当前位置: X-MOL 学术Plant Biotech. J. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Auxin promotes fiber elongation by enhancing gibberellic acid biosynthesis in cotton
Plant Biotechnology Journal ( IF 10.1 ) Pub Date : 2021-12-31 , DOI: 10.1111/pbi.13771
Liping Zhu 1 , Bin Jiang 1 , Jiaojie Zhu 1 , Guanghui Xiao 1
Affiliation  

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).

Details are in the caption following the image
Figure 1
Open in figure viewerPowerPoint
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).



中文翻译:

生长素通过增强棉花赤霉酸生物合成促进纤维伸长

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)。

详细信息在图片后面的标题中
图1
在图形查看器中打开微软幻灯片软件
生长素通过增强 GA 生物合成来促进纤维伸长。(a) 用 0.5 μ m GA 1、1 μ m PAC、5 μ m IAA、1 μ m NPA、0.5 μ m GA 1  + 1 μ m NPA培养的 10 日龄胚珠的表型,和 5 μ m IAA + 1 μ m PAC。(b)用 IAA 或 NPA 处理的纤维的GA 1含量。(c) GhARF18在不同纤维发育阶段的相对表达。(d, e) CK、GhARF18过表达和 CRISPR/Cas9 系的纤维表型 (d) 和长度测量 (e)。(f) 大会1和大会来自 CK、GhARF18过表达和 CRISPR/Cas9 系的 5 和 10 根 DPA 纤维中的4含量。( g ) GhARF18 和赤霉素酸生物合成基因的九个启动子之间的酵母单杂交试验。P53 和p53启动子用作阳性对照。(h, i) GhGA3OX4DGhGA20OX1D-2在不同纤维发育阶段 (h) 和来自 CK、GhARF18过表达和 CRISPR/Cas9 系 (i) 的 5 根 DPA 纤维中的相对表达。(j, k) GhARF18 和pGhGA3OX4D (j) 或pGhGA20OX1D-2 (k) 启动子的烟草瞬时表达测定。(l, m) pGhGA3OX4D ::LUC (l) 和pGhGA20OX1D-2的烟草双荧光素酶测定::LUC 表达式 (m)。REN 的表达用作内部对照。(n, o) GhARF18 和GhGA3OX4D (n) 和四个GhGA20OX1D-2 (o) 片段的酵母单杂交试验。(p, q) GhARF18 和pGhGA3OX4D-P2或 AuxRE 突变的 pGhGA3OX4D-P2片段 (p) 和 GhARF18 和pGhGA20OX1D-2-P3或 AuxRE 突变的 pGhGA20OX1D-2-P3片段 (q ) 的烟草瞬时表达测定。(r, s) 完整或突变的 pGhGA3OX4D-P2 ::LUC (r) 和pGhGA20OX1D-2-P3 ::LUC (s) 表达的烟草双荧光素酶测定。给出的值是平均值 ± SD (n ≥ 5)。(t-w) GhARF18 的 EMSA 与GhGA3OX4DGhGA20OX1D-2的 AuxRE 结合发起人。含有完整 AuxRE 的启动子片段与梯度浓度的 GhARF18 蛋白 (t, u) 一起孵育。将不同浓度的完整或突变 AuxRE(冷探针)的未标记探针与 GhARF18 一起孵育,以与带有 AuxRE(v,w)的标记的天然启动子片段竞争。(x, y) GhARF18过表达系中GhGA3OX4D (x) 和GhGA20OX1D-2 (y) 启动子片段的ChIP-qPCR。(z)分别用 5 μ m IAA、1 μ m NPA、0.5 μ m GA 1和 1 μ m PAC培养的GhARF18过表达和 CRISPR/Cas9 系的胚珠的表型。(ab) 的相对表达GhGA3OX4DGhGA20OX1D-2在它们的转基因纤维中。(ac, ad, ae ) 来自 CK、 GhGA3OX4DGhGA20OX1D-2-过表达系的 10 根 DPA 纤维 (ac) 的GA 1和 GA 4含量、成熟纤维长度 (ad) 和纤维表型 (ae)。(af) 示意图模型。CK,非转基因植物。d,开花后天数(DPA)。酒吧 = 1 厘米。显示了每个比较的统计显着性(t检验):*,P  ≤ 0.05,**,P  ≤ 0.01,***,P  ≤ 0.001。

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GhGA3OX4DGhGA20OX1D-2的启动子结合(图1g)。GhGA3OX4DGhGA20OX1D-2的转录本在早期纤维发育阶段丰富(图1h)。GhGA3OX4DGhGA20OX1D-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GhGA3OX4DGhGA20OX1D-2的转录。GhARF18在次生细胞壁积累阶段高表达,而GhGA3OX4DGhGA20OX1D-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GhGA3OX4DGhGA20OX1D-2的转录。

我们接下来研究了 GhARF18 调节GhGA3OX4DGhGA20OX1D-2转录的机制。GhGA3OX4DGhGA20OX1D-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GhGA3OX4DGhGA20OX1D-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GhGA3OX4DGhGA20OX1D-2启动子中的 AuxRE 结合。

为了探索GhGA3OX4DGhGA20OX1D-2的功能,我们构建了 GhGA3OX4DGhGA20OX1D-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GhGA3OX4DGhGA20OX1D-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GhGA3OX4DGhGA20OX1D-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GhGA3OX4DGhGA20OX1D-2的转录以促进纤维伸长来调节 GA 生物合成(图 1af)。

更新日期:2021-12-31
down
wechat
bug