Regulation in tobacco callus of enzyme activities of the nicotine pathway

Feth, Friedhelm; Wagner, Roland; Wagner, Karl

doi:10.1007/bf00392368

Cited by 56 publications

(24 citation statements)

References 21 publications

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“…This step is considered to be the ®rst committed step in tropane alkaloid production (Robins and Walton 1993), and the activity of putrescine N-methyltransferase (PMT) declines in parallel with the inhibition of hyoscyamine production when D. stramonium is treated with NAA and kinetin (Robins et al 1991c). An equally strong correlation has been found between PMT activity and nicotine biosynthesis in transformed root cultures (Rhodes et al 1989) and suspension cultures (Feth et al 1986) of tobacco, indicating that PMT is a key enzyme in alkaloid biosynthesis in this species as well.…”

Section: Introductionmentioning

confidence: 77%

In vivo nuclear-magnetic-resonance analysis of polyamine and alkaloid metabolism in transformed root cultures of Datura stramonium L.: evidence for the involvement of putrescine in phytohormone-induced de-differentiation

Ford

Ratcliffe

Robins

1998

Planta

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The utilisation and accumulation of 15 Nlabeled metabolites by a 15 N-labeled transformed root culture of Datura stramonium L. was investigated by in vivo 15 N-nuclear-magnetic-resonance (NMR) spectroscopy. After resuspension in spent growth medium, the pools of [ 15 N]glutamate and [ 15 N]glutamine were rapidly depleted and there was an increase in the 15 N-NMR signals from conjugated putrescines and hyoscyamine. The signal from the conjugated putrescines passed through a maximum 2 d after the roots were resuspended, and it was concluded that putrescine could be stored as putrescine conjugates prior to its utilisation in other pathways. The transient accumulation of 15 Nlabel in the hydroxy-cinnamoylputrescines was reduced when the de-dierentiation of the root cultures into a suspension culture was initiated by exposure to a medium containing a-napthaleneacetic acid and kinetin. This led to the hypothesis that phytohormone-induced de-dierentiation of the root cultures required the presence of free polyamines, and this was tested using two potent inhibitors of putrescine biosynthesis, DL-a-di¯uor-omethylarginine and DL-a-di¯uoromethylornithine. Invivo 15 N-NMR spectra of roots grown in 15 N-enriched medium supplemented with these inhibitors showed that the 15 N-labelling of the conjugated polyamines and hyoscyamine was markedly reduced. DL-a-di¯uoro-methylarginine also prevented the phytohormone induced de-dierentiation of the root cultures, and this eect could be reversed by the supply of exogenous putrescine. Thus the supply of putrescine appears to play a crucial role in mediating the phytohormone induced de-dierentiation of the root culture.

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Section: Introductionmentioning

confidence: 77%

In vivo nuclear-magnetic-resonance analysis of polyamine and alkaloid metabolism in transformed root cultures of Datura stramonium L.: evidence for the involvement of putrescine in phytohormone-induced de-differentiation

Ford

Ratcliffe

Robins

1998

Planta

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“…Root-specific expression of the PMT gene (Figure 10) further confirms the site of nicotine synthesis in tobacco. Downregulation of the PMT gene by auxin also reflects the inhibitory effects of auxin on nicotine formation in tobacco callus (Ohta et al, 1978;Feth et al, 1986).…”

Section: Pmt Has Evolved From Spermidine Synthasementioning

confidence: 99%

Gene expression in tobacco low-nicotine mutants.

et al. 1994

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Two nuclear genes, Nicl and Nic2, regulate nicotine levels in tobacco. nicl and nic2 are semidominant mutations in Burley 21 that reduce leaf nicotine levels and the activities of multiple enzymes in the nicotine pathway and simultaneously increase polyamine levels in cultured roots. Cultured roots homozygous for both mutations were used to isolate two cDNAs by subtraction hybridization; the transcript levels of these two cDNAs were much lower in the mutant roots than in the wild-type roots. The A411 gene encodes a 41-kD protein with considerable homology to mammalian spermidine synthase, whereas the A622 gene encodes a 35-kD protein with high homology to isoflavone reductase. When these genes were expressed in Escherichia coli, A411 had no spermidine synthase activity but did show putrescine N-methyltransferase activity, which is the first enzyme committed to the nicotine biosynthetic pathway, and A622 did not show isoflavone reductase activity. Both the methyltransferase and A622 genes are predominantly expressed in the root, and their expression levels in cultured roots are coordinately decreased by the nic mutations in the order of wild type > nic2 > nicl > nicl nic2. Removal of tobacco flower heads and young leaves rapidly and coordinately induced both genes in the root. Further, exogenous supply of auxin down-regulated both genes in cultured tobacco roots. These results suggest that N i c l and Nic2 are regulatory genes for nicotine biosynthesis.

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“…(Dawson 1942;Baldwin 1989Baldwin , 1991Baldwin et al 1993Baldwin et al , 1994b. Moreover, the key regulatory enzyme in nicotine biosynthesis, putrescine N-methyltransferase and the mRNA coding for this enzyme, is wound-inducible and localized in the roots (Mizusaki et al 1973;Saunders and Bush 1979;Feth et al 1986;Hibi et al 1994). The large spatial separation between the site of stimulus (leaves) and response (roots) make this system useful for differentiating between local and systemic transduction pathways.…”

Section: Introductionmentioning

confidence: 99%

Quantification, correlations and manipulations of wound-induced changes in jasmonic acid and nicotine in Nicotiana sylvestris

Baldwin

Zhang

Diab

et al. 1997

Planta

283

201

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Jasmonic acid (JA) is thought to be part of a signal-transduction pathway which dramatically increases de-novo nicotine synthesis in the roots and increases whole-plant (WP) nicotine pools in response to the wounding of the leaves in Nicotiana sylvestrisSpegazzini and Comes (Solanaceae). We report the synthesis of a doubly labeled JA ([1, 2-13 C]JA) and use it as an internal standard to quantify by gas chromatography-mass spectrometry the changes in root and shoot JA pools in plants subjected to differing amounts of standardized leaf wounding. Wounding increased JA pools 10-fold locally in damaged leaves within 90 min and systemically in the roots (3.5-fold) 180 min after wounding. If JA functions as an intermediary between stimulus and response, quantitative relationships among the stimulus, JA, and the response should exist. To examine these relationships, we varied the number of punctures in four leaves and quantified both the resulting JA in damaged leaves after 90 min and the resulting WP nicotine concentration after 5 d. We found statistically significant, positive relationships among number of leaf punctures, endogenous JA, and WP nicotine accumulation. We used two inhibitors of wound-induced nicotine production, methyl salicylate and indole-3-acetic acid, to manipulate the relationships between wound-induced changes in JA and WP nicotine accumulation. Since wounding and the response to wounding occur in widely separated tissues, we applied inhibitors to different plant parts to examine their effects on the local and systemic components of this response. In all experiments, inhibition of the wound-induced increase in leaf JA 90 min after wounding was associated with the inhibition of the nicotine response 5 d after wounding. We conclude that wound-induced increases in leaf JA are an important component of this longdistance signal-transduction pathway.Abbreviations: JA = jasmonic acid; WP = whole plant; MSA = methyl salicylate

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Regulation in tobacco callus of enzyme activities of the nicotine pathway

Cited by 56 publications

References 21 publications

In vivo nuclear-magnetic-resonance analysis of polyamine and alkaloid metabolism in transformed root cultures of Datura stramonium L.: evidence for the involvement of putrescine in phytohormone-induced de-differentiation

In vivo nuclear-magnetic-resonance analysis of polyamine and alkaloid metabolism in transformed root cultures of Datura stramonium L.: evidence for the involvement of putrescine in phytohormone-induced de-differentiation

Gene expression in tobacco low-nicotine mutants.

Quantification, correlations and manipulations of wound-induced changes in jasmonic acid and nicotine in Nicotiana sylvestris

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