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

Abstract

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.

Keywords: Ubiquitin-conjugating enzyme (UBC) gene; Vacuolar ATPase subunit G (VAG) gene; gene expression; heat shock protein; in vitro culture; quantitative real-time PCR (qPCR).

Figure 1

(a) Amplified products of the candidate reference genes (lane 1—PEP; lane 2—UBC, and lane 3—VAG) and of the target gene HSP17.9A (lane 4) visualised after electrophoresis on 2% agarose gels. In each gel, the molecular weight marker GeneRuler^TM 100 bp DNA Ladder Plus (#SM0321, Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) was loaded. (b) Dissociation curves of the candidate reference genes and the HSP17.9A target gene (identified in the image).

Figure 2

Relative gene expression in arbitrary units (a.u.) of the target gene, HSP17.9A, determined for the in vitro TF and TN plants at the end of each experimental phase (Ph1 to Ph4), relative to the control plants. Different lowercase letters among bars represent statistically significant differences (p ˂ 0.05). Notes: CTF and CTN—control plants of TF and TN varieties, respectively; Ph1—32 °C for 3 h (heat acclimation); Ph2—42 °C for 1 h (severe HS); Ph3—32 °C for 3 h (first recovery period); and Ph4—24 °C during 24 h (second recovery period).