| Reactive oxygen species (ROS) are byproducts of metabolism. However, environmental stresses, like drought, cause increased ROS production. This triggers oxidative stress and leads to oxidative damage resulting in cellular components including lipid membranes, nucleic acids and key proteins being damaged or destroyed. Higher plants have developed the ascorbate-glutathione pathway to aid in the deactivation of cellular ROS. This pathway involves the enzymes ascorbate peroxidase (APX), superoxide dismutase (SOD) and glutathione reductase (GR) and is induced when ROS production exceeds the scavenging capabilities of the pathway.; Alfalfa (Medicago sativa cv. Malone) was analyzed to determine cellular response during drought. The protein level or enzyme activity of APX, MnSOD and GR were found to be higher in droughted alfalfa. Transcripts of genes encoding key enzymes in the leaves [glutamine synthetase (GS 2)] and nodules [leghemoglobin (Lb), nitrogenase (nifH), glutamine synthetase (GS1)] decreased in droughted alfalfa, while the transcript level of ferritin, a gene induced by drought, was found to increase. In addition, the level of lipid peroxidation (measuring thiobarbituric acid reactive substances or TBARS) was higher in droughted alfalfa compared to control. This suggests that even though the activity level of enzymes associated with ROS scavenging increases, the increase is not sufficient to protect cellular components like lipid membranes and nucleic acids from oxidative damage.; To increase the ROS scavenging capabilities of alfalfa, constructs containing either an MnSOD or APX cDNA, independently driven by the CaMV 35S constitutive promoter, were used to transform alfalfa (Medicago sativa cv. Regen SY). Independent transformants were identified by expression of their respective transgene.; A non-transformed (control) and three transformed lines were subjected to moderate drought (ΨW = −1.5 MPa). While there was no significant difference in the ΨW between the transformed and control lines during drought, the transformed lines had less wilting than control. Northern analysis of RuBisCO and GS2, in the leaves, and GS1, Lb, nifH and sucrose synthase (SuSy), in the nodules, revealed lower level of transcripts in the control line during moderate drought. GS specific activity was also found to be lower in non-transformed nodules during drought. In addition, non-transformed nodules had a 1.5-fold increase in TBARS while transformed nodules had less than a 0.5-fold increase in TBARS.; The results suggest that the transformed lines retain higher cellular function than non-transformed alfalfa during moderate drought and that increasing ROS scavenging capabilities, through genetic engineering, may aid in increasing drought tolerance in alfalfa. |
