Heavy metal hyperaccumulating plants: how and why do they do it? And what makes them so interesting?

THE JOURNAL OF BIOLOGICAL CHEMISTRY: The Biochemical Behavior of LeadL I. Influence of Calcium, Phosphorus, and Vitamin D on Lead in Blood and Bone

Plant cell cultures: Chemical factories of secondary metabolites

Transition metals such as copper are essential for many physiological processes yet can be toxic at elevated levels. Other metals (e.g. lead) are nonessential and potentially highly toxic. Plants – like all other organisms – possess homeostatic mechanisms to maintain the correct concentrations of essential metal ions in different cellular compartments and to minimize the damage from exposure to nonessential metal ions. A regulated network of metal transport, chelation, trafficking and sequestration activities functions to provide the uptake, distribution and detoxification of metal ions. Some of the components of this network have now been identified: a number of uptake transporters have been cloned as well as candidate transporters for the vacuolar sequestration of metals. Chelators and chaperones are known, and evidence for intracellular metal trafficking is emerging. This recent progress in the molecular understanding of plant metal homeostasis and tolerance is reviewed.

Molecular mechanisms of plant metal tolerance and homeostasis.

Chemistry and biology of monoterpene indole alkaloid biosynthesis

Hyperaccumulation, complexation and distribution of nickel in Sebertia acuminata

Using serpentine fluorescence as an indicator of alkaloid production in cultured CATHARANTHUS ROSEUS cells, 6 cell lines producing alkaloid in excess of 300 mg/l were selected from more than 2 × 10 5 individual colonies and their alkaloid production was monitored over a period of 8 years. Rapid loss of productivity invariably occurred during the first few months of cultivation, and spontaneous recovery of the initial production rates was never observed. Production of the indole alkaloid precursor, secologanin, followed the same pattern. Recovery of high alkaloid yielding strains was, however, possible at any time by repetition of the clonal selection procedure, but these strains were again instable. Clonal selection of high yielding plant cell strains apparently favours an inherent instability.

Instability of Indole Alkaloid Production in Catharanthus roseus Cell Suspension Cultures.

-Plant cell cultures rather than differentiated plants are the preferred material for studying secondary product formation and elucidate the enzyme pathways responsible for isoquinoline alkaloid formation. Using callus and cell suspension cultures of different BwberiJ species, we studied the incorporation ofearly precursors like tyrosine, dopa, tyramine, and dopamine into jatrorrhizine. Besides tyrosine, tyramine and, to a lesser extent, dopamine were incorporated into the aldehyde precursor moiety of the isoquinoline molecule. The use of cellfree extracts of Berberis cultures has allowed us to isolate and characterize all of the eight enzymes involved in berberine biosynthesis. (S)-Tetrahydroprotoberberine oxidase was successfully immobilized and could be used for the quantitative conversion of (S)-tetrahydroprotoberberines through their dehydroderivatives and their racemic reduction products into the pure (R)-enantiomer. This latter enzyme and the berberine bridge enzyme are localized exclusively in a new vesicle with the density 6 = 1.14; all six other enzymes of the pathway are cytoplasmic. Using highly labeled (R)and (S)-reticuline as well as scoulerine, it could be demonstrated that the transport of these compounds into vacuoles of Fumaria capreolata does not proceed by an ion-trap mechanism, but rather involves an active ATP-requiring, stereospecific carrier system acting only on the (S)and not on the @?)-enantiomer. The benzylisoquinoline alkaloid series shows an unusually rich variety of structural types ( 1,2). Many useful biogenetic schemes have been suggested (summarized in 3 ) to account for the biosynthesis of these molecules. Remarkable progress was made in the 1960s and 1970s in elucidating these pathways using living differentiated whole plants and various precursor molecules labeled with radioactive or stable isotopes. However, as was pointed out already by I.D. Spenser as early as 1968 (4 ) , more reliable information on the actual biosynthetic sequences can only be obtained by in-vitro work with the purified enzymes of biosynthesis. Tracer work only suggests probable biosynthetic sequences; the ultimate proof, however, can only be given by the isolation and characterization of the respective catalysts. In addition, more information has to be sought and gained on the compartmentation of enzymes involved in the biosynthetic chain leading to secondary plant products and on the mechanisms by which these products and their precursors are transported and stored. Because of the structural diversity expressed in the benzylisoquinoline group, and due to the pharmaceutical value that some of its members (e.g., codeine, berberine, galanthamine, hydrastine, morphine, narcotine, tetrandrine, a-tubocurarine) represent, biosynthetic work on these compounds is justified in addition to its heuristic value. CELL CULTURES.Xel1 suspension as well as callus cultures of several species of the families Berberidaceae, Ranunculaceae, Menispermaceae, Papaveraceae, and Fumariaceae produce isoquinoline alkaloids in substantial amounts (5). The most diverse isoquinoline alkaloid spectrum for a cell suspension culture thus far published has been found to occur in Fumaria capreolata (6). The structural types of benzylisoquinoline alkaloids found in cell cultures indicating also their biogenetic relationship are shown ‘Presented as a plenary lecture at the “Biologically Active Nitrogen-Containing Natural Products: Structure, Biosynthesis, and Synthesis” Symposium of the International Research Congress on Natural Products at the University of North Carolina, Chapel Hill, North Carolina, July 7-12, 1985. 726 Journal of Natural Products [Vol. 48, No. 5 in Figure 1. All of these structures have been isolated and identified in callus and/or suspension cultures except for cularineand pavine-type alkaloids, which have not yet been found to occur in undifferentiated plants. Plant cell cultures capable of producing the desired alkaloids are excellent sources of enzyme systems (7). Cells can be grown under exactly defined conditions on a large scale, thus providing any desired amount of homogeneous plant material as an enzyme source independent of seasons. As a rule, levels of enzyme activity are considerably higher in suspension cells as compared with whole plants because the time required for product synthesis under cell culture conditions is much shorter than in field-grown plants. In differentiated plants, stationary levels of enzymes involved in secondary metabolism seem to be very low in general. The high content of phenolics and other undesirable cell constituents tends to inactivate enzymes during the isolation procedure. By using plant cell cultures, however, it has been possible to gain a deeper insight into the cell-free biosynthesis of benzylisoquinoline alkaloids (7,8). Maphinandionow / Benzylisoquinolim Aporphine Pavine Benrophemthridino Protobutmino RDtopinc FIGURE 1. Structural types of benzylisoquinoline alkaloids found in plant cell cultures indicating also their biogenetic relationship. Cularine and pavine type alkaloids have not been detected so far in cell cultures. THE DISTANT PRECURSORS.-AS early as 1910, Winterstein and Trier (9) suggested that two molecules of 3,4-dihydroxyphenylalanine (DOPA) might be modified in the plant to yield dopamine and 3,4-dihydroxyphenylacetaldehyde, which in turn could condense and yield norlaudanosoline, recognized already at that time as a potential precursor for more complex isoquinoline alkaloids. Gear and Spenser (10) were the first to observe that singly-labeled tyrosine (a known precursor of DOPA) can serve as a specific precursor of hydrastine and that the two units forming this benzylisoquinoline alkaloid bear labels to a different extent. Further work, (4) established beyond doubt that tyrosine contributes to the upper (isoquinoline) as well as to the lower (benzyl) portion of these alkaloids, whereas DOPA, formed from tyrosine, is exclusively incorporated into the isoquinoline moiety (Figure 2). The nature of the true precursors during the formation of the benzylisoquinoline system has not been fully Sep-Oct 19851 Zenk et af. : Benzylisoquinoline Biosynthesis 727

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Benzylisoquinoline Biosynthesis by Cultivated Plant Cells and Isolated Enzymes

Biosynthesis of cyclic bis(bibenzyls) in Marchantia polymorpha

From differentiated plants of Catharanthus roseus (L.) G. Don, a specific enzyme was isolated and named acetyl-CoA : 17-O-deacetylvindoline 17-O-acetyltransferase, acting on the biosynthetic formation of the Aspidosperma type alkaloid vindoline.

Brigitte Deus-Neumann

Papers

Hyperaccumulation, complexation and distribution of nickel in Sebertia acuminata

Instability of Indole Alkaloid Production in Catharanthus roseus Cell Suspension Cultures.

Benzylisoquinoline Biosynthesis by Cultivated Plant Cells and Isolated Enzymes

Biosynthesis of cyclic bis(bibenzyls) in Marchantia polymorpha

Late enzymes of vindoline biosynthesis. Acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyl-transferase.