Next Generation Sequencing of Mislocalized POPEYE Enables Quantitative Analysis of Arabidopsis Root Transcriptomes in Iron Deficient Conditions. Next Generation Sequencing of Mislocalized POPEYE Enables Quantitative Analysis of Arabidopsis Root Transcriptomes in Iron Deficient Conditions

Purpose: Identify differences in the trascriptional landscape of Arabidopsis roots under iron deficiency stress where POPEYE, a non cell autonomous iron responsive transcription factor, is localized to specific cell files. Method: RNA-Seq was performed on 7 day old roots (4d +Fe then transferred for 3d - Fe) of cell specific localization lines (WT;+ iron, WT, pye-1, PPL, SHR23, PEP45, WER13, WER65) - triplicates. RNA was extracted using RNAeasy Plant RNA Purification Kit (Qiagen). cDNA synthesis and amplification were performed using the NEBNext® Poly(A) mRNA Magnetic Isolation Module followed by the NEBNext® Ultra™ II Directional RNA Library Prep and NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1) and sequenced using an Illumina HiSeq 2500 sequencing machine, with 125bp single end reads. Adapters and low quality reads were filtered out using fastQC and fastq-mcf. Clean reads were mapped against the TAIR 10 reference genome using the tophat2. RPKM were acquired using RSubread, followed by edgeR to identify differentially expressed genes (DEGs), using a maximum false discovery rate (FDR) of 0.05 and minimum log fold change threshold of 0.75. Results: We identified 4414 DEGs between WT (+iron) and WT (-iron). WT minus was used as the baseline to compare transcriptome landscape between cell specific localization lines. Compared to WT minus iron, pye-1 had 3878 DEGs, SHR23 had 3697 DEGs, PEP45 had 3678 DEGs, WER13 had 4696 DEGs, and WER65 had 3920 DEGs. Conclusion: Transcriptomic profiles indicated that localization of PYE to the vasculature and endodermis (SHR23;V-EN) increased genes involved in metal aquisition, while localization expanded to the cortex (PEP45;C-EN-V) helps regulate overall iron bioavailability, and the expression of genes associated with various aspects of photosynthesis and carbon metabolism. Overall design: RNA-Seq of 7 day old roots (4d +Fe then transferred for 3d - Fe) of cell specific localization lines (WT;+ iron, WT, pye-1, PPL, SHR23, PEP45, WER13, WER65) triplicates

研究目的：明确缺铁胁迫下，定位于特定细胞列的非细胞自主型缺铁响应转录因子POPEYE（POPEYE）所影响的拟南芥根转录组景观差异。
实验方法：以7日龄拟南芥根（先在加铁培养基培养4天，随后转移至缺铁培养基培养3天）为材料，对8种细胞特异性定位株系（加铁野生型WT、野生型WT、pye-1、PPL、SHR23、PEP45、WER13、WER65）进行RNA测序（RNA-Seq），每组设置3次生物学重复。使用Qiagen公司RNAeasy植物总RNA纯化试剂盒提取总RNA；采用NEBNext® Poly(A) mRNA磁珠分离试剂盒、NEBNext® Ultra™ II定向RNA文库制备试剂盒以及适配Illumina®的NEBNext®多重寡核苷酸（索引引物组1）完成cDNA合成与扩增；随后使用Illumina HiSeq 2500测序仪进行测序，读长为125bp单端。使用fastQC与fastq-mcf软件过滤接头序列及低质量读段；将clean reads比对至TAIR 10参考基因组，比对工具为TopHat2。利用RSubread包计算每百万reads中来自基因的每千碱基序列的reads数（RPKM），随后通过edgeR包鉴定差异表达基因（DEGs），筛选阈值为最大错误发现率（FDR）≤0.05，最小对数倍变化阈值为0.75。
实验结果：本研究在加铁野生型与缺铁野生型样本间鉴定出4414个差异表达基因。以缺铁野生型作为参照基线，用于比较各细胞特异性定位株系的转录组景观。与缺铁野生型相比，pye-1突变体存在3878个差异表达基因，SHR23株系存在3697个，PEP45株系存在3678个，WER13株系存在4696个，WER65株系存在3920个。
研究结论：转录组分析结果显示，当POPEYE定位于维管束与内皮层（SHR23；V-EN）时，会上调金属吸收相关基因的表达；而当其定位范围扩展至皮层（PEP45；C-EN-V）时，则有助于调控整体铁的生物可利用性，并影响光合作用与碳代谢各相关通路的基因表达。
实验整体设计：对7日龄拟南芥根（先在加铁培养基培养4天，随后转移至缺铁培养基培养3天）的8种细胞特异性定位株系（加铁野生型WT、野生型WT、pye-1、PPL、SHR23、PEP45、WER13、WER65）进行RNA测序，每组设置3次生物学重复。