Brassinosteroids enhance gibberellic acid biosynthesis to promote cotton fibre cell elongation

Cotton serves as not only a crucial natural textile crop, with cotton fibre accounting for approximately 95% of fibre usage in the textile industry but also a valuable model for the investigation of plant cell elongation (Cao et al., 2020; Wang et al., 2019). The plant hormones brassinosteroid (BR) and gibberellic acid (GA) promote fibre cell development (He et al., 2024; Huang et al., 2021; Shan et al., 2014; Zhu et al., 2023). Despite the positive role of BR and GA in fibre cell development that has been reported, the cross-talk between BR and GA biosynthesis pathway and signalling pathway in fibre growth remains largely unknown. In this study, our results reveal that BR stimulates GA biosynthesis during fibre elongation in cotton. BR and GA considerably promote cotton fibre development, whereas their respective inhibitors, brassinazole (BRZ, a BR biosynthesis inhibitor) and paclobutrazol (PAC, a GA biosynthesis inhibitor), impede fibre growth (Yang et al., 2023; Zhu et al., 2022). To explore the potential regulatory mechanisms between BR and GA, we treated wild-type (WT) ovules to with BR, BRZ, GA3, and PAC using an in vitro ovule culture system. Our observations reveal that BR and GA improved fibre development, and BRZ and PAC impeded it. In addition, GA3 mitigated the inhibitory effects of BRZ on fibre development, whereas PAC treatment considerably inhibited the fibre-promoting effect of BR Figure 1a,b. Moreover, the GA levels were increased after the BR treatment and decreased after the BRZ treatment (ovule with fibres; Figure 1c). BES1 (Gh_D02G0939) is the critical regulator in BR signalling (Zhu et al., 2023). Overexpression of BES1 notably stimulated the GA content in fibres (Figure 1d), accompanied with the considerably increased fibre length (Figure 1e,f and S1a,b). PAC significantly inhibited the promotion of fibre length after BES1 overexpression (Figure S2a,b). These results suggest that BR acts upstream of GA in the context of fibre development. In upland cotton, we identified 26 GA synthesis genes and 15 of them harboured BES1 binding site (E-box cis-element) in their promoters. The interaction between BES1 and 15 candidate gene promoters was investigated using yeast one-hybrid assay. As a result, BES1 was able to interact with two gene promoters (pGA20OX1D and pGA3OX1D) (Figure 1g). The tobacco dual-luciferase assay demonstrated that BES1 activated the promoters of GA20OX1D and GA3OX1D, which resulted in enhanced expression of LUC gene (Figures 1h,i and S3a,b). The promoters of GA20OX1D and GA3OX1D were segmented into three fragments based on the distribution of E-box cis-elements. BES1 was found to specifically bind to the P2 and F3 fragments of the GA20OX1D and GA3OX1D promoters, respectively (Figures S4 and S5a–d). Notably, this binding interaction was abolished upon mutation of the first E-box within the P2 or F3 fragments (Figures S4 and S5e–h). Furthermore, the electrophoretic mobility shift assay revealed the specific binding affinity of BES1 to pGA20OX1D-L1 and pGA3OX1D-L2 fragments with the E-box (Figure 1j,k). In addition, competitive binding probes, without biotin, considerably reduced the binding of BES1 protein to pGA20OX1D-L1 and pGA3OX1D-L2, respectively (Figure 1l,m). Moreover, chromatin immunoprecipitation (ChIP) followed by sequencing and ChIP-quantitative PCR (qPCR) analysis demonstrated that BES1 was selectively recruited to the promoter fragments that contain the E-box (Figure S6a–d). The expression levels of GA20OX1D and GA3OX1D in fibres were significantly increased after BR treatment or overexpression of BES1, and decreased after BRZ treatment or knockout of BES1 (Figure S7a–d). Furthermore, the expression levels of GA20OX1D and GA3OX1D were increased during cotton fibre development, suggesting the functional roles of these genes in fibre cell development (Figure S7e,f). To further investigate the roles of GA20OX1D and GA3OX1D in cotton fibre development, GA20OX1D and GA3OX1D transgenic cotton plants were generated (Figure S8a–f). Furthermore, we detected the GA content in GA20OX1D and GA3OX1D transgenic cotton fibres and found that overexpression of GA20OX1D or GA3OX1D increased GA accumulation (Figure S9a,b). The fibre length was substantially increased in GA20OX1D or GA3OX1D overexpression plants and significantly decreased in knockout lines (Figure 1n–q). In addition, the cell wall thickness of fibres was largely enhanced in GA3OX1D overexpression lines and reduced in GA3OX1D knockout lines (Figures 1r–u and S10a,b). However, the cell wall thickness of fibres from GA20OX1D transgenic lines was comparable with that from WT plants (Figure S10c–h). More importantly, exogenous application of GA3 successfully rescued the short fibre phenotype resulted from the mutation of GA20OX1D or GA3OX1D. Conversely, PAC inhibited the promotion of fibre elongation led by the overexpression of GA20OX1D or GA3OX1D (Figure S11a–d). Previous studies indicate that GA facilitates cotton fibre elongation by enhancing the biosynthesis of very long-chain fatty acids (VLCFAs) (He et al., 2024; Tian et al., 2022; Xiao et al., 2016). We speculate that GA20OX1D and GA3OX1D may enhance fibre elongation by regulating the biosynthesis of VLCFAs. Collectively, our results illustrate that BR modulates the transcription of GA20OX1D and GA3OX1D via BES1, which in turn regulates GA biosynthesis to facilitate fibre development (Figure 1v). This work was supported by the National Key Research and Development Program of China (2022YFF1002000), the National Natural Science Foundation of China (32270578, 32070549, 32200444 and 32470574), the Xinjiang Production and Construction Corps Key Field Science and Technology Research and Development Plan (KC00310501), the Science and Technology Innovation Team of Shaanxi Provincial Department of Science and Technology (2024RS-CXTD-72), the Scientific Research Project of Shaanxi Academy of Basic Sciences (22JHQ086), the Hong Kong Scholars Program (XJ2023014) and the Natural Science Basic Research Program of Shaanxi Province (2024JC-YBQN-0222, 2024JC-YBQN-0228). Shaanxi Postdoctoral Research Funding Project (2023BSHEDZZ206). The authors declare no conflict of interest. XGH conceived the research. HLY carried out the experiments. HLY, ZLP, HMM and LYF analysed the data. ZLP, HLY and XGH prepared the manuscript. All authors read and approved of the manuscript. The data that supports this study are available in the Supplementary Information of this article. Figure S1 Identification of the BES1 overexpression lines. Figure S2 Phenotype of cotton fibers from wild type and BES1 overexpression plants treated with GA3 or PAC. Figure S3 Quantification of dual-luciferase assays of LUC expression. Figure S4 Systematic yeast one-hybrid assay showing the interaction between BES1 and GA20OX1D or GA3OX1D gene promoter. Figure S5 BES1 directly binds to GA20OX1D promoter and GA3OX1D promoter. Figure S6 ChIP-seq and ChIP-qPCR showing the binding activities of BES1 to the promoters of GA20OX1D and GA3OX1D. Figure S7 Relative expression of GA20OX1D and GA3OX1D. Figure S8 Identification of GA20OX1D and GA3OX1D transgenic lines. Figure S9 GA1 and GA4 content in fibers from wild type (WT), GA20OX1D and GA3OX1D overexpression (OE) and knockout (KO) lines. Figure S10 Cell wall thickness of mature fibers from wild type (WT), overexpression (OE) and knockout (KO) lines. Figure S11 Phenotype of cotton fibers from wild type (WT), overexpression (OE) and knockout (KO) lines treated with GA3 or PAC. Table S1 All primers used in this work. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.