Identification of reference genes and analysis of heat shock protein gene expression (Hsp90) in arta (Calligonum comosum L.) leaf under heat stress

In southern Tunisia, plants are subjected to high temperatures and disturbances, causing the degradation of soils and plants. Studying wild plants and their response to heat stress can facilitate their conservation and help ecosystem rehabilitation. Understanding heat stress responses has gradually become a hotspot in desert plant research. The adaptive responses of Calligonum comosum species to high temperature were examined in this regard. For C.comosum gene expression studies, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is the best technique, in addition to the use of appropriate reference genes. So, in our research, we analyzed four different housekeeping genes to select the most stable one during heat stress conditions. To analyze the results, we applied various statistical programs, including GeNorm, Normfinder, Bestkeeper, and the comparative ΔCt method, to identify the most appropriate reference genes for normalizing gene expression under heat stress conditions. Ultimately, our analysis revealed that α-tubulin (α-tub) emerged as the most stable reference gene. Additionally, we subjected C. comosum plants to thermal stress to elucidate the expression patterns of heat shock protein (hsp90) genes. Fourteen-day-old plants were exposed to a temperature of 60 °C for a period ranging from 30 to 240 min. The highest level of hsp90 gene expression was realized upon exposure to heat for 180 min, underscoring the upregulation of this protein in response to thermal stress. This study is the first systematic investigation for selecting reference genes for qPCR in C. comosum under heat stress. These findings will substantially benefit gene expression studies conducted under similar challenging conditions, contributing to our understanding of desert plant responses to heat stress.