Auxin promotes fiber elongation by enhancing gibberellic acid biosynthesis in cotton.

Cotton is an important cash crop grown worldwide, (Huang et al., 2021; Ma et al., 2018; Wang et al., 2019), which fibers are regulated by multiple phytohormones (Liu et al., 2020; Shan et al., 2014; Zhang et al., 2011). Targeted expression IAA biosynthesis gene enhances both fiber yield and quality (Zhang et al., 2011). Exogenous application of GA also improved fiber length (Shan et al., 2014). However, mechanism by which auxin and GA promote cotton fiber development and whether there is cross‐talk between them remains unclear. Our study shows that auxin promotes fiber development by enhancing GA biosynthesis.

To investigate the regulatory relationships between auxin‐ and GA‐ regulated fiber development, we observed fiber phenotype after exogenous application of IAA and GA1 and corresponding inhibitors. The results showed that IAA and GA1 promoted fiber elongation, N‐1‐naphthylphthalamic acid (NPA, a polar auxin transport inhibitor), and paclobutrazol (PAC, a GA biosynthesis inhibitor) inhibited fiber development. GA1 rescues fiber elongation inhibited by NPA, whereas IAA did not rescue shortened fibers by PAC treatment (Figure 1a), suggesting that GA may function downstream of IAA in regulating fiber development. Consistently, GA1 content was increased and decreased after IAA and NPA treatment, respectively (Figure 1b).

Figure 1

Auxin promotes fiber elongation by enhancing GA biosynthesis. (a) Phenotypes of 10 day‐old ovules cultured with 0.5 μm of GA1, 1 μm of PAC, 5 μm of IAA, 1 μm of NPA, 0.5 μm of GA1 + 1 μm of NPA, and 5 μm of IAA + 1 μm of PAC. (b) GA1 content of fibers treated with IAA or NPA. (c) Relative expression of GhARF18 in different fiber development stages. (d, e) Fibers phenotype (d) and length measurement (e) of CK, GhARF18 over‐expression and CRISPR/Cas9 lines. (f) GA1 and GA4 content in 5 and 10 DPA fibers from CK, GhARF18 over‐expression and CRISPR/Cas9 lines. (g) Yeast one‐hybrid assay between GhARF18 and nine promoters of gibberellin acid biosynthesis genes. P53 and p53 promoter were used as positive control. (h, i) Relative expression of GhGA3OX4D and GhGA20OX1D‐2 in different fiber development stages (h) and in 5 DPA fibers from CK, GhARF18 over‐expression and CRISPR/Cas9 lines (i). (j, k) Tobacco transient expression assay of GhARF18 and pGhGA3OX4D (j) or pGhGA20OX1D‐2 (k) promoters. (l, m) Tobacco dual‐luciferase assay of pGhGA3OX4D::LUC (l) and pGhGA20OX1D‐2::LUC expression (m). Expression of REN was used as internal control. (n, o) Yeast one‐hybrid assay of GhARF18 and GhGA3OX4D (n) and four GhGA20OX1D‐2 (o) fragments. (p, q) Tobacco transient expression assay of GhARF18 and pGhGA3OX4D‐P2 or AuxRE‐mutated pGhGA3OX4D‐P2 fragment (p) and GhARF18 and pGhGA20OX1D‐2‐P3 or AuxRE‐mutated pGhGA20OX1D‐2‐P3 fragment (q). (r, s) Tobacco dual‐luciferase assay of intact or mutated pGhGA3OX4D‐P2::LUC (r) and pGhGA20OX1D‐2‐P3::LUC (s) expression. Values given are mean ± SD (n ≥ 5). (t–w) EMSA of GhARF18 binding to AuxREs from GhGA3OX4D and GhGA20OX1D‐2 promoters. Promoter fragments containing intact AuxRE were incubated with gradient concentrations of GhARF18 protein (t, u). Different concentrations of unlabeled probes of intact or mutated AuxRE (cold probe) were incubated with GhARF18 to compete with labeled native promoter fragments with AuxRE (v, w). (x, y) ChIP‐qPCR of GhGA3OX4D (x) and GhGA20OX1D‐2 (y) promoter fragments in GhARF18 over‐expression line. (z) Phenotypes of ovules from GhARF18 over‐expression and CRISPR/Cas9 lines cultured with 5 μm of IAA, 1 μm of NPA, 0.5 μm of GA1, and 1 μm of PAC, respectively. (ab) Relative expression of GhGA3OX4D and GhGA20OX1D‐2 in their transgenic fibers. (ac, ad, ae) GA1 and GA4 content of 10 DPA fibers (ac), mature length of fibers (ad), and phenotypes of fibers (ae) from CK, GhGA3OX4D and GhGA20OX1D‐2‐over‐expression lines. (af) Schematic model. CK, nontransgenic plants. d, day post anthesis (DPA). Bar = 1 cm. Statistical significance for each comparison is indicated (t‐test): *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001.

Our previous work showed that auxin responsive factor 18 (GhARF18) was a key regulator in fiber development (Xiao et al., 2018) and GhARF18 transcripts were accumulated in early and secondary cell wall accumulation stage of fibers (Figure 1c). To explore the function of GhARF18, we constructed GhARF18‐overexpression and ‐knockout transgenic plants. Compared with nontransgenic plants (CK), overexpressing of and knocking out GhARF18 enhanced and reduced fiber length, respectively (Figure 1d,e). GA1 and GA4 content was increased and decreased in fibers from GhARF18 overexpression and knock‐out lines, respectively (Figure 1f), suggesting that GhARF18 regulates GA1 and GA4 biosynthesis in fiber elongation stage.

GA 3‐beta‐hydroxylases (GA3OX) and GA 20‐oxidase (GA20OX) are two key enzymes involved in GA biosynthesis. To explore whether auxin regulates GA biosynthesis through GhARF18, we identified 14 GhGA20OX and 12 GhGA3OX genes in Gossypium hirsutum genome (Zhang et al., 2015). Five GhGA20OX and four GhGA3OX genes contain AuxREs (ARF‐binding site) in promoters. We next examined whether these genes are regulated by GhARF18 and found that GhARF18 bind to promoters of GhGA3OX4D and GhGA20OX1D‐2 (Figure 1g). Transcripts of GhGA3OX4D and GhGA20OX1D‐2 are abundant in early fiber development stage (Figure 1h). Transcripts of GhGA3OX4D and GhGA20OX1D‐2 were increased and decreased in fibers from GhARF18 overexpression and knockout lines, respectively (Figure 1i). GhARF18 activates pGhGA3OX4D::LUC and pGhGA20OX1D‐2::LUC reporter genes (Figure 1j–m), indicating that GhARF18 activates transcription of GhGA3OX4D and GhGA20OX1D‐2. GhARF18 was highly expressed in secondary cell wall accumulation stage, but GhGA3OX4D and GhGA20OX1D‐2 have lower transcripts in this stage, suggesting that unknown factors may exist and inhibit the transcription of GhGA3OX4D and GhGA20OX1D‐2 during secondary cell wall accumulation stage of fibers.

We next investigated the mechanism by which GhARF18 regulates the transcription of GhGA3OX4D and GhGA20OX1D‐2. Promoters of GhGA3OX4D and GhGA20OX1D‐2 were divided into three fragments each. GhARF18 interacted with the fragments containing AuxRE, and this interaction disappeared once AuxRE was mutated (Figure 1n,o). Co‐expression of GhARF18 activated the transcription of LUC reporter genes driven by AuxRE‐containing fragments, and this activation was abolished once AuxRE was mutated (Figure 1p–s). Furthermore, electrophoretic mobility shift assay showed that GhARF18 had significant binding affinity for GhGA3OX4D and GhGA20OX1D‐2 fragments containing AuxREs (Figure 1t–w). Chromatin immunoprecipitation assay showed that GhARF18 specifically recruited to promoter fragments containing AuxREs (Figure 1x,y). IAA treatment resulted in longer fibers in GhARF18 overexpression lines than that in GhARF18 knock‐out lines. NPA cannot inhibit fiber elongation of GhARF18 overexpression lines. GA1 treatment enhanced and PAC treatment inhibited fiber length from GhARF18 transgenic lines, respectively (Figure 1z). These results suggest that GhARF18 binds directly to AuxRE in GhGA3OX4D and GhGA20OX1D‐2 promoters.

To explore the function of GhGA3OX4D and GhGA20OX1D‐2, we constructed GhGA3OX4D and GhGA20OX1D‐2 overexpression cotton plants and detected their transcripts in overexpression plants (Figure 1ab). Compared with CK, overexpression of GhGA3OX4D and GhGA20OX1D‐2 not only enhanced GA1 and GA4 content of fibers (Figure 1ac), but also promoted length of mature fiber (Figure 1ad,ae), suggesting that GhGA3OX4D and GhGA20OX1D‐2 promote fiber elongation by increasing GAs content.

Our results demonstrate that auxin regulates GA biosynthesis through GhARF18 regulating the transcription of GhGA3OX4D and GhGA20OX1D‐2 to promote fiber elongation (Figure 1af).

Conflict of interest

The authors declare no conflict of interest.

Author contributions

B.J. and J.Z. performed the experiments; L.Z. and B.J. analyzed data; G.X. and L.Z. wrote the paper.