Functional expression and subcellular localization of the Nectria haematococca Mak1 phytoalexin detoxification enzyme in transgenic tobacco.

Medicarpin and maackiain are antifungal pterocarpan phytoalexins produced by many legumes, and are thought to be important components of the defense response of these legumes to certain fungal pathogens. The Mak1 gene from the fungal pathogen Nectria haematococca encodes an FAD-dependent mono-oxygenase, known to specifically hydroxylate the phytoalexins medicarpin and maackiain, converting them to less fungitoxic derivatives. Two binary vector constructs were made containing the coding regions from two fungal clones, a Mak1 cDNA (intronless) and a genomic (including three fungal introns) clone, regulated by an enhanced cauliflower mosaic virus 35S promoter. The constructs were introduced into tobacco to check for expression of active fungal enzyme in plant cells and for splicing of fungal introns. Leaves of tobacco plants transformed with the Mak1 cDNA construct readily metabolized infiltrated medicarpin to 1a-hydroxymedicarpin, indicating high levels of active enzyme. RT-PCR analysis of tobacco plants transformed with the Mak1 genomic construct indicated no processing of Mak1 introns, and no Mak1 activity was detected in these plants. When using plants containing the Mak1 cDNA construct, immunolocalization with a Mak1-specific antibody together with cellular fractionation indicated that Mak1 protein accumulated in the plant cytoplasm, associated with endoplasmic reticulum membranes; medicarpin biosynthetic enzymes have been localized to the same subcellular region. The Mak1 cDNA construct is therefore suitable for use in studies to selectively eliminate medicarpin accumulation to assess the relative importance of medicarpin in the antifungal defense mechanisms of alfalfa and other legumes.

Tryptophan decarboxylase is encoded by two autonomously regulated genes in Camptotheca acuminata which are differentially expressed during development and stress.

Camptothecin (CPT) is a valuable anti-cancer monoterpene alkaloid produced by the Chinese tree Camptotheca acuminata. Tryptophan decarboxylase (TDC) supplies tryptamine for the indole moiety of CPT and its derivatives, and is considered a key step in monoterpene indole alkaloid biosynthesis as it links primary and secondary metabolism. This report describes the isolation and characterization of tdc1 and tdc2, two autonomously regulated TDC genes from Camptotheca. When expressed in Escherichia coli, the products of each gene could decarboxylate tryptophan, but were inactive against tyrosine, phenylalanine and 3,4-dihydroxyphenylalanine (dopa), tdc1 was developmentally regulated, having its highest expression level in the apex, young stem and bark, tissues which also contain the highest levels of CPT. Expression of tdc1 also increased during seedling development and was correlated with alkaloid accumulation during germination. tdc2 expression was induced in Camptotheca leaf discs and cell suspension cultures treated with fungal elicitor or methyl jasmonate, treatments which did not affect tdc1 expression. Unlike tdc1, tdc2 expression was not detected in any unstressed Camptotheca tissues nor in developing seedlings. These data suggest that tdc1 may be part of a developmentally regulated chemical defense system in Camptotheca, while tdc2 serves as part of a defense system induced during pathogen challenge.

Sustained harvest of camptothecin from the leaves of Camptotheca acuminata.

Over a 12-week period, new growth was collected at different intervals from Camptotheca acuminata trees to determine whether a leaf harvest strategy would be an efficient means for the production of the alkaloid camptothecin. Because camptothecin accumulates in young leaves and because the harvesting of young tissue stimulates axillary bud outgrowth, this strategy increased the harvestable amount of camptothecin from trees in a nondestructive manner.

Molecular analysis of a new member of the opium poppy tyrosine/3,4-dihydroxyphenylalanine decarboxylase gene family.

An aromatic amino acid decarboxylase DNA fragment was generated from opium poppy (Papaver somniferum L.) genomic DNA by the PCR using primers designed from conserved amino acid sequences of other aromatic amino acid decarboxylase genes. Using this fragment as a probe, a genomic clone was isolated that encodes a new member of the opium poppy tyrosine/3,4-dihydroxyphenylalanine decarboxylase gene family (TyDC5). The predicted TyDC5 amino acid sequence shares extensive identity with other opium poppy tyrosine/3,4-dihydroxyphenylalanine decarboxylases (84%), and when expressed in Escherichia coli, it is active against tyrosine and to a lesser extent against 3,4-dihydroxyphenylalanine. Ribonuclease protection assays indicate that TyDC5 is expressed primarily in the roots of mature poppy plants. A peak of TyDC5 expression was also observed during germination, coincident with the emergence of the radicle from the seed coat. Parallel results were obtained in transgenic tobacco using a TyDC5 promoter fragment (-2060) translationally fused to the beta-glucuronidase reporter gene (GUS). IN TyDC5::GUS tobacco, GUS activity transiently appeared in all parts of the seedling during germination, but was limited to the roots in older plants. These results indicate that TyDC5 expression is transcriptionally regulated and suggest that the TyDC5 enzyme may play an important role in providing precursors for alkaloid synthesis in the roots and germinating seedlings of opium poppy.

Sites of accumulation of the antitumor alkaloid camptothecin in Camptotheca acuminata.

Camptothecin is an anticancer and anti-viral alkaloid produced by the Chinese tree Camptotheca acuminata (Nyssaceae). Despite previous reports of low levels of anticancer activity in leaves of Camptotheca acuminata, we have discovered that camptothecin accumulates to approximately 0.4% of the dry weight of young leaves. This level is 1.5-fold higher than that of the seeds and 2.5-fold higher than that of the bark, the two currently used sources of the drug. As the leaves mature, the concentration and absolute amount of camptothecin decreases rapidly. The high levels of camptothecin in young leaves could provide an easily harvested, non-destructive source of this important drug.