Overexpression of DfRaf from Fragrant Woodfern (Dryopteris fragrans) Enhances High-Temperature Tolerance in Tobacco (Nicotiana tabacum)

Abstract

Heat stress seriously affects medicinal herbs’ growth and yield. Rubisco accumulation factor (Raf) is a key mediator regulating the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which plays important roles in carbon assimilation and the Calvin cycle in plants. Raf has been studied in many plants, but has rarely been studied in the important medicinal plant fragrant woodfern (Dryopteris fragrans). The aim of this study was to analyze the effects of Raf on carbohydrate metabolism and the response to heat stress in medicinal plants. In this study, high temperature treatment upregulated the expression of DfRaf, which was significantly higher than that of phosphoribokinase (DfPRK), Rubisco small subunits (DfRbcS), Rubisco large subunits (DfRbcL) and Rubisco activase (DfRCA). The subcellular localization showed that the DfRaf proteins were primarily located in the nucleus; DfPRK, DfRbcS, DfRbcL and DfRCA proteins were primarily located in the chloroplast. We found that overexpression of DfRaf led to increased activity of Rubisco, RCA and PRK under high-temperature stress. The H2O2, O2− and MDA content of the DfRaf-OV-L2 and DfRaf-OV-L6 transgenic lines were significantly lower than those of WT and VC plants under high-temperature stress. The photosynthetic pigments, proline, soluble sugar content and ROS-scavenging ability of the DfRaf-OV-L2 and DfRaf-OV-L6 transgenic lines were higher than those of WT and VC plants under high-temperature stress. The results showed that overexpression of the DfRaf gene increased the Rubisco activity, which enhanced the high-temperature tolerance of plants.