Transcriptomic temperature stress responses in Australian plants: supplementary data and RNA-seq files

To improve our understanding of transcriptomic stress responses in plants to hot and cold temperature shocks a selection of 20 native Australian species were sequenced. The 20 study species were selected from three ecosystems that represent the three contrasting biomes of arid, alpine, and coastal temperate environments in Australia (Harris et al., 2024). The arid biome represents an extreme environment that is known for its high maximum temperatures, but many regions also experience freezing nights during winter. The alpine biome represents extreme cold in the cooler months, but the exposed slopes of mountains can also experience high temperatures during summer. Coastal temperate environments represent a more stable climate, with lower temperature fluctuations within a day or across a year. The 20 study species spanned 13 families and 16 genera (see “DAP_LOTE_metadata.xlsx” for metadata tables). \nSeeds for the study species were obtained from conservation seed banks, including the Australian National Botanic Gardens Seed Bank and the Australian Botanic Gardens Plant Bank. A single accession of seed was used for each species (Harris et al., 2024).\nPlants were grown in a temperature controlled glass house (25°C day 15°C night) before being moved to the Australian Plant Phenomics Facility at CSIRO Black Mountain laboratories, where they were kept in Conviron growth chambers for an acclimatisation period of 1-2 days prior to temperature treatments. Temperature treatments were run for three days with the heat treatment being 40°C days to 15°C nights and the cold treatment being 25°C days to -2°C nights, relative to a control group that experienced no change in temperature from benign growing conditions (25°C day 15°C night). On the morning after the third day, leaf samples were taken between 9:00am and 10:30am for RNA extraction (see below) and photosynthetic thermal tolerance measurements.\n\nThis collection contains Supplementarty information and RNA-seq libraries used for all data analyses presented in "LOTE_results_240703.html" and our paper. The Rmarkdown file "LOTE_Rcode_240703.Rmd" and the .rds file "LOTE_full_221026.rds" included here can be used to replicate the results. the .rds file includes binaries for the main data frames used for analyses. For more details on data files see “DAP_LOTE metadata.xlsx”.\n\nHarris, R. J., Alvarez, P. R., Bryant, C., Briceño, V. F., Cook, A. M., Leigh, A., & Nicotra, A. B. (2024). Acclimation of thermal tolerance in juvenile plants from three biomes is suppressed when extremes co-occur. Conservation Physiology, 12(1). https://doi.org/10.1093/conphys/coae027\n\nLineage: Leaf samples for RNA extraction were taken the morning after three days of treatment. Leaf samples were snap frozen with liquid nitrogen before being transported on dry ice to a -80 °C freezer immediately after sampling.\nFor extracting total RNA from leaf 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 mortar and pestle was cleaned with bleach between samples. 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. \nAfter 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 fully described in Paten et al., (2022). Two plates of 96 libraries were prepared using custom barcodes. Samples were sequenced on a single NovaSeq S2 flowcell (300 cycles, 2x150bp), using a lane splitter kit to split the two sample pools onto one lanes each (i.e. set A on lane 1 and set B on lane 2). 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.\n\nPaten, A. M., Colin, T., Coppin, C. W., Court, L. N., Barron, A. B., Oakeshott, J. G., & Morgan, M. J. (2022). Non-additive gene interactions underpin molecular and phenotypic responses in honey bee larvae exposed to imidacloprid and thymol. Science of the Total Environment, 814. https://doi.org/10.1016/j.scitotenv.2021.152614\n