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

ABSTRACT: 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.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE207619 | GEO | 2022/07/12

REPOSITORIES: GEO

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Project description:Transcriptional profile of whole roots of wild-type and pye-1 mutants exposed to 24 hours -Fe were generated Global population increases and climate change underscore the need for better comprehension of how plants acquire and process nutrients such as iron. A systems biology approach was taken to elucidate novel regulatory mechanisms involved in plant responses to iron deficiency (-Fe). Using cell-type specific transcriptional profiling we identified a pericycle-specific iron deficiency response, and a previously uncharacterized transcription factor, POPEYE (PYE), that plays an important role in this response. Functional analysis of PYE suggests that it positively regulates growth and development under iron deficient conditions. ChIP-on-chip analysis and transcriptional profiling reveal that PYE helps maintain iron homeostasis by directly and indirectly regulating the expression of ferric reductases, metal ion transporters, iron storage proteins, and other key iron homeostasis genes. In addition to PYE, we also identified a second protein BRUTUS (BTS), which appears to negatively regulate the response to iron deficiency. BTS is a unique putative E3 ligase protein, with metal ion binding and DNA binding domains. PYE and BTS are tightly co-regulated and physically interact with PYE paralogs, one of which is thought to positively regulate expression of genes involved in iron homeostasis. We propose that iron content is sensed within the pericycle where PYE, perhaps in conjunction with BTS and other regulatory proteins, is then activated to control a regulatory network involved in maintaining proper iron distribution in plants. Keywords: Expression analysis To determine how loss of PYE expression affects the transcriptional profile of whole roots, pye-1 mutants and wild-type seeds were germinated under standard growth conditions then transferred to standard media (control, MS media) or iron deficient media (-Fe, 0.3mM Ferrozine in MS media containing no ferrous sulfate). After 24 hours of exposure to +Fe or -Fe whole roots were collected and analyzed.