The aim of the study was to identify DEGs in pistils from plants grown at high ambient temperature and the involvement of the PhyB PIF4 signaling pathway.
Plants were grown in a Fytoscope growth chamber (FS WI, Plant Systems Instruments (PSI), Czech Republic) under growth conditions with a longday regime (16 h light and 8 h dark), LED illumination with an intensity of 150 umol.m-2.s-1, and 35% 45% humidity. For normal conditions (nAT), the temperature was set at 21oC during the day and 18oC at night. For high ambient temperature conditions (hAT), the temperature wat set at 28oC during the day and 24oC at night. Plants were grown at nAT until flowering is induced and moved to hAT.
Gynoecium samples from flowers at stages 11 and 12 (before anthesis) were collected from wild type, phyb and 35S::PIF4 plants grown at nAT and hAT.
Total RNA was extracted from 100 mg of ovules using the RNeasy Plant Mini Kit (Qiagen) following the manufacturer's protocol. RNA isolates were treated with rDNAse Macherey Nagel) to remove traces of contaminant DNA and purified using a RNeasy MinElute Cleanup Kit (Qiagen). RNA quality was assessed using a NanoDrop2000 spectrophotometer and agarose gel electrophoresis. Samples, four biological replicates each, were sent to the Novogene Genomic Sequencing Labs (Cambridge Sequencing Center) for sequencing. All samples passed Novogene quality control threshold for library preparation and RNA-seq. mRNAseq libraries were constructed by Novogene, starting with 100 ng of high-quality RNA per sample. mRNA purification was performed using oligo(dT) attached magnetic beads, followed by fragmentation and first-strand cDNA synthesis. Second strand cDNA synthesis, end repair, adapter ligation, and size selection were performed. PCR enrichment yielded in the final cDNA library. Sequencing was conducted on the Illumina NovaSeq platform, generating 150bp 150bp paired-end read. Less...