Selection of Reference Genes and HSP17.9A Expression Profiling in Heat-Stressed Grapevine Varieties.

BACKGROUND: "Touriga Franca" (TF) and "Touriga Nacional" (TN) are grapevine varieties cultivated in the 'Douro Superior' subregion (Northern Portugal) that experience stressful environmental conditions during the summer. OBJECTIVES: Aiming to profile the expression of stress-responsive genes by quantitative real-time PCR (qPCR) in TF and TN plants growing naturally, three candidate reference genes were first tested under controlled conditions. METHODS: To simulate a summer's day, TF and TN in vitro plants were exposed to 32 °C-3 h (heat acclimation) and 42 °C-1 h (severe heat stress, HS) followed by two recovery periods (32 °C-3 h and 24 °C-24 h). Leaf samples were collected at the end of each phase. Control plants were kept at 24 °C. RESULTS: Among the candidate reference genes, the UBC and VAG pair showed the highest stability. The suitability of these genes for qPCR was validated by heat shock protein 17.9A (HSP17.9A) gene profiling. The HSP17.9A expression was up-regulated in both varieties and all experimental phases except in TF control plants. TN showed the highest HSP17.9A relative expression ratio after severe HS. CONCLUSIONS: TN responded faster than TF to the induced heat shocks. The UBC, VAG, and HSP17.9A genes revealed to be suitable for further qPCR assays in TF and TN grapevine varieties.