Data from: Systems and trans-system level analysis identifies conserved iron deficiency responses in the plant lineage

We surveyed the iron nutrition-responsive transcriptome of Chlamydomonas reinhardtii using RNA-Seq methodology. Presumed primary targets were identified in comparisons between visually asymptomatic iron-deficient versus iron-replete cells. This includes the known components of high-affinity iron uptake as well as candidates for distributive iron transport in C. reinhardtii. Comparison of growth-inhibited iron-limited versus iron-replete cells revealed changes in the expression of genes in chloroplastic oxidative stress response pathways, among hundreds of other genes. The output from the transcriptome was validated at multiple levels: by quantitative RT-PCR for assessing the data analysis pipeline, by quantitative proteomics for assessing the impact of changes in RNA abundance on the proteome, and by cross-species comparison for identifying conserved or universal response pathways. In addition, we assessed the functional importance of three target genes, VITAMIN C 2 (VTC2), MONODEHYDROASCORBATE REDUCTASE 1 (MDAR1), and CONSERVED IN THE GREEN LINEAGE AND DIATOMS 27 (CGLD27), by biochemistry or reverse genetics. VTC2 and MDAR1, which are key enzymes in de novo ascorbate synthesis and ascorbate recycling, respectively, are likely responsible for the 10-fold increase in ascorbate content of iron-limited cells. CGLD27/At5g67370 is a highly conserved, presumed chloroplast-localized pioneer protein and is important for growth of Arabidopsis thaliana in low iron.

本研究采用RNA测序（RNA-Seq）技术，对莱茵衣藻（Chlamydomonas reinhardtii）的铁营养响应转录组开展了系统性分析。通过对比表观无症状的铁缺乏细胞与铁充足细胞，我们鉴定出了推定的初级响应靶标，涵盖了已知的高亲和力铁摄取相关组分，以及莱茵衣藻中分布式铁转运的候选蛋白。对比生长受抑制的铁受限细胞与铁充足细胞，我们发现除数百个其他基因外，叶绿体氧化应激响应通路中的基因表达亦发生显著变化。本转录组测序的产出结果通过多维度方式完成验证：采用定量反转录PCR（quantitative RT-PCR）评估数据分析流程的可靠性，通过定量蛋白质组学分析RNA丰度变化对蛋白组的影响，并通过跨物种比较鉴定保守或通用的响应通路。此外，本研究借助生物化学或反向遗传学手段，评估了三个靶标基因的功能重要性，分别为维生素C合成酶2（VITAMIN C 2, VTC2）、单脱氢抗坏血酸还原酶1（MONODEHYDROASCORBATE REDUCTASE 1, MDAR1）以及绿色谱系与硅藻保守蛋白27（CONSERVED IN THE GREEN LINEAGE AND DIATOMS 27, CGLD27）。VTC2与MDAR1分别是从头抗坏血酸合成与抗坏血酸循环的关键酶，它们大概率是铁受限细胞中抗坏血酸含量提升10倍的核心原因。CGLD27/At5g67370是一种高度保守的推定叶绿体定位先驱蛋白，对拟南芥（Arabidopsis thaliana）在低铁环境下的生长具有关键调控作用。