To identify the signaling mechanisms and components that are involved in regulation of a promoter for a gene involved in a secondary pathway I studied the nicotinic alkaloid biosynthetic pathway using various N. tabacum tissues. Nicotine and tropane alkaloids are widely known to be synthesized predominantly in the roots of species that produce pyrrolinium ring containing alkaloids. Putrescine Nmethyltransferase (PMT) catalyzes the first committed step in the biosynthesis of these alkaloid secondary products and earlier studies have indicated that PMT gene expression is restricted to root tissue in Solanaceae plants. To further elucidate the factors that govern the regulation of alkaloid synthesis, expression patterns dictated by the 5'-flanking region of one of the members of the PMT -gene family, NsPMT3, using the b-glucuronidase (GUS) reporter gene were examined. Various treatments were used to characterize the nature of signaling in various tissues of seedlings, whole plants and callus. High expression levels were detected in root tissue and no expression was detected in leaves, in agreement with previous studies. However, mechanically wounded leaves resulted in highly localized PMT expression. This wound-induced expression was transient, with maximum levels occurring immediately after wounding and diminishing after approximately 24 h. RT-PCR analysis of mRNA isolated from wild-type plants also indicated upregulation of PMT expression in leaves upon wounding as well as very low transcript levels in unwounded leaves. Low levels of PMT activity were detected in leaf tissue, and this activity did not increase significantly upon wounding. Transgenic callus material showed strong repression of PMT promoter activity in the presence of light and auxin, whereas dark conditions and the absence of auxin upregulated PMT promoter activity. Reactive oxygen species have been implicated in signaling. When treated with the scavengers of reactive oxygen species (ROS), dimethylthiourea (DMTU) or catalase, tobacco callus tissue, which displays highly repressed alkaloid synthesis under normal culture conditions in the light, exhibited significant induction of PMT promoter activity and alkaloid accumulation. It is thought that light repression signals through an ROS intermediate to affect changes in alkaloid pathway gene expression. Upregulation of PMT-promoter activity was observed upon treatment with JA (jasmonic acid) or darkness in roots of very young transgenic seedlings. Treatment with auxin, salicylic acid (SA) and H2O2, on the other hand, was found to highly repress PMT promoter activity. Action of other ROS such as nitric oxide and superoxide radicals on PMT expression is not clear but probably play less of a role, compared to H2O2. Consistent with this content ion, treatment with light or glucose oxidase (GOX) and glucose to generate H2O2, also repressed alkaloid accumulation, and treatment of seedlings to dark conditions, the ROS scavenger DMTU, or jasmonic acid resulted in alkaloid accumulation. Long distance signaling from leaves to roots is also suspected to involve ROS, as leaves treated with GOX and glucose exhibited repressed PMT promoter activity in roots. The responses of the PMT promoter to auxin, salicylic acid and H2O2 treatments were conserved as sho wn by similar responses of the N. tabacum PMT promoter when examined in transgenic Arabidopsis, thereby suggesting that these molecules signal through a conserved mechanism. Thus, ROS is strongly implicated in acting as an intermediate in these signaling processes with H2O2 proposed as a major signaling component.

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