Effect of drought and combined drought and heat stress on polyamine metabolism in proline-over-producing tobacco plants

The roles of proline and polyamines (PAs) in the drought stress responses of tobacco plants were investigated by comparing the responses to drought alone and drought in combination with heat in the upper and lower leaves and roots of wild-type tobacco plants and transformants that constitutively over-express a modified gene for the proline biosynthetic enzyme Δ1-pyrroline-5-carboxylate synthetase (P5CSF129A; EC 2.7.2.11/1.2.1.41). In both genotypes, drought stress coincided with a decrease in relative water content (RWC) that was much less severe in the upper leaves than elsewhere in the plant. The drought also increased proline levels in both genotypes. A brief period of heat stress (2 h at 40 °C) at the end of the drought period did not significantly influence the proline levels in the upper leaves and roots but caused a further increase in the lower leaves of both genotypes. The rate at which these elevated proline levels returned to normal during the post-stress recovery period was slower in the transformants and plants that had been subjected to the combined stress. In both genotypes, drought stress significantly reduced the levels of spermidine (Spd) and putrescine (Put) in the leaves and roots relative to those for controls, and increased the levels of spermine (Spm) and diaminopropane (Dap, formed by the oxidative deamination of Spd and Spm). Spd levels may have declined due to its consumption in Spm biosynthesis and/or oxidation by polyamine oxidase (PAO; EC 1.5.3.11) to form Dap, which became more abundant during drought stress. During the rewatering period, the plants' Put and Spd levels recovered quickly and the activity of the PA biosynthesis enzymes in their leaves and roots increased substantially; this increase was more pronounced in transformants than WT plants. The high levels of Spm observed in drought stressed plants persisted even after the 24 h recovery and rewatering phase. The malondialdehyde (MDA) contents of the lower leaves of WTs increased substantially during the drought stress period; a less pronounced increase occurred in the transformants and after the application of the combined stress. After the post-stress recovery period, the MDA contents in the leaves of both genotypes were higher than those in the corresponding controls. The MDA contents of the upper leaves in plants of both genotypes remained relatively constant throughout, indicating that these leaves are preferentially protected against the adverse effects of oxidat