Phase separation of Rht8-derived RNHL1 integrates ethylene and gibberellin signaling to regulate wheat internode elongation.

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Learn more: PMC Disclaimer | PMC Copyright Notice Plant Cell . 2026 Mar 19;38(4):koag081. doi: 10.1093/plcell/koag081 Search in PMC Search in PubMed View in NLM Catalog Add to search Phase separation of Rht8 -derived RNHL1 integrates ethylene and gibberellin signaling to regulate wheat internode elongation Chaoqun Dong Chaoqun Dong 1 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China Find articles by Chaoqun Dong 1, # , Xiliu Cheng Xiliu Cheng 2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R. China Find articles by Xiliu Cheng 2, # , Meng Yuan Meng Yuan 3 State Key Laboratory of Crop Stress Resistance and High-Efficiency Production, College of Agronomy, Northwest A&F University, Yangling 712199, P. R. China Find articles by Meng Yuan 3, # , Zhe Zhang Zhe Zhang 4 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China Find articles by Zhe Zhang 4 , Yiqing Wang Yiqing Wang 5 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China Find articles by Yiqing Wang 5 , Jing Liu Jing Liu 6 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China Find articles by Jing Liu 6 , Long Song Long Song 7 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China Find articles by Long Song 7 , Haoran Wang Haoran Wang 8 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China Find articles by Haoran Wang 8 , Yujie Jiang Yujie Jiang 9 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China Find articles by Yujie Jiang 9 , Xiangqing Liu Xiangqing Liu 10 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China Find articles by Xiangqing Liu 10 , Jianhui Wu Jianhui Wu 11 State Key Laboratory of Crop Stress Resistance and High-Efficiency Production, College of Agronomy, Northwest A&F University, Yangling 712199, P. R. China Find articles by Jianhui Wu 11 , Yingyin Yao Yingyin Yao 12 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China Find articles by Yingyin Yao 12 , Zhongfu Ni Zhongfu Ni 13 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. 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China Find articles by Jie Liu 16, ✉, 4 Author information Article notes Copyright and License information 1 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China 2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R. China 3 State Key Laboratory of Crop Stress Resistance and High-Efficiency Production, College of Agronomy, Northwest A&F University, Yangling 712199, P. R. China 4 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China 5 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China 6 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China 7 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China 8 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China 9 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China 10 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China 11 State Key Laboratory of Crop Stress Resistance and High-Efficiency Production, College of Agronomy, Northwest A&F University, Yangling 712199, P. R. China 12 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China 13 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China 14 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China 15 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China 16 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China ✉ Corresponding authors: Jie Liu, State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China. Email: [email protected] ; Lingling Chai, State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China. Email: [email protected] # Chaoqun Dong, Xiliu Cheng and Meng Yuan contributed equally to this article. 3 The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors ( https://academic.oup.com/plcell/pages/General-Instructions ) is Jie Liu ( [email protected] ). 4 Conflicts of interest: No conflict of interest is declared. Received 2025 Aug 19; Accepted 2026 Jan 26; Collection date 2026 Apr. © The Author(s) 2026. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence ( https://creativecommons.org/licenses/by-nc-nd/4.0/ ), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact [email protected] for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact [email protected]. PMC Copyright notice PMCID: PMC13069570  PMID: 41855183 Abstract Semidwarf varieties of wheat ( Triticum aestivum L.) carrying Reduced height ( Rht ) genes revolutionized wheat production. Rht8 , a widely deployed semidwarfing allele, encodes RNHL1 (ribonuclease H-Like 1); yet, the molecular mechanisms underlying its growth regulation remain unclear. Here, we uncover a liquid–liquid phase separation (LLPS)-mediated transcriptional pathway by which RNHL1 controls plant height. We show that RNHL1 forms nuclear biomolecular condensates via its intrinsically disordered regions (IDR1 and IDR3) and physically interacts with the ethylene signaling transcription factor TaEIL1 (ethylene insensitive3-like 1) to establish functional transcriptional hubs. These RNHL1–TaEIL1 condensates directly bind and repress the ethylene response factor gene TaERF1 ( Ethylene response factor 1 ), and TaERF1 suppresses the gibberellin (GA) biosynthetic gene TaGA3ox2 ( Gibberellin 3-beta dioxygenase 2 ). Genetic analyses demonstrate that both RNHL1 and TaEIL1 positively regulate plant height, with loss-of-function mutants exhibiting similar dwarf phenotypes and convergent transcriptomic profiles. Importantly, we establish TaERF1 as a direct repressor of TaGA3ox2 , completing a regulatory cascade in which RNHL1–TaEIL1 condensates modulate GA-mediated internode elongation. Our findings reveal an integration point between ethylene and GA signaling orchestrated by RNHL1–TaEIL1 phase separation and highlight RNHL1's roles in nucleic acid metabolism and transcriptional regulation. This study provides fundamental insights into LLPS-mediated growth control in crops and identifies specific protein domains as potential targets for wheat improvement. RNHL1 phase separation with TaEIL1 integrates ethylene and gibberellin signaling to control wheat internode elongation by repressing TaERF1 expression and GA biosynthesis. Introduction Wheat ( Triticum aestivum L.) is one of the most widely cultivated cereal crop globally, serving as the primary source of nutrition for over 35% of the world's population and contributing approximately 20% of total caloric intake ( Dubcovsky and Dvorak 2007 ). The Green Revolution of the mid-20th century transformed global wheat production through the introduction of semidwarf varieties carrying Reduced height-B1b ( Rht-B1b ) and Rht-D1b ( Peng et al. 1999 ; Hedden 2003 ; Van De Velde et al. 2021 ). These genes encode truncated DELLA proteins that confer gibberellin (GA) insensitivity, effectively reducing plant height while increasing harvest index ( Peng et al. 1999 ; Achard et al. 2006 ). However, this GA insensitivity comes with an agronomic tradeoff: impaired coleoptile elongation that compromises seedling establishment, particularly in moisture-limited environments requiring deep sowing ( Richards 1992 ; Rebetzke et al. 1999 ; Rebetzke et al. 2001 ; Ellis et al. 2004 ). The discovery of alternative dwarfing genes that maintain GA sensitivity has therefore become a major focus of wheat improvement programs. Among these, Rht8 has emerged as particularly valuable, conferring 10% to 15% height reduction without compromising coleoptile development or seedling vigor ( Worland et al. 1998 ; Ellis et al. 2004 ; Grover et al. 2018 ). Recent molecular characterization identified RNHL1 ( Ribonuclease H-Like 1 ) as the causal gene underlying Rht8 ( Chai et al. 2022 ; Xiong et al. 2022 ). RNHL1 encodes a nuclear protein containing both an RNA:DNA hybrid-binding domain (HBD) and catalytic RNase H domain, suggesting potential roles in nucleic acid metabolism. However, its precise molecular function in plant growth regulation remains enigmatic, particularly given its apparent divergence from classical DELLA-mediated GA signaling pathways. GAs are diterpenoid hormones that regulate virtually all aspects of plant growth and development ( Yamaguchi 2008 ). The GA biosynthetic pathway involves multiple oxidation steps catalyzed by 2-oxoglutarate-dependent dioxygenases, with GA 3-oxidase (GA3ox) performing the final activation steps to produce bioactive GA 1 and GA 4 ( Yamaguchi 2008 ). In wheat, alterations in GA metabolism through either biosynthesis (eg, TaGA3ox2 and TaGA20ox1) or catabolism (eg, TaGA2oxA9) can significantly impact plant height ( Appleford et al. 2006 ; Yamaguchi 2008 ; Ford et al. 2018 ; Sun et al. 2019 ; Buss et al. 2020 ; Tian et al. 2022 ). However, the regulatory networks controlling these enzymes, particularly through noncanonical mechanisms, remain poorly understood. Recent advances in cell biology have revealed the fundamental importance of liquid–liquid phase separation (LLPS) in organizing cellular processes ( Hyman et al. 2014 ; Hnisz et al. 2017 ; Boija et al. 2018 ; Wang et al. 2023 ). LLPS enables the formation of membraneless organelles through multivalent interactions between proteins containing IDRs or modular interaction domains ( Li et al. 2012 ; Banani et al. 2017 ). In plants, LLPS has been implicated in diverse processes including temperature sensing ( Jung et al. 2020 ; Zhu et al. 2021 ), flowering time regulation ( Fang et al. 2019 ), inflorescence development ( Huang et al. 2021 , 2025 ), and chlorophyll homeostasis ( Jiang et al. 2023 ). However, its potential roles in hormone signaling and crop plant height regulation remain largely unexplored. In this study, we elucidate a molecular mechanism whereby RNHL1 regulates wheat growth through LLPS-mediated transcriptional control. We demonstrate that RNHL1 undergoes nuclear phase separation through its IDR domains (IDR1 and IDR3) and interacts with ETHYLENE INSENSITIVE3 (EIN3)-Like 1 (TaEIL1) to form functional biomolecular condensates. These RNHL1–TaEIL1 condensates specifically bind to and repress the transcription of ethylene response factor gene TaERF1 , which further regulates the expression of GA biosynthetic gene TaGA3ox2 . Through comprehensive phenotypic analysis of transgenic wheat lines, we establish “RNHL1–TaEIL1–TaERF1–TaGA3ox2” regulatory module in controlling wheat plant height by modulating bioactive GA levels, representing a previously unknown integration point between ethylene and GA signaling pathways. Our findings not only provide evidence for LLPS-mediated growth regulation in crops but also reveal RNHL1's remarkable dual functionality in both nucleic acid metabolism and transcriptional control. Results RNHL1 positively regulates plant height and internode elongation in wheat In our previous study, we generated the rnhl1 null mutant line ( rnhl1-aabbdd ), which exhibited significantly reduced plant height, shortened internodes, and decreased spike length ( Fig. 1a–e ) ( Chai et al. 2022 ). To further confirm the essential role of RNHL1 in internode elongation, we overexpressed the D-subgenome-derived RNHL-D1 gene (GFP-tagged) in the Fielder background ( RNHL1-OE ; Fig. S1a ). Consistent with its proposed function, all RNHL1-OE lines showed significantly increased plant height, spike length, and internode length compared with the wild-type ( Fig. 1f–i ). Together, these results demonstrate that RNHL1 acts as a positive regulator of culm and spike elongation in wheat. Figure 1. Open in a new tab Phenotypic characterization of rnhl1 mutants and RNHL1 overexpression plants. (a) Comparative analysis of whole-plant architecture at the reproductive phase shows significant height reduction in rnhl1 ( rnhl1-aabbdd ) mutants compared with wild-type (WT) Fielder plants. Scale bar, 30 cm. Images were linearly adjusted for brightness and contrast. (b) Spikes and internodes of WT (left) and rnhl1 mutants (right). Scale bar, 10 cm. Images were linearly adjusted for brightness and contrast. (c) Schematic representation of wheat tiller structure, highlighting measured parameters (created with BioRender.com ). (d, e) Quantitative measurements confirming significant reductions in plant height (D), spike length and internode lengths (e) in rnhl1 mutants relative to WT (** P < 0.01, Student's t -test). (f) RNHL1 -overexpressing ( RNHL1-OE ; lines #1 and #2) plants exhibit increased stature compared to WT at the reproductive phase. Scale bar, 30 cm. Images were linearly adjusted for brightness and contrast. (g) Spikes and internodes of WT (left) and RNHL1-OE lines (#1 and #2, middle and right). Scale bar, 10 cm. Images were linearly adjusted for brightness and contrast. (h, i) Statistical analysis demonstrating significant increases in plant height (H), spike length and internode lengths (i) in RNHL1-OE plants relative to WT (** P < 0.01, Student's t -test). See source data for detailed statistical parameters. RNHL1 physically interacts with TaEIL1 To elucidate the molecular mechanism by which RNHL1 regulates culm and spike elongation, we screened a wheat young spike cDNA library using yeast two-hybrid (Y2H) assays to identify RNHL1-interacting proteins. Among the candidate interactors ( Supplementary Data Set S1 ), we identified TaEIL1 (TraesCS4D02G177000), an ETHYLENE-INSENSITIVE3 (EIN3)-LIKE (EIL) transcription factor involved in ethylene signaling. Notably, OsEIL1a , the rice ortholog of TaEIL1 , has been reported to regulate internode elongation by activating SD1 expression and promoting GA 4 biosynthesis ( Kuroha et al. 2018 ). Based on clues from rice research, we focused our investigation on TaEIL1 in this study. Using firefly luciferase complementation imaging (LCI), in vivo co-immunoprecipitation (Co-IP), in vitro pull-down, and biomolecular fluorescence complementation (BiFC) assays, we confirmed a direct physical interaction between RNHL1 and TaEIL1 ( Fig. 2a, c, d, e ). Truncation analysis revealed that the N-terminal (NT) domain of RNHL1 mediates this interaction ( Fig. 2b ). Furthermore, subcellular colocalization assays showed that RNHL1-GFP and mCherry-TaEIL1 fluorescence signals overlapped in the nucleus ( Fig. 2f ), supporting their direct in vivo association. Collectively, these results demonstrate a functional linkage between RNHL1 and TaEIL1 in regulating wheat development. Figure 2. Open in a new tab Physical interaction between RNHL1 and TaEIL1. (a) Luciferase (LUC) complementation imaging (LCI) assays demonstrating physical interaction between RNHL1 and TaEIL1 in Nicotiana benthamiana leaf cells. (b) Quantitative analysis revealing the N-terminal domain (NT, 1 to 272 aa) of RNHL1 shows the strongest interaction with TaEIL1, compared to middle (MD, 273 to 549 aa) and C-terminal (CT, 550 to 808 aa) domains. Data represent mean ± SD (n = 4 independent assays; ** P < 0.01, Student's t -test). Schematic illustrates RNHL1 domain architecture. (c) Bimolecular fluorescence complementation (BiFC) assays visualize RNHL1-TaEIL1 interaction complexes in plant nuclei. Scale bars, 20 μm. (d) Co-immunoprecipitation (Co-IP) assays in wheat leaf protoplasts showing that TaEIL1-Flag specifically interacts with RNHL1-GFP (red arrows), but not with the GFP control (blue arrow). (e) In vitro pull-down assays confirming the direct interaction between recombinant RNHL1 and TaEIL1 proteins. (f) Confocal microscopy reveals colocalization of RNHL1-GFP and TaEIL1-mCherry in nuclear condensates. Merged channels show overlapping fluorescence signals. Right panels display fluorescence intensity profiles along indicated lines, demonstrating specific colocalization of RNHL1-GFP with TaEIL1-mCherry (top) but not with mCherry alone (middle) or GFP with TaEIL1-mCherry (bottom). Scale bars, 10 μm. RNHL1 and TaEIL1 form biomolecular condensates Fluorescence colocalization analysis revealed that RNHL1 and TaEIL1 coassembled into distinct nuclear puncta with complete signal overlap ( Fig. 2f ), suggesting potential liquid–liquid phase separation (LLPS) behavior. To investigate this possibility, we first analyzed their protein sequences using PONDR ( https://www.pondr.com/ ), which predicted multiple intrinsically disordered regions (IDRs) in both proteins (PONDR score >0.5; Fig. 3a, b ). We then performed in vitro LLPS assays using purified MBP-RNHL1-GFP and MBP-mCherry-TaEIL1 proteins. Both proteins formed spherical droplets upon addition of 10% PEG8000 (PEG) ( Fig. 3c ), a crowding agent known to promote phase separation ( Hondele et al. 2019 ). To examine condensate dynamics in vivo, we conducted fluorescence recovery after photobleaching (FRAP) assays in Nicotiana benthamiana epidermal cells. As expected, both RNHL1-GFP and mCherry-TaEIL1 exhibited rapid fluorescence recovery in bleached regions ( Fig. 3d–f ), indicating liquid-like properties of these condensates. Figure 3. Open in a new tab Liquid–liquid phase separation (LLPS) properties of RNHL1 and TaEIL1. (a, b) Prediction of intrinsically disordered regions (IDRs) in RNHL1 (a) and TaEIL1 (b) using PONDR algorithms. Top show disorder probability profiles from VL3 (blue) and VSL2 (purple) predictions. Bottom illustrate domain organization, with overlapping VSL3/VSL2 predictions highlighted in orange. (c) In vitro phase separation assays demonstrating that RNHL1-GFP and mCherry-TaEIL1 proteins could form condensate droplets. Purified MBP-RNHL1-GFP and MBP-mCherry-TaEIL1 (each expressed separately in E. coli ) were co-incubated at 1 mg/mL each in the presence of 10% PEG 8,000 to induce phase separation. The mixture was visualized by confocal microscopy. Insets show higher magnification views of representative droplets. Scale bars, 10 μm. (d) Fluorescence recovery after photobleaching (FRAP) analysis of RNHL1-GFP and TaEIL1-GFP in N. benthamiana nuclei. Bleached areas (white circles) show rapid fluorescence recovery, indicating liquid-like properties of the condensates. Scale bars, 2 μm. (e, f) Quantitative analysis of FRAP recovery kinetics shown in (D). Normalized fluorescence intensity measurements (mean ± SD, n = 10 nuclei) demonstrate dynamic exchange of both RNHL1 (e) and TaEIL1 (f) within nuclear condensates, with similar recovery half-times. Collectively, these results demonstrate that RNHL1 and TaEIL1 possess the capacity to form dynamic, liquid-like condensates both in vitro and in vivo, likely mediated by their intrinsic disordered regions. IDR domains are essential for RNHL1 phase separation and TaEIL1 interaction Given that RNHL1 primarily interacts with TaEIL1 through its N-terminal region (NT; Fig. 2b ) and that its IDRs are also localized to the NT ( Fig. 3a ), we investigated whether these IDR domains mediate RNHL1-TaEIL1 association. We first mapped 3 distinct IDRs within the NT: IDR1 (amino acids 1 to 56 amino acids), IDR2 (120 to 189 aa), and IDR3 (199 to 264 aa) ( Fig. 4a ). To characterize their functions, we generated a series of RNHL1 deletion mutants (RNHL1-ΔIDR1, −ΔIDR2, and −ΔIDR3) and analyzed their subcellular localization patterns as GFP fusion proteins. Figure 4. Open in a new tab Functional characterization of RNHL1 intrinsically disordered regions (IDRs). (a) Schematic representation of RNHL1 domain architecture highlighting 3 major IDRs (IDR1, IDR2, and IDR3) and the prion-like domain (PrLD) and glycine-rich tandem motif (GGG) within IDR2. (b) Subcellular localization analysis demonstrating the essential roles of IDR1, IDR3 and PrLD in facilitating RNHL1-GFP phase condensation. Fluorescence intensity profiles (bottom) quantify distribution differences using coefficient of variation (cV = SD/mean). Scale bars, 5 μm. (c) Subcellular localization assay revealing that the G-rich tandem motif in IDR2 is essential for nuclear localization. Scale bars, 10 μm. (d, e) LCI quantification showing that deletion of IDR1, IDR3, and PrLD impairs RNHL1-TaEIL1 interaction. NLS, nuclear localization signal. Data represent mean ± SD (n = 8; ** P < 0.01, Student's t -test vs control, denoted as sample 1). Notably, deletion of either IDR1 or IDR3 substantially impaired RNHL1's phase separation capacity, as evidenced by the loss of characteristic punctate structures in RNHL1-ΔIDR1 and RNHL1-ΔIDR3 mutants ( Fig. 4b ). Intriguingly, IDR2 deletion completely abolished nuclear import of RNHL1, resulting in cytoplasmic retention of RNHL1-ΔIDR2 ( Fig. 4c ). Bioinformatics analysis using cNLS Mapper revealed no classical nuclear localization signal (NLS) within IDR2, suggesting that RNHL1 nuclear targeting depends on this non-canonical IDR2 motif rather than traditional NLS sequences. The IDR2 region contains 2 functionally distinct elements: a proline-rich prion-like domain (PrLD, 120 to 142 aa) and a glycine-rich tandem motif (composed of 7 consecutive glycine residues, 175 to 181 aa)—both known to promote liquid–liquid phase separation ( do Amaral et al. 2023 ). Functional dissection showed that while PrLD deletion (ΔPrLD) specifically impaired nuclear phase separation without affecting nuclear localization ( Fig. 4b ), ablation of the glycine-rich domain (ΔGGG) or its replacement with alanine (GGG→AAA) caused complete or partial nuclear exclusion ( Fig. 4c ), thereby implicating distinct roles of the PrLD and glycine-rich domains in phase separation and nuclear trafficking. We also explored the potential role of the highly conserved RNase H domain in RNHL1 phase separation, despite the absence of predicted IDRs within this domain ( Fig. 3a ). To address this, we generated a truncated RNHL1 variant (RNHL1-ΔRNase H) by deleting the RNase H domain (amino acids 400 to 627) and fused it with GFP to produce the RNHL1-ΔRNase H-GFP construct. Surprisingly, when expressed in plant cells, this truncated protein—similar to the IDR1- or IDR3-deleted variants—exhibited a diffuse nuclear distribution and failed to form condensates ( Fig. S1b, c ). This result demonstrates that, in addition to IDR1 and IDR3, the RNase H domain is essential for stabilizing RNHL1 phase separation. We next assessed how IDR deletions affect RNHL1–TaEIL1 interaction using LCI. Strikingly, while IDR1 deletion weakened the interaction, IDR3 deletion enhanced RNHL1–TaEIL1 binding ( Fig. 4d ), despite both deletions similarly impairing phase separation. To control for nuclear localization effects in IDR2 mutants, we introduced an exogenous NLS. This revealed that only PrLD deletion (which specifically disrupts phase separation) significantly reduced RNHL1–TaEIL1 association, whereas glycine-rich domain modifications (ΔGGG or GGG→AAA) had minimal effect ( Fig. 4e ). These findings establish that IDR-mediated phase separation is fundamentally required for proper RNHL1–TaEIL1 interaction, while demonstrating functional specialization among different IDR domains in regulating this process. TaEIL1 positively regulates internode elongation in a manner similar to RNHL1 To determine whether TaEIL1 plays a functional role in wheat internode elongation, we generated TaEIL1 -knockout mutants ( eil1 , lines #1 and #2; Fig. S2 ) and transgenic plants overexpressing the D-subgenome-derived TaEIL1 (Flag-tagged TaEIL1-OE , lines #1 and #2; Fig. S3 ). Phenotypic characterization revealed that eil1 mutants exhibited a significant reduction in spike length and plant height, along with shortened internodes—particularly in INL1 to INL3—compared with wild-type plants ( Fig. 5a–d ). In contrast, TaEIL1 overexpression produced only marginal effects on plant height: while line #1 showed a modest increase in plant height, line #2 displayed no significant difference from wild-type plants ( Fig. S3 ). These results demonstrate that TaEIL1 is essential for normal internode elongation and plant height establishment in wheat. Figure 5. Open in a new tab Phenotypes of TaEIL1 editing mutant lines eil1 . (a) Whole-plant comparison at the reproductive phase reveals significant height reduction in eil1 mutants compared with WT. Scale bar, 30 cm. Images were linearly adjusted for brightness and contrast. (b) Spikes and internodes of WT (left) and 2 eil1 mutant lines (#1 and #2, middle and right). Scale bar, 10 cm. Images were linearly adjusted for brightness and contrast. (c, d) Quantitative analysis confirming significant reductions in plant height (c) and spike and internode lengths (d) in eil1 mutants compared to WT. Data represent mean ± SD (n = 20; * P < 0.05, ** P < 0.01; Student's t -test). We further examined whether TaEIL1 gene participates in ethylene signaling in wheat. Under dark-grown conditions, eil1 mutants exhibited reduced sensitivity to 1-aminocyclopropane-1-carboxylic acid (ACC, the ethylene precursor) treatment compared with wild-type Fielder, whereas TaEIL1-OE plants displayed enhanced ACC sensitivity ( Fig. S4 ), confirming the essential role of TaEIL1 in wheat ethylene signaling. Together, the phenotypic parallels between TaEIL1 and RNHL1, along with their physical interaction, support a cooperative role for these 2 proteins in regulating plant height. Transcriptome profiling reveals coregulated gene networks downstream of RNHL1 and TaEIL1 To elucidate the shared signaling pathways regulated by RNHL1 and TaEIL1, we performed RNA sequencing (RNA-seq) analysis using young spike tissues collected from rnhl1 and eil1 null mutants. Quality control confirmed high reproducibility across biological replicates ( Fig. S5 ). Comparative transcriptomics identified 8,559 differentially expressed genes (DEGs) in rnhl1 (|FC| > 1, P < 0.05; Supplementary Data Set S2 ) and 19,589 DEGs in eil1 ( Supplementary Data Set S3 ). Venn diagram analysis revealed 1,103 co-upregulated and 1,545 co-downregulated genes in both mutants ( Fig. 6a ), indicating substantial overlap in their transcriptional regulatory networks. Figure 6. Open in a new tab Transcriptomic analysis reveals coregulated gene networks downstream of RNHL1 and TaEIL1 . (a) Venn diagram showing 1,103 co-upregulated and 1,545 co-downregulated genes in both rnhl1 and eil1 mutants. (b) Gene ontology (GO) enrichment analysis of coregulated genes showed distinct functional categories. (c) Heatmap analysis of selected hormone-related genes reveals consistent expression changes in both rnhl1 and eil1 mutants. (d, e) qRT-PCR validation confirming the expression levels of TaERF1 and TaGA3ox2 in young spike tissues in rnhl1 and eil1 (line #2) mutant plants. Data represent mean ± SD (n = 3; ** P < 0.01; Student's t -test). Gene ontology (GO) analysis showed that co-upregulated genes were significantly enriched in abscisic acid (ABA)- and ethylene-activated signaling pathways, as well as responses to hydrogen peroxide ( Fig. 6b ; Supplementary Data Set S4 ). In contrast, co-downregulated genes were associated with light responses, photosynthesis, auxin signaling, and GA metabolism ( Fig. 6b ; Supplementary Data Set S5 ). Strikingly, among these coregulated DEGs, we detected key hormonal regulators, including TaERF1 ( Ethylene-response-factor 1 , TraesCS2A02G417300), an ethylene-responsive transcription factor gene, and TaGA3ox2 ( Gibberellin 3-beta dioxygenase 2 , TraesCS3D02G124500), encoding a GA biosynthetic enzyme ( Fig. 6c ). Quantitative real-time PCR (qRT-PCR) validation confirmed that TaERF1 was transcriptionally activated in the young spike and internode tissues of both rnhl1 and eil1 mutants ( Fig. 6d ; Fig. S6a ), whereas the expression of TaGA3ox2 was significantly suppressed ( Fig. 6e ; Fig. S6b–c ). Consistent with the downregulation of TaGA3ox2 , we detected substantially lower levels of endogenous bioactive GAs—including GA 1 , GA 3 , GA 4 , and GA 7 —in the internodes of NIL- rnhl1 (a near isogenic line carrying the loss-of-function RNHL1 allele from the D subgenome) compared with NIL- RNHL1 (carrying the wild-type functional RNHL1 from the D subgenome; Fig. S7 ), suggesting that RNHL1 not only regulates TaGA3ox2 expression but also bioactive GA homeostasis in vivo. Together, these results demonstrate that RNHL1 and TaEIL1 coregulate overlapping transcriptional programs, converging on ethylene and GA pathways to modulate plant height and spike length. The coordinated repression of TaERF1 and activation of TaGA3ox2 suggests that these genes function synergistically to fine-tune hormone-mediated growth regulation. Direct association of TaERF1 by both RNHL1 and TaEIL1 In the canonical ethylene signaling pathway, EIN3/EIL1 family transcription factors are known to directly regulate ERF transcription factor genes ( Yang et al. 2015 ). To investigate whether TaERF1 serves as a direct target of both RNHL1 and TaEIL1, we conducted chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) using young spike tissues from RNHL1-OE (GFP-tagged) and TaEIL1-OE (Flag-tagged) transgenic plants. Immunoprecipitation with anti-GFP and anti-Flag antibodies revealed distinct binding patterns across the TaERF1-A/-B/-D gene loci: RNHL1-GFP showed significant enrichment specifically at the 5′-untranslated region (UTR; P3), while TaEIL1-Flag bound both the promoter region (P2) and 5′-UTR (P3) ( Fig. 7a ; Fig. S8a, b ), demonstrating their direct association with TaERF1 in vivo. Figure 7. Open in a new tab Synergistic regulation of TaERF1 expression by RNHL1 and TaEIL1. (a) Chromatin immunoprecipitation (ChIP) analysis reveals distinct but overlapping binding patterns of RNHL1 and TaEIL1 at the TaERF1 gene locus. While TaEIL1 shows enrichment at both promoter (P2) and 5′-UTR (P3) regions, RNHL1 specifically binds the 5′-UTR (P3). Quantitative PCR demonstrates significant enrichment of the 2 proteins at their respective binding sites. Data are mean ± SD (n = 3; ** P < 0.01; Student's t -test). nd, not detected. (b) Schematic representation of RNHL1 domain organization, highlighting the N-terminal hybrid-binding domain (HBD, blue) and C-terminal RNase H domain (green). Below are shown the 3 nucleic acid probes designed from the TaERF1 5′-UTR (P3) region: single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and RNA:DNA hybrid structures. (c) Electrophoretic mobility shift assays (EMSAs) demonstrate RNHL1's substrate specificity. The recombinant MBP-RNHL1 protein shows strong binding to RNA-containing substrates, with greatest affinity for the RNA:DNA hybrid (lane 6), intermediate binding to ssRNA (lane 5), and minimal interaction with ssDNA (lane 4). Control reactions with MBP alone (lanes 1 to 3) show no detectable binding, confirming the observed interactions are mediated by RNHL1. (d) EMSA analysis confirms the binding of TaEIL1 to the TaERF1 promoter region. Competition assays with unlabeled probes show dose-dependent reduction of TaEIL1-DNA complex formation, demonstrating binding specificity. (e) Dual-luciferase assays with a 2-kb TaERF1 promoter fragment reveal that RNHL1 and TaEIL1 synergistically suppress TaERF1 promoter activity (n = 8). (f, g) A truncated 500-bp promoter retains regulatory capacity of TaEIL1 on TaERF1 expression (F), while mutation of TaEIL1 binding sites (g) abolishes responsiveness (n = 3). (h) Deletion of either the IDR1 or IDR3 domain in RNHL1 compromises its ability to repress TaERF1 expression (n = 3). (i) Deletion of the RNase H domain in RNHL1 attenuates its repressive function on TaERF1 expression (n = 6). For (e) to (I), data are mean ± SD. Significant differences were determined by 1-way ANOVA with Tukey's HSD post hoc test ( P < 0.05). Different lowercase letters denote statistically significant differences between groups. Bioinformatic analysis using PlantTFDB ( https://planttfdb.gao-lab.org/prediction.php ) identified P2 as a putative TaEIL1-binding site, which was experimentally confirmed by electrophoretic mobility shift assay (EMSA) showing TaEIL1's specific interaction with a biotin-labeled P2 probe ( Fig. 7d ). Structural characterization of RNHL1 predicted it contains 2 functional domains, ie, an HBD domain (61 to 119 aa) and an RNase H domain (400 to 627 aa) ( Fig. 7b ; Fig. S9 ). Given that RNHL1 bound specificity to single-stranded RNA (ssRNA) and RNA:DNA heteroduplexes but not single-stranded DNA (ssDNA) ( Fig. S9 ), we designed ssDNA, ssRNA, and RNA/DNA hybrid probes derived from the P3 sequence ( Fig. 7b ). EMSA confirmed that RNHL1 directly binds the 5′-UTR sequence of TaERF1 in its ssRNA and RNA:DNA heteroduplex forms but not the ssDNA counterpart ( Fig. 7c ). Collectively, these results provide evidence that both RNHL1 and TaEIL1 directly bind the 5′-UTR of TaERF1, with TaEIL1 additionally targeting the promoter region. The distinct but overlapping binding patterns suggest a potential mechanism for their cooperative regulation of TaERF1 expression. Synergistic transcriptional repression of TaERF1 by RNHL1 and TaEIL1 To investigate the regulatory effects of RNHL1 and TaEIL1 on TaERF1 expression, we constructed a TaERF1-A pro-2kb ::LUC reporter, in which the firefly luciferase ( LUC ) gene was driven by the 2-kb native promoter of the A-subgenome-derived TaERF1 , while the Renilla luciferase ( REN ) gene, under the control of the 35S promoter, served as an internal control. Transient expression assays revealed that individual expression of either RNHL1-GFP or TaEIL1-GFP significantly reduced LUC activity, confirming their repressive effects on TaERF1 expression ( Fig. 7e ). Strikingly, co-expression of RNHL1-GFP and TaEIL1-GFP resulted in a stronger repression than either protein alone, indicating additive effects between RNHL1 and TaEIL1 in suppressing TaERF1 transcription ( Fig. 7e ). To further dissect the regulatory mechanism, we generated truncated reporter constructs: TaERF1-A pro-500bp ::LUC , containing a 500-bp TaERF1 promoter fragment, and TaERF1-A pro-500bp-mut ::LUC , in which the TaEIL1-binding site was mutated ( Fig. 7f, g ; Fig. S10a, b ). The TaERF1-A pro-500bp ::LUC reporter recapitulated the repression pattern observed with the full-length promoter in TaERF1-A pro-2kb ::LUC ( Fig. 7f ). However, mutation of the TaEIL1-binding site ( TaERF1-A pro-500bp-mut ::LUC ) not only partially impaired TaEIL1-mediated repression but also abolished the additive effect of RNHL1 and TaEIL1 ( Fig. 7g ). Together, these results suggest that the regulatory effects of RNHL1 may depend, at least in part, on TaEIL1 function. We next investigated whether the phase separation ability of RNHL1 contributes to its regulatory function. Disruption of RNHL1's IDRs through ΔIDR1 or ΔIDR3 mutations—which impair phase separation and RNHL1–TaEIL1 interaction—significantly weakened RNHL1-mediated repression of TaERF1 ( Fig. 7h ). Furthermore, deletion of the RNase H domain in RNHL1, which similarly disrupts phase separation of RNHL1 as observed in IDR1 and IDR3 deletions ( Fig. S1b, c ), also compromised the repression of TaERF1 ( Fig. 7i ). Taken together, these results demonstrate that RNHL1 and TaEIL1 act synergistically to repress TaERF1 expression. This cooperative repression depends on both the binding of TaEIL1 to the TaERF1 promoter and the phase separation capacity of RNHL1, underscoring the essential role of their physical association and functional interplay in the transcriptional regulation of TaERF1 . TaERF1 reduces plant height by repressing TaGA3ox2 expression To investigate the biological function of TaERF1 in plant height regulation, we generated GFP-tagged TaERF1 -overexpressing transgenic lines ( TaERF1-OE #1 and #2) in the Fielder background ( Fig. S11 ). Phenotypic characterization demonstrated that TaERF1-OE plants exhibited a modest but statistically significant reduction in plant height, primarily due to decreased internode length ( Fig. 8a–d ). Notably, spike length remained unchanged in transgenic plants ( Fig. 8b, c ), indicating that TaERF1 specifically regulates plant height without affecting spike elongation. Figure 8. Open in a new tab TaERF1 directly represses TaGA3ox2 to regulate wheat plant height. (a) Whole plants of WT and TaERF1 -overexpressing TaERF1-OE lines at the reproductive phase. Scale bar, 30 cm. (b) Spikes and peduncles/internodes of WT (left) and TaERF1-OE lines. Scale bar, 10 cm. Images were linearly adjusted for brightness and contrast. (c, d) Comparison of plant height in (C; n = 20), and spike length and internode lengths (INLs) in (D; n = 20) between WT and TaERF1-OE . (e) qRT-PCR shows the expression of TaGA3ox2 in TaERF1-OE lines relative to in WT (n = 3). (f) Dual-luciferase assays confirming TaERF1-mediated repression of TaGA3ox2 promoter activity (n = 5). (g) Transient assays show TaERF1 suppresses TaGA3ox2 promoter-driven luminescence (n = 6). (h) EMSA confirming direct binding of recombinant TaERF1 protein to the TaGA3ox2 promoter region (probe designed from 86 to 116 bp upstream of ATG). (i) ChIP-qPCR analysis confirming the in vivo enrichment of TaERF1 on the 5′-UTR of TaGA3ox2 gene. Top: Schematic of TaGA3ox2 promoter region showing primer sets (blue; P1-P4) for ChIP-qPCR; bottom: ChIP-qPCR analysis of TaERF1 enrichment in TaERF1-OE plants (n = 3). (j) TaGA3ox2 knockout mutant plants show severe dwarfing phenotype. Scale bar,10 cm. Images were linearly adjusted for brightness and contrast. In (C)-(G) and (I), data represent mean ± SD (* P < 0.05, ** P < 0.01; Student's t -test). Given that TaERF1 functions as a common downstream effector of both RNHL1 and TaEIL1, we hypothesized that TaERF1-mediated regulation of downstream signaling networks would partially overlap with those controlled by RNHL1 and TaEIL1. To test this hypothesis, we examined TaGA3ox2 expression as a molecular marker, since this gene showed strong repression in both rnhl1 and eil1 mutant lines ( Fig. 6e ). Consistent with our hypothesis, qRT-PCR analysis revealed that TaERF1 overexpression ( TaERF1-OE ) similarly downregulated TaGA3ox2 expression ( Fig. 8e ), mirroring the effects observed in the rnhl1 and eil1 mutant backgrounds. To further investigate the direct regulatory relationship, we constructed a TaGA3ox2-D pro ::LUC reporter containing the 2-kb promoter region from the D-subgenome TaGA3ox2 allele. Using both dual-luciferase assay ( Fig. 8f ) and transient transcriptional activation assay ( Fig. 8g ), we consistently observed that TaERF1-GFP expression significantly reduced TaGA3ox2-D promoter activity. These complementary in vitro findings demonstrate that TaERF1 overexpression phenocopies the genetic effects of RNHL1 / TaEIL1 loss-of-function mutations, particularly in regulating downstream targets such as TaGA3ox2 . More intriguingly, in silico analysis using PlantTFDB ( https://planttfdb.gao-lab.org/prediction.php ) revealed multiple high-confidence TaERF1-binding motifs in the promoters of all 3 TaGA3ox2 homoeologs (A, B, and D subgenomes; Fig. 8f ; Fig. S12a ), strongly suggesting TaGA3ox2 as a direct transcriptional target of TaERF1. Experimental validation through EMSA confirmed that purified His-TaERF1 protein specifically binds to the predicted cis-element in vitro ( Fig. 8h ). Further supporting this regulation in planta , ChIP-qPCR analysis of young spike tissues from TaERF1-OE plants demonstrated significant TaERF1-GFP enrichment across all tested genomic regions of all 3 TaGA3ox2 homoeologs—including promoter segments (P1, P2), 5′-UTR (P3), and coding region (P4) —with peak binding observed at the 5′-UTR region (more than 6-fold enrichment over wild-type controls; Fig. 8i ; Fig. S12b ). These complementary biochemical and genetic approaches collectively establish TaERF1 as a direct regulator of TaGA3ox2 expression through physical association with its genomic loci. TaGA3ox2 knockout recapitulates the dwarfing phenotype To establish the functional connection between TaERF1-mediated TaGA3ox2 repression and plant height reduction, we generated CRISPR-Cas9 knockout lines ( Taga3ox2 , #1, #2 and #3; Fig. S13 ). Strikingly, loss-of-function mutations in TaGA3ox2 resulted in extreme dwarfism, with near-complete inhibition of culm elongation ( Fig. 8j ). Together with our biochemical evidence demonstrating TaERF1's direct repression of TaGA3ox2 expression, these genetic findings establish a mechanistic pathway whereby TaERF1 regulates wheat culm development through modulation of GA biosynthesis via TaGA3ox2 . Discussion RNHL1 represents an RNase H-like protein integrating ribonuclease activity and transcriptional regulation The ribonuclease H-like (RNHL) protein family comprises diverse members with distinct structural and functional features ( Majorek et al. 2014 ). Canonical RNHL proteins function as endonucleases that specifically cleave RNA within RNA:DNA hybrids, playing pivotal roles in R-loop [RNA:DNA hybrid and a displaced single-stranded DNA (ssDNA)] resolution and genome stability maintenance ( Cerritelli and Crouch 2009 ; Ohle et al. 2016 ; Yang et al. 2017 ). Structurally, RNase H1 proteins contain 2 conserved domains: An N-terminal hybrid-binding domain (HBD) for nucleic acid recognition and a C-terminal RNase H domain mediating RNA degradation. Intriguingly, the wheat dwarfing gene Rht8 was recently identified as encoding an RNase H-like protein (annotated as RNHL1), though its biochemical functions remained uncharacterized ( Chai et al. 2022 ; Xiong et al. 2022 ). Here, we systematically investigated RNHL1's molecular functions through in vivo and in vitro analyses. Sequence alignment confirmed the presence of a conserved RNase H domain (400 to 627aa). Functional assays demonstrated that RNHL1 binds RNA:DNA hybrids ( Fig. 7c ; Fig. S9b ) and degrades the RNA component within these structures ( Fig. S9c ). Strikingly, deletion of the RNase H domain (RNHL1mut) abolished catalytic activity ( Fig. S9c ), unequivocally establishing the C-terminal domain's essential role in RNA cleavage. These results define RNHL1 as a bona fide RNase H-like protein with dual nucleic acid-binding and degradation capabilities, broadening the functional spectrum of this protein family. Phase separation orchestrates the multifunctional integration of RNHL1 Beyond its canonical enzymatic activity, we discovered that RNHL1 undergoes LLPS to form biomolecular condensates ( Fig. 2f ). Bioinformatics analysis identified 3 high-confidence intrinsically disordered regions (IDR1–3) within the N-terminus ( Fig. 4a ). Truncation studies demonstrated that IDR1, IDR3, and the PrLD motif in IDR2 are all critical for condensate formation ( Fig. 4b ), while the G-rich domain within IDR2 is essential for its nuclear localization ( Fig. 4c ). Interestingly, the structured RNase H domain—despite lacking predicted disordered sequences ( Fig. 3a )—also proved necessary for stabilizing RNHL1's phase separation behavior ( Fig. S1b, c ), although no putative IDR region was predicted within this region ( Fig. 3a ). We speculate that this contribution may arise from intramolecular interactions between the RNase H domain and IDRs, which could modulate the LLPS propensity of RNHL1; however, this hypothesis remains to be validated experimentally. Notably, RNHL1 condensates do not exhibit typical spherical morphology but instead form fine punctate structures with notably small diameters. Previous studies have indicated that the size and shape of biomolecular condensates—ranging from spherical assemblies to mesh-like networks—can be highly dynamic and functionally significant, influencing properties such as molecular exchange and condensate maturation ( Ma et al. 2021 ; Gao et al. 2022 ). Whether the morphology of RNHL1 condensates varies in response to intra- or extracellular cues represents an intriguing direction for future studies, which may further elucidate the biochemical and functional properties of RNHL1. In particular, the agronomic importance of Rht8 ( RNHL1 ) in drought-prone areas has been well confirmed ( Worland and Law 1986 ; Gasperini et al. 2012 ), further analysis linking RNHL1 LLPS dynamics to drought or other abiotic stress adaptation would significantly enhance the impact of the current work. Importantly, the phase separation capability facilitates the interaction of RNHL1 with TaEIL1, mirroring the established role of biomolecular condensates in organizing functional cellular compartments ( Hyman et al. 2014 ). Remarkably, our studies reveal a striking functional division within distinct regions of RNHL1: while the C-terminus mediates nucleic acid binding/degradation, the N-terminus drives protein-protein interactions (eg, with TaEIL1) via phase separation. Transient transcriptional assays demonstrated that both RNHL1's intrinsic repressive activity and its synergistic repression with TaEIL1 (targeting genes like TaERF1 ) require an intact N-terminal region ( Fig. 7h ). These findings illustrate how spatially segregated protein domains can cooperatively execute complex biological functions through modular activity integration. A “RNHL1–TaEIL1–TaERF1–TaGA3ox2” transcriptional regulatory cascade Previous studies have shown that loss-of-function mutations in RNHL1 in wheat (representing the plant height reduction conferred by the Rht8 locus) or its orthologs in maize and Arabidopsis consistently result in semidwarf architecture and shortened inflorescences ( Chai et al. 2022 ; Xiong et al. 2022 ), indicating a conserved role for RNHL1 in promoting internode/cell elongation across plant species. However, the molecular mechanism underlying RNHL1-mediated cell elongation remains unclear. In this study, we demonstrate that RNHL1 physically interacts with TaEIL1, a transcription factor involved in ethylene response regulation ( Fig. 2 ). Furthermore, RNHL1 and TaEIL1 form biomolecular condensates, suggesting they function as a cooperative complex to regulate downstream signaling networks, including ABA-, ethylene-, and auxin-activated pathways. These findings support the notion that RNHL1 broadly influences multiple phytohormone signaling cascades, primarily through direct association with key regulators such as TaEIL1. Notably, in rice, SMALL GRAIN2 (SG2), another RNase H-like protein, interacts with GSK2, a repressor of brassinosteroid signaling, to modulate plant stature and grain size ( Huang et al. 2022 ), further underscoring the functional versatility of this protein family. By analyzing genes coregulated by RNHL1 and TaEIL1, we identified TaERF1 , encoding an AP2/ERF transcription factor, as a direct target. Both RNHL1 and TaEIL1 suppress TaERF1 expression in vitro and in vivo ( Fig. 7 ). Intriguingly, RNHL1 primarily binds the 5′-UTR of TaERF1 , whereas TaEIL1 localizes to its promoter and 5′-UTR, with overlapping occupancy in the 5′-UTR. Although RNHL1 and TaEIL1 interact via phase separation, they act additively—rather than independently—to regulate TaERF1 expression ( Fig. 7f–h ). We propose that TaEIL1, as a canonical transcription factor, represses TaERF1 by binding to cis-elements within the promoter of TaERF1 ; in contrast, RNHL1—an RNase H-like protein—likely targets R-loops within the 5′-UTR of TaERF1 , leading to degradation of newly synthesized pre-mRNAs associated with these structures and consequently modulating TaERF1 mRNA abundance in a manner independent of cis-elements ( Wahba et al. 2011 ; Niehrs and Luke 2020 ; Shibata et al. 2020 ; Zhou et al. 2022 ). These distinct regulatory mechanisms are integrated through the physical association of RNHL1 and TaEIL1 within shared biomolecular condensates ( Fig. 2 ). What is the biological significance of this condensation? Despite their divergent biochemical activities, RNHL1 and TaEIL1 converge on common downstream genes and signaling networks ( Fig. 6 ). Their co-condensation into a shared phase-separated compartment represents a sophisticated regulatory mechanism that enhances transcriptional control efficiency. Although RNHL1 and TaEIL1 do not strictly depend on each other for phase separation, their colocalization creates a synergistic effect—enabling 2 otherwise independent regulatory events (the RNA-processing activity of an RNase H1-like protein and the DNA-binding capacity of a classical transcription factor) to be coordinated within a single biomolecular condensate. This spatial integration not only increases local protein concentration, thereby enhancing their individual activities, but also ensures precise and synchronized regulation of shared downstream pathways ( Hnisz et al. 2017 ). Thus, the RNHL1–TaEIL1 complex exemplifies how phase-separated multimolecular assemblies can amplify transcriptional regulation—not only by facilitating activation ( Boija et al. 2018 ; Sabari et al. 2018 ) or repression in isolation but also by integrating both functions within a unified condensate to achieve more efficient and dynamic gene expression control. Conserved GA-centric regulation of plant height by ERF transcription factors in cereals In rice, ethylene-responsive AP2/ERF transcription factors such as SNORKEL1/2 ( SK1/2 ), SUBMERGENCE 1A ( SUB1A ), and OsEATB regulate internode elongation in opposing ways ( Fukao et al. 2006 ; Xu et al. 2006 ; Hattori et al. 2009 ; Qi et al. 2011 ), underscoring the complex role of ethylene-responsive ERFs in culm development. For instance, SK1/2 enhances rice adaptability to deepwater conditions by promoting dramatic internode elongation via GA ( Hattori et al. 2009 ), whereas SUB1A suppresses internode elongation during submergence, functioning as a plant height repressor ( Fukao et al. 2006 ; Xu et al. 2006 ). SUB1A achieves this inhibition by dampening ethylene-promoted GA signaling, largely through the accumulation of GA signaling repressors SLENDER RICE-1 (SLR1) and SLR1-LIKE1 (SLRL1) ( Fukao and Bailey-Serres 2008 ). Similarly, OsEATB (for ERF protein associated with tillering and branching) restricts internode elongation by downregulating the GA biosynthetic gene ENT-COPALYL DIPHOSPHATE SYNTHASE 2 ( OsCPS2 ) ( Qi et al. 2011 ). Additionally, OsAP2-39 overexpression reduces biomass and seed yield by activating the ABA biosynthetic gene OsNCED1 and the GA catabolic gene ELONGATED UPPERMOST INTERNODE1 ( OsEUI1 ) ( Yaish et al. 2010 ). In wheat, TaERF1 functionally mirrors SUB1A and OsEATB as a negative regulator of plant height ( Fig. 8 ). Like SUB1A and OsEATB —which impair GA biosynthesis or sensitivity in rice ( Fukao et al. 2006 ; Qi et al. 2011 )— TaERF1 directly represses the GA biosynthetic gene TaGA3ox2 , thereby inhibiting bioactive GA production ( Fig. 8e–g ). This aligns with the observed suppression of TaGA3ox2 expression in Rht8 -carrying wheat ( Chai et al. 2022 ). Strikingly, whether acting as positive ( SK1/2 ) or negative ( SUB1A , OsEATB , and TaERF1 ) regulators, these ERF genes ultimately converge on GA metabolism or signaling, establishing GA as a central regulatory node for plant height in cereals. In summary, our study elucidates a “RNHL1–TaEIL1–TaERF1–TaGA3ox2” regulatory module that integrates ethylene signaling with GA metabolism to shape wheat plant architecture ( Fig. 9 ). These findings uncover a previously unrecognized mechanism of biomolecular condensate-mediated growth regulation in cereals. From a translational perspective, our results suggest multiple strategies for precise manipulation of plant architecture, including: (1) engineering RNHL1 condensate properties through modification of its IDRs; (2) tuning TaERF1 expression or transcriptional activity; and (3) directly regulating TaGA3ox2 expression. Further efforts to generate rnhl1 eil1 or rnhl1 eil1 erf1 multiple mutants will help clarify the genetic relationships among these pathway components and facilitate their pyramiding in a common genetic background—paving the way for breeding high-yielding wheat varieties with optimized hormonal coordination. Further investigation should also focus on the environmental sensitivity of the RNHL1–TaEIL1 module, its interplay with other hormone pathways, and its evolutionary conservation across cereal species. Figure 9. Open in a new tab Proposed working model of the RNHL1–TaEIL1–TaERF1–TaGA3ox2 regulatory module in wheat. This study reveals that the ribonuclease H-like protein RNHL1 interacts with ethylene-responsive transcription factor TaEIL1 to form functional biomolecular condensates, which regulate downstream gene expression. (a) In wild-type plants: The RNHL1–TaEIL1 module represses TaERF1 expression, thereby releasing its inhibition on the gibberellin biosynthesis gene TaGA3ox2 . This leads to increased GA production, promoting plant growth and resulting in taller plants that are more prone to lodging. (b) In RNHL1–TaEIL1 deficient plants: The loss-of-functional RNHL1–TaEIL1 module results in upregulation of TaERF1, which strongly suppresses TaGA3ox2 expression. Consequently, reduced GA levels lead to shorter plant stature and improved lodging resistance. This model demonstrates how ethylene signaling integrates with GA metabolism through the RNHL1–TaEIL1–TaERF1–TaGA3ox2 regulatory cascade to control wheat plant architecture. Blunt arrows (┴) in the figure indicate inhibition, while sharp arrows (↗) indicate promotion effects; the thickness of the arrows corresponds to the relative strength of inhibition/promotion effects. Materials and methods Plant materials and growth conditions The near-isogenic recombinant lines NIL- RNHL1 (previously NIL- rht8 ) and NIL- rnhl1 (previously NIL- Rht8 ) were described in detail in our previous report ( Chai et al. 2022 ). Genetic transformations and gene-editing experiments were conducted using the spring hexaploid wheat ( Triticum aestivum ) cultivar “Fielder.” Transgenic wheat plants were grown in greenhouse facilities at China Agricultural University (Beijing, P. R. China) under controlled environmental conditions with 60% relative humidity, day/night temperatures maintained at 25/20 °C, and illumination provided by Master GreenPower (Philips) lights at 3,000 lux intensity. For transient expression assays, N. benthamiana plants were cultivated in separate greenhouse compartments under a 16-h photoperiod at constant 22 °C temperature with supplemental lighting providing 150 μmol m −2 s −1 photosynthetic photon flux density. Gene transformation in bread wheat For overexpression constructs, full-length coding sequences (CDS) of RNHL1 , TaEIL1 , and TaERF1 were amplified from wheat cDNA and cloned into the plant binary vector pWMB110 using Bam HI-restriction sites. CRISPR/Cas9-mediated knockout constructs were designed by selecting specific sgRNA targets within the coding sequences of RNHL1 (A/B/D homoeologs), TaEIL1 (A/B/D homoeologs), and TaGA3ox2 (A/B/D homoeologs) using the E-CRISPR design platform ( http://www.e-crisp.org ). The MT1T2 vector was modified by inserting these sgRNA sequences through PCR amplification with target-specific primers, followed by BsaI digestion and T4 ligase-mediated insertion into the pBUE411 CRISPR/Cas9 backbone vector. All constructs were transformed into Fielder via Agrobacterium tumefaciens -mediated transformation using strain EHA105 (Weidi Bio, AC1010). Transgenic T 0 plants were screened through molecular characterization: overexpression lines were verified by both genomic DNA PCR to confirm transgene integration and qRT-PCR to assess transcript levels, while knockout lines were analyzed by DNA sequencing to identify targeted mutations in the respective gene loci. Phenotypic evaluation and statistical analysis Phenotypic characterization was conducted following grain filling, when plant height had stabilized under greenhouse conditions. A comprehensive assessment of agronomic traits was performed on transgenic lines, including: (1) total plant height (from soil surface to panicle apex), (2) spike length, and (3) internode lengths (specifically measuring the peduncle and subsequent 4 basal internodes, designated as internodes I–IV from apex to base). For each transgenic line, measurements were collected from 20 independent T 3 plants with 3 biological replicates. Statistical analysis was performed using 1-way ANOVA with Tukey's post hoc test ( P < 0.05) to determine significant differences between transgenic and wild-type plants. Y2H assay To identify proteins interacting with RNHL1, we performed a yeast two-hybrid screening using a full-length CDS of RNHL1 lacking autoactivation activity as bait. The corresponding coding sequence was cloned into the pGBKT7 vector (Clontech Laboratories, Inc., PT3024-1) and transformed into Y2HGold yeast strain for bait validation. A cDNA library prepared from Fielder wheat seedlings was introduced into Y187 yeast strain for prey construction. Following the manufacturer's protocol (Matchmaker™ Gold Yeast Two-Hybrid System), the bait and prey strains were mated and selected on high-stringency quadruple dropout medium (SD/-Ade/-His/-Leu/-Trp) to eliminate false positives. After selection, positive colonies were isolated and the interacting prey plasmids were rescued for sequencing identification. Split-luciferase complementation imaging (split-LCI) assay The CDS of RNHL1 and its mutant variants, along with TaEIL1 , were amplified and individually cloned into the KpnI/SalI -digested pCAMBIA1300-nLUC ( nLUC ) and KpnI/BamHI -digested pCAMBIA1300-cLUC ( cLUC ) vectors, respectively. The resulting constructs were transformed into Agrobacterium tumefaciens strain GV3101. Bacterial cultures harboring the nLUC - and cLUC -fusion constructs were co-infiltrated into N. benthamiana leaves. Approximately 36 h post-infiltration, the LUC activity in the infiltrated regions was quantified using a NightSHADE LB 985 (Berthold Technologies) plant imaging system. For each assay, 4 independent leaf samples were analyzed for statistical validation. Western blotting Total cellular proteins were extracted by direct lysis in 2× Laemmli buffer (100 mM Tris-HCl [pH 6.8], 20% [v/v] glycerol, 4% [w/v] SDS, 2% [v/v] β-mercaptoethanol) and denatured by boiling at 100 °C for 5 min. Protein samples were loaded onto 10% or 12% SDS-polyacrylamide gel (SDS-PAGE). Electrophoresis was initiated at 80 V for 30 min, followed by 120 V for approximately 1 h. Following electrophoresis, proteins were transferred onto a nitrocellulose membrane in transfer buffer at 300 mA for 2 h at 4 °C. The membrane was blocked with 5% (w/v) nonfat dry milk in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 h at room temperature with gentle agitation. For immunodetection, the membrane was incubated at 4 °C with primary antibodies, including anti-β-actin antibody (CW0096, CoWin Biotech Corp), anti-GFP antibody (ab32146, Abcam), anti-Flag antibody (F1804, Sigma-Aldrich) with gentle shaking. After TBST washes, the membrane was incubated with HRP-conjugated secondary antibodies, including antirabbit IgG-HRP, and antimouse IgG-HRP (Sigma-Aldrich) for 40 min at room temperature. Following 3 additional TBST washes (5 min each), protein bands were visualized using Immobilon Western chemiluminescent HRP substrate (WBKLS, Millipore) under a fluorescence chemiluminescence imaging system (ChemiScope 6100). Co-IP assay in wheat protoplasts For the Co-IP assay, the full-length CDS of RNHL1 and TaEIL1 were cloned into plant expression vectors S1300-GFP and 1300-Flag to generate C-terminal fusions with GFP and Flag tags, respectively. Approximately 16 h after transfection, total protein was extracted from the protoplasts using SDS-loading buffer (200 mM Tris-HCl, pH 6.8, 40% glycerol, 8% SDS, and 20% β-mercaptoethanol). TaEIL1-Flag was immunoprecipitated using Anti-Flag Magnetic Beads (M8823, Millipore). The immunoprecipitated proteins were then detected by immunoblotting with anti-Flag (1:5,000; F3165, Sigma-Aldrich) and anti-GFP (1:5,000; ab32146, Abcam) antibodies. In vitro pull-down assay The RNHL1 CDS was amplified and cloned into the EcoRI/XbaI sites of the pMAL-c2x vector (NEB, E8000S), generating an MBP-RNHL1 fusion protein. For His-tagged TaEIL1, the TaEIL1 CDS was cloned into the EcoRI/SalI sites of pET-32a (Novagen). Recombinant proteins were expressed in Escherichia coli Rosetta (DE3) cells induced with 0.5 mM IPTG in lysogeny broth (LB) overnight at 16 °C and purified as previously described ( Han et al. 2022 ). For the pull-down assay, bacterial lysates containing MBP-tagged or His-tagged proteins were incubated with high-affinity amylose resin (NEB, E8021L) in binding buffer (25 mM Tris-HCl [pH 7.5], 50 mM NaCl, 1 mM DTT) for 5 h at 4 °C with gentle rotation. The resin was washed 6 times with 1 mL wash buffer (binding buffer + 1% [v/v] Triton X-100) and eluted in 100 μL 2× Laemmli buffer (100 mM Tris-HCl [pH 6.8], 20% [v/v] glycerol, 4% [w/v] SDS, 10% [v/v] β-mercaptoethanol) by boiling at 100 °C for 5 min. Eluted proteins were resolved by 10% SDS-PAGE and analyzed by immunoblotting using anti-MBP (1:5,000; TransGen Biotech, HT701-01) or anti-His (1:5,000; TransGen Biotech, HT501-01) antibodies. BiFC assay The CDS of RNHL1 was cloned into the BamHI/XbaI sites of the C-terminal yellow fluorescent protein (cYFP) vector, while the full-length TaEIL1 was cloned into the BamHI/XbaI sites of the N-terminal YFP (nYFP) vector. The recombinant constructs were transformed into Agrobacterium tumefaciens strain GV3101. Bacterial cultures (OD 600 = 0.5) containing the nYFP- and cYFP-fusion constructs were co-infiltrated into 4-week-old N. benthamiana leaves. After 48 h of incubation under normal growth conditions (22 °C, 16/8 h light/dark cycle), YFP fluorescence signals were detected at excitation/emission wavelengths of 488 nm/526 nm (YFP) and 546 nm/576 nm (autofluorescence control) using a LSM880 confocal laser scanning microscope (Carl Zeiss, Germany) with a 20× objective lens. Three independent biological replicates were performed. Subcellular localization analysis The CDS of RNHL1 and its mutant variants, along with TaEIL1 , were cloned into pCAMBIA1300-GFP to generate C-terminal GFP fusion constructs, while TaEIL1 was separately cloned into pCAMBIA1300-mCherry to create an mCherry-tagged version. These constructs were transformed into Agrobacterium tumefaciens strain GV3101 and transiently expressed in N. benthamiana epidermal cells either individually or in combination through agroinfiltration (OD 600 = 0.5). After 48 h post-infiltration, fluorescence was examined using an LSM880 confocal microscope (Carl Zeiss) with GFP excitation at 488 nm (emission 500 to 550 nm) and mCherry excitation at 546 nm (emission 560 to 620 nm). All experiments were repeated in 3 independent biological replicates with consistent results. In vitro phase-separation assay Purified MBP-RNHL1-GFP and MBP-EIL1-mCherry fusion proteins were incubated with TEV protease overnight at 4 °C to remove the MBP tags, followed by collection of the cleaved RNHL1-GFP and EIL1-mCherry proteins. The purified proteins were then mixed in phase-separation buffer (25 mM HEPES pH 7.4, 150 mM NaCl, 1 mM DTT) containing 10% (w/v) PEG8000 (Sigma-Aldrich) as a crowding agent to promote droplet formation. The reaction mixture was immediately transferred to a 35 mm glass-bottom confocal dish (15 mm diameter, MatTek Corporation) and allowed to equilibrate for 5 min at room temperature. Liquid–liquid phase separation was monitored in real time using a Zeiss LSM 880 confocal laser-scanning microscope equipped with a 63× oil-immersion objective, with GFP excitation at 488 nm (emission collected at 500 to 550 nm) and mCherry excitation at 561 nm (emission collected at 570 to 620 nm). FRAP analysis FRAP experiments were conducted in living cells using a Zeiss LSM 880 confocal laser-scanning microscope equipped with a 40× water immersion objective. A defined region of interest containing fluorescent protein condensates was photobleached using a 488 nm laser at 100% power. Time-lapse images of fluorescence recovery were taken every 3 s for at least 60 s. Fluorescence recovery was monitored at 488 nm excitation with emission collected between 500 and 550 nm. Quantitative analysis of fluorescence recovery kinetics was performed using Zeiss ZEN 3.0 software for initial processing, followed by detailed intensity measurements. Three independent biological replicates were analyzed, with at least 10 individual cells measured per experiment to ensure statistical significance. Total RNA extraction and qRT-PCR Total RNA was isolated from frozen tissue samples using TRIzol Reagent (15,596,018, Thermo Fisher Scientific) following the manufacturer's protocol, with an additional DNase I (EN0521, Thermo Fisher Scientific) treatment to eliminate genomic DNA contamination. RNA concentration and purity were determined spectrophotometrically (NanoDrop One, Thermo Fisher Scientific). First-strand cDNA synthesis was performed using 1 μg total RNA with HiScript II Q RT SuperMix (R223, Vazyme Biotech) in a 20 μL reaction volume under the following conditions: 50 °C for 15 min followed by 85 °C for 5 s. qRT-PCR was carried out using AceQ qPCR SYBR Green Master Mix (Q121, Vazyme Biotech) on a QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific) with the following cycling parameters: 95 °C for 3 min; 40 cycles of 95 °C for 20 s, 60 °C for 20 s, and 72 °C for 20 s; followed by a melt curve analysis (65 to 95 °C, increment 0.5 °C/s). Gene-specific primers were listed in Supplementary Data Set S6 . β-actin was used as the reference gene for normalization, and relative gene expression was calculated using the 2-ΔΔCt method. Three biological replicates were analyzed for each sample, with each replicate containing 3 technical replicates. RNA-seq analysis Young spikes at the W6.5 developmental stage were collected ( Waddington et al. 1983 ). Total RNA was extracted using TRIzol Reagent (15,596,018, Thermo Fisher Scientific). RNA integrity was verified by (specify method, eg, Agilent Bioanalyzer) with all samples showing RIN values >6. RNA-seq libraries were prepared using the (specify kit) and sequenced on an Illumina NovaSeq 6,000 platform (Illumina, San Diego, CA) to generate 150-bp paired-end reads. Raw reads were quality-trimmed using Trimmomatic (v0.39), and high-quality clean reads were aligned to the Triticum aestivum Chinese Spring reference genome (IWGSC RefSeq v1.1) using STAR (v2.7.10a) with default parameters ( Dobin et al. 2013 ). Differential expression analysis was performed using DESeq2 (v1.38.3) with thresholds of |log 2 (fold change)| ≥ 1 and P value <0.05 ( Love et al. 2014 ). Significantly enriched GO categories of up- and downregulated genes [adjusted P value (FDR) < 0.05] were identified using ClusterProfler ( Yu et al. 2012 ). Visualization was performed using ggplot2 (v3.4.2), including heatmaps of normalized expression values (z-scores) for differentially expressed genes (DEGs) and bubble charts displaying significantly enriched GO terms ( Ito and Murphy 2013 ). ChIP-qPCR assays ChIP was performed using approximately 2 g of young spike tissues per biological replicate following an established protocol ( Yang et al. 2016 ) with modifications. Tissues were vacuum-infiltrated with 1% formaldehyde in PBS buffer for 10 min at room temperature for cross-linking, followed by quenching with 0.25 M glycine for 5 min. Chromatin was isolated and sonicated using a Bioruptor Plus (Diagenode) to obtain DNA fragments averaging 100 to 1,000 bp in size. Immunoprecipitations were performed overnight at 4 °C with either anti-Flag antibody (M185 to 3L, MBL; 5 μg per reaction), anti-GFP antibody (ab290; 5 μg per reaction), or no-antibody control, using Magna ChIP™ Protein A + G Magnetic Beads (16 to 663). Beads were sequentially washed with (1) low-salt buffer (50 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100), (2) high-salt buffer (500 mM NaCl), (3) LiCl buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.25 M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate), and (4) TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Immunoprecipitated DNA was eluted in elution buffer (1% SDS, 0.1 M NaHCO 3 ) and reverse cross-linked at 65 °C overnight. DNA was analyzed by qRT-PCR using AceQ qPCR SYBR Green Master Mix (Q121, Vazyme) on a QuantStudio 6 Flex system (Thermo Fisher Scientific). Specific primer sets (Supplemental Data 1) were designed to amplify: (1) the TaERF1 promoter region (GFP and Flag immunoprecipitations) and (2) the TaGA3ox2 promoter region (GFP immunoprecipitation). Enrichment was calculated as percentage of input using the 2-ΔΔCt method, normalized to the no-antibody control samples. Three biological replicates were analyzed. Electrophoretic mobility shift assay Recombinant His-tagged TaEIL1 and TaERF1 proteins were expressed and purified for DNA-binding analysis, while RNHL1 protein was examined for RNA:DNA hybrid-binding activity. For DNA-binding assays, biotin-labeled double-stranded DNA probes ( Supplementary Data Set S6 ) were prepared by annealing complementary oligonucleotides in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 100 mM NaCl through heating to 95 °C followed by gradual cooling. Binding reactions containing 5 ng labeled probe, purified protein (0 to 500 nM), 1× binding buffer (10 mM Tris, 50 mM KCl, 1 mM DTT, pH 7.5), 5% glycerol, 5 mM MgCl 2 , 0.05% NP-40, and 50 μg/mL poly (dI-dC) competitor DNA were incubated at 25 °C for 20 min. For RNHL1 binding to RNA:DNA hybrids, equimolar biotin-labeled single-stranded DNA and RNA were first annealed to form heteroduplexes before incubation with protein. Protein-nucleic acid complexes were resolved on 6% native polyacrylamide gels in 0.5× TBE buffer at 100 V for 60 min at 4 °C, then transferred to positively charged nylon membranes for chemiluminescent detection using the LightShift EMSA Kit (Thermo Fisher). Specificity was confirmed through competition assays with 50 to 200-fold molar excess of unlabeled probes (competitors). Three independent experiments were performed with consistent results. Transcriptional activation assays in N. benthamiana Transcriptional activation activity was investigated using both single (firefly luciferase) and dual-luciferase reporter systems in N. benthamiana leaves. For the firefly luciferase system, the 2-kb promoter region of TaGA3ox2 was cloned into pGWB35 vector ( Nakagawa et al. 2007 ) via Gateway® recombination to create TaGA3ox2pro::LUC reporter, while effector constructs expressing TaERF1-GFP fusion were generated in pCAMBIA1300-GFP . The dual-luciferase system employed pGreenII 0800-LUC vectors ( Hellens et al. 2005 ) containing 2-kb or 500-bp promoter fragments of TaERF1 and TaGA3ox2 as reporters, with effector constructs expressing RNHL1 variants, TaEIL1 or TaERF1 as GFP fusions in pCAMBIA1300-GFP . All constructs were transformed into Agrobacterium tumefaciens GV3101, and bacterial cultures (OD 600 = 0.5) containing reporter:effector mixtures (1:1 ratio) were co-infiltrated into 4-week-old N. benthamiana leaves . After 36 h incubation under standard growth conditions (22 °C, 16/8 h light/dark), transcriptional activity was assessed either by quantifying firefly luciferase activity using NightSHADE LB 985 imaging system or by performing dual-luciferase assays where firefly LUC activity was normalized to 35S-driven Renilla LUC activity. Each experiment included at least 10 individual leaves and 3 biological replicates. All primer sequences used for construct generation are listed in Supplementary Data Set S6 . Statistical analysis Statistical analyses were performed to evaluate differences between genotypes and treatments using appropriate parametric and non-parametric methods. For pairwise comparisons, a 2-tailed Student's t -test was employed to assess statistical significance. When analyzing multiple genotypes or treatment groups, data distribution was first evaluated using the Shapiro–Wilk normality test. For normally distributed data, one-way analysis of variance (ANOVA) was conducted followed by Tukey's honestly significant difference (HSD) post hoc test for multiple comparisons. In cases where the normality assumption was violated, the non-parametric Kruskal–Wallis test was performed instead, with Dunnett's post hoc test used for subsequent group comparisons. Statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software). See source data for detailed statistical parameters. Measurement of endogenous GA levels To compare the endogenous GA levels between the NIL- RNHL1 and NIL- rnhl1 lines, the first internode below the spike was collected from plants at the booting stage, immediately frozen in liquid nitrogen, and ground into a powder. Each sample, comprising a pool of 15 individuals, was divided into 3 technical replicates for analysis, with 4 independent biological replicates performed. Briefly, endogenous GAs were extracted with 90% aqueous methanol spiked with deuterium-labeled GA internal standards. The extracts were then purified and fractionated using MAX and MCX solid-phase extraction cartridges (Waters Corporation, Milford, USA) prior to UPLC-MS/MS analysis. The quantification of GAs was performed using a UPLC system (Waters, Milford, MA, USA) coupled to a QTRAP 6,500 hybrid mass spectrometer (AB SCIEX, Foster City, CA) equipped with an electrospray ionization source. 1-aminocyclopropane-1-carboxylic acid (ACC) sensitivity assay A 100 mM aqueous stock solution of ACC (Sigma-Aldrich) was prepared. Two-day-old seedlings of the Fielder cultivar, the eil1 mutant (#1), and the TaEIL1-OE line (#1) were subjected to treatment by soaking in aqueous ACC solutions of specified concentrations. Seedlings soaked in an equivalent volume of water served as the mock control. Seedling and root lengths were measured 7 d post-treatment. All experiments were performed with 3 independent biological replicates. Supplementary Material koag081_Supplementary_Data koag081_supplementary_data.zip (8.1MB, zip) Acknowledgments We thank Na Song (China Agricultural University) for technical assistance with wheat transformation. Contributor Information Chaoqun Dong, State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China. Xiliu Cheng, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R. China. Meng Yuan, State Key Laboratory of Crop Stress Resistance and High-Efficiency Production, College of Agronomy, Northwest A&F University, Yangling 712199, P. R. China. Zhe Zhang, State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China. Yiqing Wang, State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China. Jing Liu, State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China. Long Song, State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China. Haoran Wang, State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China. Yujie Jiang, State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China. Xiangqing Liu, State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China. Jianhui Wu, State Key Laboratory of Crop Stress Resistance and High-Efficiency Production, College of Agronomy, Northwest A&F University, Yangling 712199, P. R. China. Yingyin Yao, State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China. Zhongfu Ni, State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China. Qixin Sun, State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China. Lingling Chai, State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China. Jie Liu, State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing 100193, P. R. China. Author contributions Jie L., Q.S., and Z.N. conceived and designed the study. Jie L. and L.C. supervised the research. C.D. performed the majority of the experiments. C.D. and X.C. performed phase separation analyses. M.Y., Jing L., L.S., and H.W. carried out genotypic and phenotypic characterization of wheat materials. Z.Z. performed electrophoretic mobility shift assays. Y.W. analyzed the transcriptome data. C.D., X.C., M.Y. Y.J., X.L., J.W., and Y.Y. contributed to data analysis and interpretation. J.L., C.D., and L.C. wrote the manuscript. Q.S. and Z.N. supervised the project and edited the manuscript. All authors reviewed and approved the final version. Supplementary material Supplementary material is available at The Plant Cell online. Funding This research was supported by grants from the National Natural Science Foundation of China (32372120, 32101767), Pinduoduo-China Agricultural University Research Fund (PC2023A01003), the Biological Breeding-National Science and Technology Major Project (2023ZD0402402), the National Key Research and Development Program of China (2022YFF1003401), and the 2115 Talent Development Program of China Agricultural University. Data availability All genes' information in this article can be found in the GeneBank/EMBL data libraries ( http://plants.ensembl.org/index.html ) under the following accession numbers: RNHL1 (TraesCS2A02G059900, TraesCS2B02G073600, TraesCSU02G024900), TaEIL1 (TraesCS4A02G12940, TraesCS4B02G175100, TraesCS4D02G177000), TaERF1 (TraesCS2A02G417300, TraesCS2B02G436300, TraesCS2D02G414600), TaGA3ox2 (TraesCS3A02G122600, TraesCS3B02G141800, TraesCS3D02G124500). Files of raw sequence reads generated in this study were deposited in the BIG Data Center ( https://ngdc.cncb.ac.cn/gsub/ ) under BioProject ID PRJCA043871. Dive Curated Terms The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper: EIL1 Gramene: AT2G27050 EIL1 Araport: AT2G27050 SUB1A Gramene: DQ011598 SUB1A Araport: DQ011598 EMSA Gramene: Electrophoretic mobility shift assay EMSA Araport: Electrophoretic mobility shift assay References Achard  P  et al.  2006. 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Supplementary Materials koag081_Supplementary_Data koag081_supplementary_data.zip (8.1MB, zip) Data Availability Statement All genes' information in this article can be found in the GeneBank/EMBL data libraries ( http://plants.ensembl.org/index.html ) under the following accession numbers: RNHL1 (TraesCS2A02G059900, TraesCS2B02G073600, TraesCSU02G024900), TaEIL1 (TraesCS4A02G12940, TraesCS4B02G175100, TraesCS4D02G177000), TaERF1 (TraesCS2A02G417300, TraesCS2B02G436300, TraesCS2D02G414600), TaGA3ox2 (TraesCS3A02G122600, TraesCS3B02G141800, TraesCS3D02G124500). Files of raw sequence reads generated in this study were deposited in the BIG Data Center ( https://ngdc.cncb.ac.cn/gsub/ ) under BioProject ID PRJCA043871. 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