Untitled

Abstract

Plants contain a large number of proteins homologous to isoflavone reductase, an NADPH-dependent reductase involved in the biosynthesis of isoflavonoid phytoalexins in legumes. Although some are bonafide isoflavone reductases, others may catalyze distinct reductase reactions. Two tobacco genes, TP7 and A622, encoding isoflavone reductase-like proteins, had been previously identified from their unique expression patterns, but their functions were not known. We show here that TP7 is a tobacco phenylcoumaran benzylic ether reductase involved in lignan biosynthesis, but that A622 is not. To gain insight into the possible function of A622, we analyzed in detail the expression patterns of the A622 gene by RNA and protein blots, immunohistochemistry, and its promoter expression in transgenic Nicotiana sylvestris roots. The A622 expression patterns were qualitatively similar to those of putrescine N-methyltransferase, the first enzyme in nicotine biosynthesis, suggesting that A622 may function in the metabolism of nicotine or related alkaloids.

A PIP-family protein is required for biosynthesis of tobacco alkaloids

“…The application of methyl jasmonate (MeJA) strongly up-regulates A622 expression, while the simultaneous addition of ethylene suppresses the MeJA-induced expression (Shoji et al 2000a(Shoji et al , 2002. Finally, the NIC regulatory loci for nicotine biosynthesis tightly control the expression of A622 (Hibi et al 1994;Shoji et al 2002). These expression profiles are very similar to those of the structural genes encoding enzymes involved in nicotine biosynthesis (Fig.…”

“…Four-day-old BY-2 cells were elicited with 50 lM MeJA (Wako Chemical, Osaka, Japan) in an auxinfree medium , whereas 1-month-old tobacco plants (cv. Burley 21) were treated with 100 lM MeJA for 1 day and the root tissues were analyzed for the up-regulated expression of nicotine biosynthesis-related genes (Shoji et al 2002). Tobacco alkaloids and their metabolic precursors were analyzed after 2 days of MeJA treatment.…”

“…Recently, two eugenol synthases were also reported to be included in this complex clade (Koeduka et al 2008). However, tobacco has an orthologous PCBER, which is distinct from A622, and recombinant A622 does not show PCBER activity (Shoji et al 2002). Since pterocarpans are phytoalexins found only in legumes, A622 does not appear to be a pterocarpan reductase either.…”

“…Expression patterns of A622 (see below) suggest that A622 is unlikely to be a synthase which produces volatile phenylpropenes, such as eugenol and isoeugenol, in many herbs (Koeduka et al 2008). A622 must be a reductase since the recombinant protein binds NADPH (Shoji et al 2002), but its enzymatic reaction remains elusive.…”

“…A622 is expressed specifically in the root, most strongly in the cortex of the root tip and in the outer cortex layer and endodermis in the differentiated region (Hibi et al 1994;Shoji et al 2002). The application of methyl jasmonate (MeJA) strongly up-regulates A622 expression, while the simultaneous addition of ethylene suppresses the MeJA-induced expression (Shoji et al 2000a(Shoji et al , 2002. Finally, the NIC regulatory loci for nicotine biosynthesis tightly control the expression of A622 (Hibi et al 1994;Shoji et al 2002).…”