Brassinosteroids enhance gibberellic acid biosynthesis to promote cotton fibre cell elongation

Abstract

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.

Figure 1

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BR enhances GA biosynthesis to promote fibre cell growth in cotton. (a) Phenotypes of fibres derived from ovules cultured with 0.5 μM GA₃, 1 μM PAC, 5 μM BR, 15 μM BRZ, 0.5 μM GA₃ + 15 μM BRZ and 5 μM BR + 1 μM PAC for 10 days. Bar = 5 mm. (b) Statistics of fibre length in (a). (c) GA₁ and GA₄ contents of fibres derived from ovule treated with BR or BRZ. (d) GA₁ and GA₄ content in wild-type and BES1 overexpression cotton fibres. (e) Mature fibre phenotype of BES1 overexpression cotton. Bar = 10 mm. (f) Statistics of fibre length in (e). (g) Y1H assay between BES1 and 15 promoters of GA biosynthesis genes. (h) and (i) Tobacco transient expression assay showing transcriptional activation of LUC reporter gene under control of GA20OX1D and GA3OX1D promoters by co-expressing BES1. (j) and (k) Electrophoretic mobility shift assay (EMSA) demonstrating BES1's direct binding to the L1 fragment of the GA20OX1D promoter (j) and the L2 fragment of the GA3OX1D promoter (k). (l) and (m) Competitive EMSA assay using a biotin-labelled L1 fragment of the GA20OX1D promoter (l) and a biotin-labelled L2 fragment of the GA3OX1D promoter (m) incubated with BES1, competing with different concentrations of cold probes (without biotin label) containing the intact or mutated binding site (L1m (l) or L2m (m)). (n)–(q) Fibres phenotype (n) and (o) and length measurement (p) and (q) of WT, GA20OX1D (n) and (p) and GA3OX1D (o) and (q) overexpression and knockout lines. Bar = 10 mm. (r) and (t) Cross-sections of paraffin-embedded mature fibres from WT, GA3OX1D overexpression and knockout lines that were stained with calcofluor white (r) or S4B (t). Bars = 20 μm. (s) and (u) Cell wall thickness measurement of fibres from WT, GA3OX1D overexpression and knockout lines. (v) Schematic model. CK, Control. WT, wild type. OE, overexpression. KO, knockout. DPA, day post-anthesis. Values given are mean ± SD with n = 10 (b), n = 20 (f), n = 3 (c) and (d). Statistical significance for each comparison is indicated (t-test) (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

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).