Comparative transcriptomic heat stress responses data for native Australian Acacia species: supplementary data and R code

Brief description

To improve our understanding of heat tolerance across related species from diverse climates, we cultivated a selection of Acacia species in a controlled environment, using seed sourced from wild populations that occupy a wide range of Australian climates. After four months of growth in control temperature conditions (ca 24 °C day and 18 °C night) plants were exposed to a four-day heatwave (38 °C day and 26 °C night). Heatwave responses were quantified with coupled measurements of the transcriptome (gene expression) and PHT acclimation. Samples for transcriptomics were taken across three days: one day prior to heatwave (control), at the onset of heatwave conditions (Day 1) and at the end of the heatwave (Day 4). Accompanying measurements of PHT were taken the day prior and at day four using basal chlorophyll fluorescence. This Collections includes RNA-seq metadata and cleaned expression data for data analysis (.rds file) and the associated Rmarkdown file (.Rmd file) to run analyses from our paper.

Lineage

Leaf samples for RNA extraction were taken the morning prior to the heatwave and on the first and fourth morning of the heatwave. Leaf samples were snap frozen with liquid nitrogen before being transported on dry ice to a -80 °C freezer immediately after sampling. For extracting total RNA from Acacia leaf and phyllode tissue, the best method for tissue homogenization proved to be grinding leaf samples with liquid nitrogen in a mortar and pestle. After grinding, samples were returned to dry ice until 16 samples were ready to start the RNA extraction. The kit used for RNA extraction was the NucleoSpin RNA Plant and Fungi Kit (Macherey-Nagel, Germany) using the standard protocol except for an adjustment to the lysis buffer. The lysis buffer aliquot per sample included 400μl of PFL and 50μl PFR buffers from the NucleoSpin Kit, 100μl Fruit-mate for RNA Purification (Takara, Japan) and 5μl of ß-mercaptoethanol. Of the 21 species grown, only 17 species were sequenced at multiple time points. After mRNA isolation with Oligo d(T)25 Magnetic Beads (New England BioLabs, Australia), strand specific RNA-seq libraries were prepared using an in-house template switching protocol. The protocol for library preps is fulling described in Paten et al., (2022). Two plates of 96 libraries were prepared using custom barcodes. Samples were sequenced on a single NovaSeq S4 flowcell (300 cycles, 2x150bp), using a XP 4-lane splitter kit to split the two sample pools onto 2 lanes each (i.e. set A on lanes 1 and 2 and set B on lanes 3 and 4). Sequencing was performed at the ACRF Biomolecular Resource Facility at John Curtin School of Medical Research at The Australian National University. Funding for sequencing costs was provided by Bioplatforms Australia. A full list of libraries and associated meta-data is provided in “Acacia_metadata_DAP_220523.xlsx” (see link = https://data.csiro.au/collection/csiro:57771). RNA-seq libraries are uploaded in two batches because of file sizes (part 1 = https://data.csiro.au/collection/csiro:57772 and part 2 = https://data.csiro.au/collection/csiro:57773). A. M. Paten, et al., Non-additive gene interactions underpin molecular and phenotypic responses in honey bee larvae exposed to imidacloprid and thymol. Sci. Total Environ. 814 (2022).