Involvement of abscisic acid-dependent and -independent pathways in the upregulation of antioxidant enzyme activity during NaCl stress in cotton callus tissue.

The role of abscisic acid (ABA) in the signal transduction pathway associated with NaCl-induced up-regulation of antioxidant enzyme activity was examined in a NaCl-tolerant cotton callus cell line treated with NaCl, ABA, paraquat, or H2O2 in the presence and absence or fluridone, an inhibitor of terpene, and therefore, ABA synthesis. Treatment with NaCl resulted in a rapid increase (within 30 minutes) in the ABA levels of the callus tissue, and the NaCl, ABA, and paraquat treatments induced rapid increases in the activities of superoxide dismutase, catalase, peroxidase, and glutathione reductase. Pre-treatment with fluridone significantly suppressed the NaCl-induced increases, but only slightly delayed the increases in tissue subjected to exogenous ABA treatment. This implies that ABA is involved in the signal transduction pathway associated with the NaCl-induced up-regulation of these antioxidant enzymes. Pre-treatment with fluridone had no effect on the paraquat-induced increases, suggesting that these enzymes can also be up-regulated by a pathway other than the one mediated by ABA. Both the NaCl and paraquat treatments produced significant increases in the superoxide levels within the callus, but the increase resulting from the paraquat treatment was significantly higher than the increase resulting from the NaCl treatment. These data suggest that NaCl stress results in the production of reactive oxygen intermediates (ROI) which signals the induction of an ABA-dependent signaling pathway. The production of very high levels of ROI, such as those that occur with paraquat treatment or perhaps during periods of prolonged or extreme stress, may induce an ABA-independent signaling pathway.

The influence of alpha-amanitin on the NaCl-induced up-regulation of antioxidant enzyme activity in cotton callus tissue.

Liquid suspensions of cotton callus tissue from a NaCl-sensitive cell line and a NaCl-tolerant cell line were subjected to the following treatments: (a) 0 and 150 mM NaCl, respectively (controls); (b) 75 and 250 mM NaCl, respectively; (c) 100 ng ml(-1) alpha-amanitin; or (d) pretreatment for 2 h with 100 ng ml(-1) alpha-amanitin followed by the respective NaCl treatments. The callus tissue was harvested at 0, 0.5, 1, 2, 4, and 8h and analyzed for antioxidant enzyme activity. In the NaCl-tolerant callus, the 250 mM NaCl treatment resulted in transient 2- to 4-fold increases above the control levels in the activities of ascorbate peroxidase, catalase, glutathione reductase, and peroxidase within 1 h after treatment, while superoxide dismutase activity increased 4-fold within 4 h. This rapid increase suggests that the up-regulation of antioxidant capacity is an early response to NaCl stress and perhaps provides protection against oxidative damage until other acclimating mechanisms can be invoked. In the control callus, peroxidase activity remained unchanged, and significant increases in the other enzymes were not observed until 8 h after treatment with 75mM NaCl. Pre-treatment with alpha-amanitin prior to the NaCl treatment completely inhibited the NaCl-induced increase in the activities of all five enzymes in both cell lines. This data supports the conclusion that the NaCl-induced up-regulation of antioxidant enzyme activity in cotton callus tissue is transcriptionally regulated, proceeding via a de novo synthesis of poly(A)+RNA and is not due to the translation of existing transcripts or the mobilization of existing enzyme pools. In addition, the results suggest that it is not only the up-regulation of antioxidant activity that bestows a degree of tolerance to environmental stress, but also the speed with which this response occurs.