High levels of tryptamine accumulation in transgenic tobacco expressing tryptophan decarboxylase

A full-length complementary DNA clone encoding tryptophan decarboxylase (TDC; EC 4.1.1.28) from Catharanthus roseus (De Luca V, Marineau C, Brisson N [1989] Proc Natl Acad Sci USA 86: 2582-2586) driven by the CaMV 35S promoter was introduced into tobacco (Nicotiana tabacum) to direct the synthesis of the protoalkaloid tryptamine from endogenous tryptophan. Young, fully expanded leaves of CaMV 35S-TDC transformed plants had from four to 45 times greater TDC activity than did controls. Tryptamine accumulated in transgenic plants to levels that were directly proportional to their TDC specific activity. Despite their increased tryptamine content, the growth and development of the CaMV 35S-TDC plants appeared normal with no significant differences in indole-3-acetic acid levels between high tryptamine and control plants. Plants with the highest TDC activity contained more than 1 milligram of tryptamine per gram fresh weight, a 260-fold increase over controls.     

Metabolic engineering of plant secondary products

Plants interact with their environment by producing a diverse array of secondary metabolites. Many of these compounds are valued for their medicinal, industrial or agricultural properties. Other secondary products are toxic or otherwise undesirable and can reduce the commercial value of crops. Gene transfer technology offers new opportunities to modify directly plant secondary product synthesis through metabolic engineering. This article reviews some of the strategies which have been used to increase or decrease the synthesis of specific plant metabolites, as well as methods for expanding the biosynthetic capabilities of individual species.     

Tryptophan Decarboxylase,Tryptamine, and Reproduction of the Whitefly

Tryptophan decarboxylase (TDC) from Catharanthus roseus (periwinkle) converts tryptophan to the indole-alkaloid tryptamine. When the TDC gene was expressed in transgenic tobacco, the 55-kD TDC enzyme and tryptamine accumulated. Bemisia tabaci (sweetpotato whitefly) reproduction on transgenic plants decreased up to 97% relative to controls. Production of tryptamine, its derivatives, or other products resulting from TDC activity may discourage whitefly reproduction and provide a single-gene-based plant protection strategy.     

Somatic embryogenesis in the opium poppy, Papaver somniferum

A procedure is described for the induction of somatic embryos in the opium poppy. Papaver somniferum L. Callus was obtained from seedling hypocotyls on an agar solidified medium [Murashige and Skoog (1962) Physiol. Plant. 15: 473–497] containing 0.25 mg/l (1.2 μ M ) kinetin and 2.0 mg/l (10.7 μ M ) naphtalene acetic acid (NAA). Suspension cultures were initiated from callus using a liquid medium in which 2.0 mg/l (9.0 μ M ) 2,4-dichlorophenoxyacetic acid was substituted for NAA. Meristemoids, spheres of closely packed cells, developed in suspensions and on the surface of a few callus cultures. Differentiation of meristemoids into somatic embryos was accomplished by removing growth regulators from the liquid medium. Embryoids appeared morphologically normal and similar to torpedo stage embryos, however, they possessed mature tracheary elements and laticifers in areas that should have contained only procambium. Whole plants have been obtained by placing embryos in the light on solid medium that also lacked growth regulators.     

Bayesian Computation Emerges in Generic Cortical Microcircuits through Spike-Timing-Dependent Plasticity

The principles by which networks of neurons compute, and how spike-timing dependent plasticity (STDP) of synaptic weights generates and maintains their computational function, are unknown. Preceding work has shown that soft winner-take-all (WTA) circuits, where pyramidal neurons inhibit each other via interneurons, are a common motif of cortical microcircuits. We show through theoretical analysis and computer simulations that Bayesian computation is induced in these network motifs through STDP in combination with activity-dependent changes in the excitability of neurons. The fundamental components of this emergent Bayesian computation are priors that result from adaptation of neuronal excitability and implicit generative models for hidden causes that are created in the synaptic weights through STDP. In fact, a surprising result is that STDP is able to approximate a powerful principle for fitting such implicit generative models to high-dimensional spike inputs: Expectation Maximization. Our results suggest that the experimentally observed spontaneous activity and trial-to-trial variability of cortical neurons are essential features of their information processing capability, since their functional role is to represent probability distributions rather than static neural codes. Furthermore it suggests networks of Bayesian computation modules as a new model for distributed information processing in the cortex.     

Camptothecin, over four decades of surprising findings

Camptothecin (CPT) is a modified monoterpene indole alkaloid produced by Camptotheca acuminata (Nyssaceae), Nothapodytes foetida, Pyrenacantha klaineana, Merrilliodendron megacarpum (Icacinaceae), Ophiorrhiza pumila (Rubiaceae), Ervatamia heyneana (Apocynaceae) and Mostuea brunonis (Gelsemiaceae), species belonging to unrelated orders of angiosperms. From the distribution of CPT and other secondary metabolites, it has been postulated that the genes encoding enzymes involved in their biosynthesis evolved early during evolution. These genes were presumably not lost during evolution but might have been "switched off" during a certain period of time and "switched on" again at some later point. The CPT derivatives, irinotecan and topotecan, are used throughout the world for the treatment of various cancers, and over a dozen more CPT analogues are currently at various stages of clinical development. The worldwide market size of irinotecan/topotecan in 2002 was estimated at about $750 million and at $1 billion by 2003. In spite of the rapid growth of the market, CPT is still harvested by extraction from bark and seeds of C. acuminata and N. foetida. All parts of C. acuminata contain some CPT, although the highest level is found in young leaves (approximately 4-5 mg g(-1) dry weight), approximately 50% higher than in seeds and 250% higher than in bark. The development of hairy root cultures of O. pumila and C. acuminata, and the cloning and characterization of genes encoding key enzymes of the pathway leading to CPT formation in plants has opened new possibilities to propose alternative and more sustainable production systems for this important alkaloid.     

L-Gulono-1,4-lactone oxidase expression rescues vitamin C-deficient Arabidopsis (vtc) mutants

Vitamin C (L-ascorbic acid) has important antioxidant and metabolic functions in both plants and animals, humans have lost the ability to synthesize it. Fresh produce is the major source of vitamin C in the human diet yet only limited information is available concerning its route(s) of synthesis in plants. In contrast, the animal vitamin C biosynthetic pathway has been elucidated since the 1960s. Two biosynthetic pathways for vitamin C in plants are presently known. The D-mannose pathway appears to be predominant in leaf tissue, but a D-galacturonic acid pathway operates in developing fruits. Our group has previously shown that transforming lettuce and tobacco with a cDNA encoding the terminal enzyme of the animal pathway, L-gulono-1,4-lactone oxidase (GLOase, EC 1.1.3.8), increased the vitamin C leaf content between 4- and 7-fold. Additionally, we found that wild-type (wt) tobacco plants had elevated vitamin C levels when fed L-gulono-1,4-lactone, the animal precursor. These data suggest that at least part of the animal pathway may be present in plants. To further investigate this possibility, wild-type and vitamin-C-deficient Arabidopsis thaliana (L.) Heynh (vtc) plants were transformed with a 35S: GLOase construct, homozygous lines were developed, and vitamin C levels were compared to those in untransformed controls. Wild-type plants transformed with the construct showed up to a 2-fold increase in vitamin C leaf content compared to controls. All five vtc mutant lines expressing GLOase had a rescued vitamin C leaf content equal or higher (up to 3-fold) than wt leaves. These data and the current knowledge about the identity of genes mutated in the vtc lines suggest that an alternative pathway is present in plants, which can bypass the deficiency of GDP-mannose production of the vtc1-1 mutant and possibly circumvent other steps in the D-mannose pathway to synthesize vitamin C.     

The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding

Carbon catabolite repression allows bacteria to rapidly alter the expression of catabolic genes in response to the availability of metabolizable carbon sources. In Bacillus subtilis, this phenomenon is controlled by the HPr kinase (HprK) that catalyzes ATP-dependent phosphorylation of either HPr (histidine containing protein) or Crh (catabolite repression HPr) on residue Ser-46. We report here that B. subtilis HprK forms homo-oligomers constituted most likely of eight subunits. Related to this complex structure, the enzyme displays strong positive cooperativity for the binding of its allosteric activator, fructose 1,6-bisphosphate, as evidenced by either kinetics of its phosphorylation activity or the intrinsic fluorescence properties of its unique tryptophan residue, Trp-235. It is further shown that activation of HPr phosphorylation by fructose 1,6-bisphosphate essentially occurs at low ATP and enzyme concentrations. A positive cooperativity was also detected for the binding of natural nucleotides or their 2'(3')-N-methylanthraniloyl derivatives, in either phosphorylation or fluorescence experiments. Most interestingly, quenching of the HprK tryptophan fluorescence by using either iodide or acrylamide revealed a heterogeneity of tryptophan residues within the population of oligomers, suggesting that the enzyme exists in two different conformations. This result suggests a concerted-symmetry model for the catalytic mechanism of positive cooperativity displayed by HprK.