Preferential induction of a 9-lipoxygenase by salt in salt-tolerant cells of Citrus sinensis L. Osbeck.

Recent findings in our laboratory suggested that in citrus cells the salt induction of phospholipid hydroperoxide glutathione peroxidase, an enzyme active in cellular antioxidant defense, is mediated by the accumulation of hydroperoxides. Production of hydroperoxides occurs as a result of non-enzymatic auto-oxidation or via the action of lipoxygenases (LOXs). In an attempt to resolve the role of LOX activity in the accumulation of peroxides we analyzed the expression of this protein under stress conditions and in cells of Citrus sinensis L. differing in sensitivity to salt. Lipoxygenase expression was induced very rapidly only in the salt-tolerant cells and in a transient manner. The induction was specific to salt stress and did not occur with other osmotic-stress-inducing agents, such as polyethylene glycol or mannitol, or under hot or cold conditions, or in the presence of abscisic acid. The induction was eliminated by the antioxidants dithiothreitol and kaempferol, thus once more establishing a correlation between salt and oxidative stresses. Analyses of both in vitro and in vivo products of LOX revealed a specific 9-LOX activity, and a very fast reduction of the hydroperoxides to the corresponding hydroxy derivatives. This suggests that one of the metabolites further downstream in the reductase pathway may play a key role in triggering defense responses against salt stress.

Regulation of stress-induced phospholipid hydroperoxide glutathione peroxidase expression in citrus

Recent findings in our laboratory showed that in citrus cells, salt treatment induced the accumulation of mRNA and a protein corresponding to phospholipid hydroperoxide glutathione peroxidase (PHGPX), an enzyme active in the cellular antioxidant system. The protein and its encoding gene, csa, were isolated and characterized, and the expected enzymatic activity was demonstrated (G. Ben-Hayyim et al., 1993, Plant Sci. 88: 129-140; D. Holland et al., 1993, Plant Mol. Biol. 21: 923-927; D. Holland et al., 1994, FEBS Lett. 337: 52-55; T. Beeor-Tzahar et al., 1995, FEBS Lett. 366: 151-155). In an attempt to find out how salt induces the expression of an antioxidant enzyme, the regulation of PHGPX in citrus cells was studied at both the mRNA transcript and the protein levels. A high and transient response at the csa mRNA level was observed after 4-7 h of exposing salt-sensitive cells to NaCl, or abscisic acid, whereas no response could be detected in the salt-tolerant cells under the same conditions. tert-Butylhydroperoxide, a substrate of PHGPX, induced csa mRNA transcripts after only 2 h, and abolished the differential response between salt-sensitive and salt-tolerant cells. On the basis of these results and those obtained under heat and cold stresses, it is suggested that csa is directly induced by the substrate of its encoded enzyme PHGPX, and that salt induction occurs mainly via the production of reactive oxygen species and hydroperoxides.

Salt and oxidative stress: similar and specific responses and their relation to salt tolerance in citrus.

Salt damage to plants has been attributed to a combination of several factors including mainly osmotic stress and the accumulation of toxic ions. Recent findings in our laboratory showed that phospholipid hydroperoxide glutathione peroxidase (PHGPX), an enzyme active in the cellular antioxidant system, was induced by salt in citrus cells and mainly in roots of plants. Following this observation we studied the two most important enzymes active in elimination of reactive oxygen species, namely, superoxide dismutase (SOD) and ascorbate peroxidase (APX), to determine whether a general oxidative stress is induced by salt. While Cu/Zn-SOD activity and cytosolic APX protein level were similarly induced by salt and methyl viologen, the response of PHGPX and other APX isozymes was either specific to salt or methyl viologen, respectively. Unlike PHGPX, cytosolic APX and Cu/Zn-SOD were not induced by exogenously added abscisic acid. Salt induced a significant increase in SOD activity which was not matched by the subsequent enzyme APX. We suggest that the excess of H2O2 interacts with lipids to form hydroperoxides which in turn induce and are removed by PHGPX. Ascorbate peroxidase seems to be a key enzyme in determining salt tolerance in citrus as its constitutive activity in salt-sensitive callus is far below the activity observed in salt-tolerant callus, while the activities of other enzymes involved in the defence against oxidative stress, namely SOD, glutathione reductase and PHGPX, are essentially similar.