The as-1 Promoter Element Is an Oxidative Stress-Responsive Element and Salicylic Acid Activates It via Oxidative Species

Abstract

The activation sequence-1(as-1)-like element found in the promoter of some glutathione S-transferase (GST) genes, has been previously described as a salicylic acid (SA)- and auxin-responsive element. In this paper, we tested the hypothesis that the activating effect of SA on the as-1 element is mediated by oxidative species. Supporting this hypothesis, our results show that the antioxidants dimethylthiourea (DMTU) and 3-t-butyl-4-hydroxy-anizole (BHA) inhibit the SA-induced transcription of genes controlled by as-1 elements in tobacco (Nicotiana tabacum) plants [i.e. GNT35gene coding for a GST and (as-1)₄ /β-glucuronidase(GUS) reporter transgene]. DMTU and BHA also inhibit SA-activated as-1-binding activity in nuclear extracts. Further support for the hypothesis that the as-1 element is activated by oxidative species comes from our result showing that light potentiates the SA-induced activation of the as-1element. Furthermore, methyl viologen, a known oxidative stress inducer in plants, also activates the as-1 element. Increasing H₂O₂ levels by incubation with H₂O₂ or with the catalase inhibitor 3-amino-1,2,5-triazole does not activate the (as-1)₄ /GUS gene. On the contrary, 3-amino-1,2,5-triazole inhibits the activating effect of SA on the (as-1)₄ /GUSgene. These results suggest that oxidative species other than H₂O₂ mediate the activation of theas-1 element by SA. Our results also suggest that even though the as-1 binding activity is stimulated by oxidative species, this is not sufficient for the transactivation of genes controlled by this element. The complex interplay between SA and reactive oxygen species in the transcriptional activation of defense genes is discussed.