title: Identifying epigenetically regulated tissue specific transcription factors in Arabidopsis thaliana
Creators: name: Armstrong, Emily May
Publisher: name: University of Glasgow
Year: 2021
Subjects: subject: arabidopsis, salt, epigenetics, rna-seq, plants, stress biology, sequencing, ion, tissue specific, roots, hydroponics, histones, fluorescence assisted cell sorting.

description: Arabidopsis thaliana roots are divided into distinct cell-types, organised in concentric rings surrounding the central vasculature. Previous large-scale sequencing datasets investigating cell-type specific expression relied on early-stage developmental markers to recover RNA from different root tissues. These datasets underpin the detailed expression maps available in the eFP browser, but they do not fully reflect the biological function of mature xylem vessels, which rely on the xylem parenchyma and pericycle to control loading/unloading of water and solutes into the transpiration stream. This project sought to address which genes are preferentially expressed in the root xylem parenchyma and pericycle, identify epigenetic processes that underpin tissue preferential expression, systematically interrogate cell type specific regulatory networks, and understand if epigenetically regulated genes with preferential expression in the xylem adjacent cells control ion accumulation in the shoot. A marker line expressing GFP under the control of the promoter of HKT1 showed GFP expression localised to mature xylem parenchyma and pericycle tissues. This transgenic line was subjected to FACS RNA-seq, which found approximately 8% of the Arabidopsis transcriptome is preferentially expressed in these cell types. Ontological analysis of preferentially expressed genes uncovered significant enrichment for ‘transcription factors’, ‘oxioreductase’ and ‘transport activity’. These data are useful for future studies into tissue-specific gene functions. To unravel the mechanisms underpinning tissue specific expression the FACS RNA-seq dataset was compared with a previously generated INTACT ChIP-Seq dataset that had identified genes with cell-type specific decrease of H3K27me3 levels specifically in the root xylem parenchyma and pericycle. From this analysis, two cell type specific transcription factors were identified as targets of the H3K27me3 demethylase REF6. RT-qPCR and ChIP-qPCR of ref6 mutants confirmed REF6 activity is required for H3K27me3 demethylation and expression of these transcription factors. RNA-seq on knockout mutants of one of the epigenetically de-repressed cell-type specific transcription factor found approximately 30% of downstream genes are also preferentially expressed in the xylem parenchyma and pericycle. These were enriched for ‘jasmonate biosynthesis’ and ‘ethylene signalling’. Promoter motif enrichment analysis of downstream genes identified numerable potential binding sequences, many of which are targeted under abiotic stress conditions. Knockouts of both identified transcription factors were subject to a 5-day salt stress to assess if they are required for controlling sodium loading/recovery from the transpiration stream. Both sets of knockout mutants hyperaccumulated sodium in the shoot, while no disturbance in potassium loading was observed. Ultimately, this project has resolved what genes are expressed in the mature xylem parenchyma and pericycle and identified an epigenetic cause of cell-type specific expression. It has allowed systematic interrogation of epigenetically regulated cell type specific regulatory networks, and identified two transcription factors that may regulate sodium exclusion from the shoot.