Reprogramming feedback strength in gibberellin biosynthesis highlights conditional regulation by the circadian clock and carbon dioxide

The phytohormone gibberellin (GA) is an important regulator of plant morphology and reproduction, and the biosynthesis and distribution of GA in planta is agriculturally relevant to past and current breeding efforts. Tools like biosensors, extensive molecular genetic resources in reference plants and mathematical models have greatly contributed to current understanding of GA homeostasis; however, these tools are difficult to tune or repurpose for engineering crop plants. Previously, we showed that a GA-regulated Hormone Activated CAS9-based Repressor (GAHACR) functions in planta (Khakhar et al., 2018). Here, we use GAHACRs to modulate the strength of feedback on endemic GA regulated genes, and to directly test the importance of transcriptional feedback in GA signaling. We first adapted existing mathematical models to predict the impact of targeting a GAHACR to different nodes in the GA biosynthesis pathway, and then implemented a perturbation predicted by the mo..., RNA-Seq For ambient carbon dioxide concentrations, T4 seedlings were cultured on plates for 4 days without drug selection, and then transplanted to LS0 plates without drug for 10 days in either ambient or carbon dioxide-supplemented Conviron (Pembina, North Dakota) growth chambers. RNA was extracted using the Illustra RNAspin Mini Kit from GE, and RNA-Seq was performed by Amaryllis Nucleics (Oakland CA). In brief, RNA was checked for quality and quantity on a Bioanalyzer, and poly(A) mRNA was purified from total RNA. The library was constructed using the Amaryllis proprietary construction kit, followed by QC by E-gel & Bioanalyzer. Libraries were pooled and run on an Illumina NextSeq 500 SR75, with a target of approximately 20 million reads per sample. Read preprocessing, mapping, and differential gene expression were performed by Amaryllis, in addition to independent differential gene expression analysis in-house using EdgeR and DESeq2. Network analysis was performed in cytoscape. ..., , # Reprogramming feedback strength in gibberellin biosynthesis highlights conditional regulation by the circadian clock and carbon dioxide [https://doi.org/10.5061/dryad.547d7wmgr](https://doi.org/10.5061/dryad.547d7wmgr) ## Description of the data and file structure **Dataset overview:** Counts tables for differential gene expression performed in the analysis of 'reprogramming feedback strength in gibberellin biosynthesis highlights conditional regulation by the circadian clock and carbon dioxide' are provided for RNA-Seq experiments performed on Arabidopsis seedlings. We performed these at both Ambient and elevated (750ppm) carbon dioxide levels, with and without the GAHACR genetic perturbation, using GAHACRs to modulate the strength of feedback on endemic GA regulated genes, and to directly test the importance of transcriptional feedback in GA signaling. In order to facilitate future researchers to most easily incorporate our RNA-seq results, we have shared the count tables genera...,植物激素赤霉素（gibberellin, GA）是调控植物形态建成与生殖发育的重要因子，其在植物体内的生物合成与分布与既往及当前的作物育种工作均具有农业生产相关性。诸如生物传感器（biosensor）、模式植物的丰富分子遗传资源以及数学模型等工具，极大推动了当前学界对赤霉素稳态（GA homeostasis）的认知；然而这些工具难以针对农作物进行定制化调试或功能改造。此前本团队已证实，一种由赤霉素调控的激素激活型基于Cas9的阻遏物（GAHACR）可在植物体内发挥功能（Khakhar et al., 2018）。本研究利用GAHACRs来调控内源赤霉素调控基因的反馈强度，并直接验证转录反馈在赤霉素信号通路中的重要性。我们首先对现有数学模型进行适配，以预测将GAHACR靶向至赤霉素生物合成通路不同节点所产生的影响，随后实施了由模型（原文此处截断）预测的扰动，相关RNA测序（RNA-Seq）实验如下： 针对环境二氧化碳浓度条件：将T4代幼苗在无药物筛选的培养板上培养4天，随后移植至无药物的LS0培养基平板上，分别置于环境空气或补充二氧化碳的康维隆（Conviron，北达科他州彭比纳）生长箱中培养10天。总RNA使用GE公司的Illustra RNAspin Mini提取试剂盒进行提取，RNA测序（RNA-Seq）由Amaryllis Nucleics（美国加利福尼亚州奥克兰）完成。简要流程如下：通过生物分析仪（Bioanalyzer）检测RNA的质量与浓度，从总RNA中纯化带有poly(A)尾的信使RNA（poly(A) mRNA）；使用Amaryllis专属建库试剂盒构建测序文库，随后通过E-凝胶与生物分析仪完成文库质量控制。将文库混合后上机至Illumina NextSeq 500平台进行单端75bp（SR75）测序，目标为每个样本获取约2000万条读段。测序读段预处理、序列比对与差异基因表达分析由Amaryllis Nucleics完成，此外本团队还使用EdgeR与DESeq2两款工具在实验室内部独立完成了差异基因表达分析。网络分析通过Cytoscape软件完成。 # 重编程赤霉素生物合成中的反馈强度揭示昼夜节律与二氧化碳的条件性调控 [https://doi.org/10.5061/dryad.547d7wmgr](https://doi.org/10.5061/dryad.547d7wmgr) ## 数据与文件结构说明 **数据集概览：** 本研究针对“重编程赤霉素生物合成中的反馈强度揭示昼夜节律与二氧化碳的条件性调控”一文的分析中所涉及的差异基因表达分析计数表，已针对拟南芥幼苗的RNA测序（RNA-Seq）实验进行了共享。本实验分别在环境二氧化碳浓度与升高的二氧化碳浓度（750ppm）条件下开展，并设置有无GAHACR遗传扰动的组别，利用GAHACRs调控内源赤霉素调控基因的反馈强度，以直接验证转录反馈在赤霉素信号通路中的重要性。为方便后续研究者便捷复用本团队的RNA测序结果，我们已共享了计数表（原文此处截断）。