Radiocarbon Flux Measurements Reveal Mechanistic Insight into Heat-Stress Induction of Nicotine Biosynthesis in Nicotiana attenuata

The effect of high-temperature (HT) stress on nicotine biosynthesis in Nicotiana attenuata was examined. Nicotine content was measured in mature leaves, young sink leaves, and in roots from well-watered plants grown at 25 degrees C as controls and from plants exposed to 38 degrees C and 43 degrees C temperatures applied for 24, 48, 72, and 96 h duration. At 38 degrees C, all leaf nicotine levels were significantly less than control plants for up to 72 h exposure but rose sharply thereafter to levels significantly greater than controls with 96 h exposure. In contrast, plants exposed to 43 degrees C never exhibited a reduction in leaf nicotine content and showed an increase in content with just 48 h exposure. Using radioactive (CO2)-C-11 and (NO3-)-N-13, we found that HT stress reduced both CO2 fixation and nitrate uptake. Furthermore, radiocarbon flux analysis revealed that 'new' carbon partitioning (as C-11) into the C-11-radiolabeled amino acid (AA) pool was significantly reduced with HT stress as were yields of [C-11]-aspartic acid, an important AA in nicotine biosynthesis, and its beta-amido counterpart [C-11]-asparagine. In contrast, [C-12]-aspartic acid levels appeared unaffected at 38 degrees C but were elevated at 43 degrees C relative to controls. [C-12]-Asparagine levels were noted to be elevated at both stress temperatures. Since HT reductions in carbon input and nitrogen uptake were noted to impede de novo AA biosynthesis, protein degradation at HT was examined as a source of AAs. Here, leaf total soluble protein (TSP) content was reduced 39% with long exposures to both stress temperatures. However, Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) which was 41% TSP appeared unaffected. Altogether, these results support the theory that plant proteins other than Rubisco degrade at elevated temperatures freeing up essential AAs in support of nicotine biosynthesis.