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).

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).

棉花是全球种植的重要经济作物（Huang et al ., 2021 ; Ma et al ., 2018 ; Wang et al ., 2019），其纤维受多种植物激素的调控（Liu et al ., 2020 ; Shan et al., ., 2014 ; 张等人., 2011 )。靶向表达IAA生物合成基因可提高纤维产量和质量（Zhang et al ., 2011）。GA 的外源性应用也提高了纤维长度（Shan et al ., 2014）。然而，生长素和GA促进棉纤维发育的机制以及它们之间是否存在串扰仍不清楚。我们的研究表明，生长素通过增强 GA 生物合成来促进纤维发育。

为了研究生长素和 GA 调节的纤维发育之间的调节关系，我们观察了外源应用 IAA 和 GA ₁和相应抑制剂后的纤维表型。结果表明，IAA 和 GA ₁促进纤维伸长，N-1-萘基邻苯二甲酸（NPA，一种极性生长素转运抑制剂）和多效唑（PAC，一种 GA 生物合成抑制剂）抑制纤维发育。GA ₁挽救了 NPA 抑制的纤维伸长，而 IAA 没有通过 PAC 处理挽救缩短的纤维（图 1a），这表明 GA 可能在 IAA 的下游发挥调节纤维发育的作用。一致地，在IAA和NPA处理后GA ₁含量分别增加和减少（图1b）。

我们之前的工作表明，生长素反应因子 18（GhARF18）是纤维发育的关键调节因子（Xiao et al ., 2018），GhARF18转录物在纤维的早期和次生细胞壁积累阶段积累（图 1c）。为了探索GhARF18的功能，我们构建了GhARF18-过表达和-敲除转基因植物。与非转基因植物（CK）相比，过表达和敲除GhARF18分别增强和减少了纤维长度（图 1d，e）。来自GhARF18的纤维中GA ₁和 GA ₄含量增加和减少分别过表达和敲除系（图1f），表明GhARF18在纤维伸长阶段调节GA ₁和GA _{4生物合成。}

GA 3-β-羟化酶 (GA3OX) 和 GA 20-氧化酶 (GA20OX) 是参与 GA 生物合成的两种关键酶。为了探索生长素是否通过 GhARF18 调控 GA 生物合成，我们在陆地棉基因组中鉴定了 14 个GhGA20OX和 12 个GhGA3OX基因（Zhang et al ., 2015）。五个GhGA20OX和四个GhGA3OX基因在启动子中包含 AuxRE（ARF 结合位点）。我们接下来检查了这些基因是否受 GhARF18 调节，发现 GhARF18 与GhGA3OX4D和GhGA20OX1D-2的启动子结合（图1g）。GhGA3OX4D和GhGA20OX1D-2的转录本在早期纤维发育阶段丰富（图1h）。GhGA3OX4D和GhGA20OX1D-2的转录本分别在来自GhARF18过表达和敲除系的纤维中增加和减少（图 1i）。GhARF18 激活 p GhGA3OX4D ::LUC 和 p GhGA20OX1D-2 ::LUC 报告基因（图 1j-m），表明 GhARF18 激活GhGA3OX4D和GhGA20OX1D-2的转录。GhARF18在次生细胞壁积累阶段高表达，而GhGA3OX4D和GhGA20OX1D-2在该阶段具有较低的转录物，表明在纤维的次生细胞壁积累阶段可能存在未知因素并抑制GhGA3OX4D和GhGA20OX1D-2的转录。

我们接下来研究了 GhARF18 调节GhGA3OX4D和GhGA20OX1D-2转录的机制。GhGA3OX4D和GhGA20OX1D-2的启动子分别分成三个片段。GhARF18 与含有 AuxRE 的片段相互作用，一旦 AuxRE 发生突变，这种相互作用就消失了（图 1n，o）。GhARF18 的共表达激活了由含有 AuxRE 的片段驱动的 LUC 报告基因的转录，一旦 AuxRE 突变，这种激活就会被取消（图 1p-s）。此外，电泳迁移率变动分析表明，GhARF18 对GhGA3OX4D和GhGA20OX1D-2具有显着的结合亲和力包含 AuxRE 的片段（图 1t-w）。染色质免疫沉淀测定显示 GhARF18 特异性募集到含有 AuxRE 的启动子片段（图 1x，y）。IAA 处理导致GhARF18过表达系中的纤维比GhARF18敲除系中的纤维更长。NPA 不能抑制GhARF18过表达系的纤维伸长。GA ₁处理增强和 PAC 处理分别抑制GhARF18转基因系的纤维长度（图 1z）。这些结果表明 GhARF18 直接与GhGA3OX4D和GhGA20OX1D-2启动子中的 AuxRE 结合。

为了探索GhGA3OX4D和GhGA20OX1D-2的功能，我们构建了 GhGA3OX4D和GhGA20OX1D-2过表达的棉花植物，并在过表达植物中检测了它们的转录本（图 1ab）。与 CK 相比，过表达GhGA3OX4D和GhGA20OX1D-2不仅增加了纤维的 GA ₁和 GA ₄含量（图 1ac），而且促进了成熟纤维的长度（图 1ad，ae），表明GhGA3OX4D和GhGA20OX1D-2促进纤维通过增加 GAs 含量的伸长率。

我们的研究结果表明，生长素通过 GhARF18 调节GhGA3OX4D和GhGA20OX1D-2的转录以促进纤维伸长来调节 GA 生物合成（图 1af）。