Integrative phenotyping approaches to unmask the Phyb-PIF4 pathway in Arabidopsis thaliana reproductive organs at high ambient temperatures

The increasing ambient temperature significantly impacts plant growth, development, and reproduction. Uncovering the temperature-regulating mechanisms in plants is of high importance, not only for boosting our plant biology knowledge but also for assisting plant breeders in improving plant resilience to these stress conditions. Numerous studies on the molecular mechanisms by which plants regulate temperature responses revealed that plants employ distinct transcription factors to regulate thermomorphogenesis specific to each tissue type. A significant discovery in this field was the identification of PHYTOCHROME-INTERACTING FACTORs (PIFs) as key regulators of thermomorphogenesis during vegetative growth. PIF4, a regulator of auxin-mediated signaling pathways, is crucial in controlling high-temperature responses. In this study, we screened the temperature responses of the wild type and several PhyB-PIF4 pathway Arabidopsis mutant lines in combined and integrative phenotyping platforms for root in soil, shoot, inflorescence, and seed. We demonstrated that high ambient temperature differentially impacts vegetative and reproductive organs through this pathway. Suppression of the PhyB-PIF4 components mimics the response to a high ambient temperature in wild-type plants. We also identified correlative responses to high ambient temperature between shoot and root tissues. This integrative and automated phenotyping was complemented by monitoring the changes in transcript levels in reproductive organs. Transcriptomic profiling of the pistils from plants grown under high ambient temperature identified key elements that may provide clues to the molecular mechanisms behind temperature-induced reduced fertilization rate, such as a downregulation of auxin metabolism, upregulation of genes involved auxin signalling, miRNA156 and miRN160 pathways, pollen tube attractants. 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.