Expression profiling by high throughput sequencing
Summary
Overexpression (OE) of phospholipase C (PLC) promotes drought stress tolerance in various plant species. How PLC contributes to this phenotype is unknown. To investigate this, transcriptomic analyses of drought-stressed Arabidopsis plants were performed in wild type (WT) and four distinct PLC-OE backgrounds. To be able to measure the earliest responses in both root- and shoot tissues, a vermiculite-based assay was developed. Subsequent transcriptomics analyses revealed that WT plants responded relatively strongly to the drought, whereas all PLC-OEs lines responded to a lesser extent. Changes in various pathways were observed that were differentially regulated in WT. These include, i) several phospholipid metabolism genes, which may counteract the overexpression of PLC activity, ii) genes involved in the circadian rhythm, shifting to a more night-like expression pattern for the PLC-OE lines, iii) downregulation of several light reaction-related genes, and iv) upregulation of genes involved in the brassinosteroid signalling pathway. In the PLC-OE lines, a subset of genes involved in auxin signalling was found to be differently regulated under drought. Similarly, genes upstream of H2O2 production were found upregulated, while ROS-induced genes were downregulated. Though unable to pin point the primary cause of the drought tolerant phenotype in PLC-OE plants, several novel connections were identified, which are highlighted and discussed in more detail.
Overall design
RNAseq of Arabidopsis WT and PLC OEs. Plants were first grown 9 days on agar, followed by 12 days of growth on vermiculite with growth media. After 7 days of either remaining in contact with growth media, or no contact with media (drought) whole roots systems and whole shoots were harvested between 1 and 5 pm, cycling between the different samples before returning to the replicates. 4 replicates were used for all WT and PLC5-OE samples, 3 replicated were used for all other PLC-OEs. A single plant was used per replicate. Samples were frozen, ground up with mortar and pestle in liquid nitrogen. RNA was isolated using RNeasy Plant Mini Kit from qiagen. Quality was checked by RIN measurements. with all root samples scoring at least 6.5 and all shoot samples at least 9. RNA was mixed with ERCC RNA spike-in Control mix 1, afterwards a poly-A enrichment was performed using Dynabeads mRNA DIRECt micro purification kit. Total RNA-Seq kit v2 and the Ion Xpress RNA-Seq barcoding kit, bar-coded RNA libraries were generated. The 2200 TapeStation was used with Agilent D1000 ScreenTapes to assess the size distribution and yield. On the Ion Chef System sequencing templates were prepared, with the Ion PI Hi-Q Chef kit. Finally, sequencing was performed using an Ion PI Chip v3 on the Ion Proton platform by the ‘Dutch Genomics Service and Support Provider’ (MAD, SILS, University of Amsterdam, The Netherlands)
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