co-immunoprecipitation (Co-IP) assays confirm the interaction in rice protoplasts co-transfected with OsELF3.1-MYC and Ghd7-eGFP. The proteins from crude lysates (upper, input) and the immunoprecipitated proteins (lower) were detected with anti-GFP and anti-MYC antibodies. GFP, Green fluorescent protein; MYC, MYC epitope tag; IB, Immunoblot; IP, Immunoprecipitation.

Fig. 8. Transcriptional activity assays. A, Illustration of constructs used in transcriptional activity assays. B, Relative LUC activity was measured in rice protoplasts with combinations of OsCATA, OsELF3.1, Ghd7, and the Ehd1 reporter. LUC and REN served as internal controls. Data are mean ± SD (n = 3). Different lowercase letters above bars indicate significant differences (P < 0.05) based on Duncan’s multiple range test. GFP, Green fluorescent protein; NOS, Nopaline synthase terminator; MYC, MYC epitope tag; LUC, Luciferase; REN, Renilla; EV, Empty vector.

Fig. 9. Pathway summarizing OsELF3.1-OsCATA-Ghd7 regulation of rice heading date.

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