Overexpression of a tryptophan decarboxylase cDNA inCatharanthus roseus crown gall calluses results in increased tryptamine levels but not in increased terpenoid indole alkaloid production

The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28) catalyses a key step in the biosynthesis of terpenoid indole alkaloids inC. roseus by converting tryptophan into tryptamine. Hardly anytdc mRNA could be detected in hormone-independent callus and cell suspension cultures transformed by the oncogenic T-DNA ofAgrobacterium tumefaciens. Supply of tryptamine may therefore represent a limiting factor in the biosynthesis of alkaloids by such cultures. To investigate this possibility, chimaeric gene constructs, in which atdc cDNA is linked in the sense or antisense orientation to the cauliflower mosaic virus 35S promoter and terminator, were introduced inC. roseus cells by infecting seedlings with an oncogenicA. tumefaciens strain. In the resulting crown gall tumour calluses harbouring thetdc sense construct, an increased TDC protein level, TDC activity and tryptamine content but no significant increase in terpenoid indole alkaloid production were observed compared to empty-vector-transformed tumour calluses. In tumour calluses containing thetdc antisense construct, decreased levels of TDC activity were measured. Factors which might be responsible for the lack in increased terpenoid indole alkaloid production in thetdc cDNA overexpressing crown gall calluses are discussed.     

Increased production of serotonin by suspension and root cultures ofPeganum harmala transformed with a tryptophan decarboxylase cDNA clone fromCatharanthus roseus

Cell suspension and root cultures ofPeganum harmala were established expressing a tryptophan decarboxylase cDNA clone fromCatharanthus roseus under the control of the cauliflower mosaic virus (CaMV) 35S promoter and terminator sequences. The tryptophan decarboxylase activity of some of the transgenic lines was greatly enhanced (25–40 pkat/mg protein) as compared to control cultures (1–5 pkat per mg protein) and remained high during the growth cycle. While the levels of tryptamine, the product of the reaction catalysed by tryptophan decarboxylase, were unchanged in the transgenic lines, their serotonin contents were enhanced up to 10-fold, reaching levels of 1.5 to 2% dry mass. Thus, tryptamine produced by the engineered reaction was apparently immediately used for enhanced serotonin biosynthesis. The yields of serotonin in transgenic lines overexpressing tryptophan decarboxylase activity were further enhanced to 3–5% dry mass by feedingl-tryptophan, while no or only minor effects were seen when control cultures were fed. These data demonstrate that the production of a plant secondary metabolite can be enhanced greatly via genetic manipulation of the level of activity of the rate-limiting enzyme. The amounts of -carboline alkaloids, the other tryptamine-derived metabolites ofP. harmala, in contrast, were not affected by the overproduction of tryptamine. The information needed for successfully predicting manipulations that enhance production of a secondary metabolite is discussed.     

Wound-inducible biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine in tryptophan and tyrosine decarboxylase transgenic tobacco lines

The wound-activated biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine was compared in untransformed and transgenic tobacco (Nicotiana tabacum) lines that express tryptophan decarboxylase (TDC), tyrosine decarboxylase (TYDC), or both activities. Transgenic in vitro-grown tobacco lines expressing TDC activity accumulated high levels of tryptamine but not hydroxycinnamic amides of tryptamine. In contrast, transgenic tobacco lines expressing TYDC accumulated tyramine as well as p-coumaroyltyramine and feruloyltyramine. The MeOH-soluble and cell wall fractions showed higher concentrations of wound-inducible p-coumaroyltyramine and feruloyltyramine, especially at and around wound sites, in TYDC and TDC xTYDC tobacco lines compared to wild-type or TDC lines. All the enzymes involved in the biosynthesis of hydroxycinnamic acid amides of tyramine were found to be similarly wound inducible in all tobacco genotypes investigated. These results provide experimental evidence that, under some circumstances, TYDC activity can exert a rate-limiting control over the carbon flux allocated to the biosynthesis of hydroxycinnamic acid amides of tyramine.     

Are tissue cultures of Peganum harmala a useful model system for studying how to manipulate the formation of secondary metabolites?

This article reviews our present knowledge on the formation of tryptophan derived secondary metabolites in tissue cultures of Peganum harmala. With the presence of -carboline alkaloids and serotonin, P. harmala contains two rather simple, interrelated biosynthetic pathways. The long term disadvantage of low and unstable productivity of P. harmala suspension culture has recently been overcome by establishing highly productive hairy root cultures. The first -carboline alkaloid biosynthetic enzymes, specific for the O-methylation of harmalol and harmol as well as for the oxidation of harmaline to harmine, have been detected in these cultures, and they should thus provide a suitable source for studying the yet unknown initial two enzymatic steps of -carboline alkaloid biosynthesis. Seedlings of P. harmala have also been successfully transformed with constructed strains of Agrobacterium, as demonstrated by the overexpression of a tryptophan decarboxylase gene from Catharanthus roseus in cultures of P. harmala. In such transgenic cultures a large overproduction of serotonin was observed. The relative simplicity of these pathways and the rather easy handling of the cultures could make P. harmala a useful and attractive model system for studying the interaction, regulation and manipulation of secondary pathways in cultured cells.Abbreviations TDC tryptophan decarboxylase - tdc gene of tryptophan decarboxylase     

Putative sites of cytokinin action during their enhancing effect on indole alkaloid accumulation in periwinkle cell suspensions

Summary The effects of cytokinins on the different branches of the indole alkaloid pathway were investigated in Catharanthus roseus cell cultures. Addition of zeatin to a 2,4-dichlorophenoxyacetic acid-containing medium decreased tryptamine levels and increased the bioconversion of secologanin to ajmalicine. Zeatin also enhanced the geraniol-10 hydroxylase activities and modified the indole alkaloid pattern. The results are discussed in the light of previous works showing that cytokinins have a positive effect on indole alkaloid accumulation in some lines of C. roseus.Abbreviations BSTFA bis(trimethylsilyl) trifluoroacetamide - CK cytokinin - 2,4-D 2,4-dichlorophenoxyacetic acid - dw dry weight - G-10H geraniol-10 hydroxylase - NAA naphthaleneacetic acid - SE standard error - TDC tryptophan decarboxylase - Z zeatin     

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.     

Subcellular localization of tryptophan decarboxylase,strictosidine synthase and strictosidine glucosidase in suspension cultured cells of Catharanthus roseus and Tabernaemontana divaricata

The subcellular localization of tryptophan decarboxylase, strictosidine synthase and strictosidine glucosidase in suspension cultured cells of Catharanthus roseus (L.) G. Don and Tabernaemontana divaricata (L.) R. Br. ex Roem. et Schult, was investigated. It was found that tryptophan decarboxylase is an extra-vacuolar enzyme, whereas strictosidine synthase is active inside the vacuole. Strong indications were obtained for the localization of strictosidine glucosidase on the outside of the tonoplast. The results suggest that tryptamine is transported into the vacuole where it is condensed with secologanin to form strictosidine, and that strictosidine passes the tonoplast and is subsequently hydrolysed outside the vacuole.Abbreviations AM -mannosidase - EDTA ethylenediaminetetraacetic acid - Hepes N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - HPLC highperformance liquid chromatography - MDH malate dehydrogenase - SG strictosidine glucosidase - SSS strictosidine synthase - TDC tryptophan decarboxylase     

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.     

Enzymatic features of serotonin biosynthetic enzymes and serotonin biosynthesis in plants

Serotonin, a pineal hormone in mammals, is found in a wide range of plant species at detection levels from a few nanograms to a few milligrams, and has been implicated in several physiological roles, such as flowering, morphogenesis and adaptation to environmental changes. Serotonin synthesis requires two enzymes, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H), with TDC serving as a rate-limiting step because of its high K_m relation to the substrate tryptophan (690 µM) and its undetectable expression level in control plants. However, T5H and downstream enzymes, such as serotonin N-hydroxycinnamoyl transferase (SHT), have low K_m values with corresponding substrates. This suggests that the biosynthesis of serotonin or serotonin-derived secondary metabolites is restricted to cellular stages when high tryptophan levels are present.Key words: feruloylserotonin, serotonin, tryptamine, tryptamine 5-hydroxylase, tryptophan, tryptophan biosynthesis, tryptophan decarboxylaseSerotonin is found in a broad range of plants and is abundant in reproductive organs, such as fruits and seeds.^1–3 Even though many physiological roles for serotonin in plants have been proposed,^2–7 its actual roles have yet to be examined in detail using molecular, biochemical and genetic approaches. In plants, serotonin is synthesized by two enzymes: tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H). TDC decarboxylates tryptophan into tryptamine, after which T5H hydroxylates tryptamine into serotonin.^8–10 TDC expresses at an undetectable level in rice leaves, whereas T5H expresses constitutively.^11,12     

Tissue-specific and ABA-regulated Maize GIN gene expression in transgenic tobacco

Summary To study the regulatory functions of the ON promoter region, a ppG1b1GUS construct, consisting of 1402 bp 5 flanking sequence ofGlbl, 1919 by GUS coding sequence, and 283 by 3 NOS terminator, was cloned into a binary vector and introduced into tobacco plants byAgrobacterium-mediated transformation. Histochemical GUS assays of T_o tobacco mature seeds indicate that theGlbl promoter drives GUS expression in ABA treated seeds. Further GUS assays of the T, seeds at different developmental stages revealed that without ABA treatment, theGibl promoter drives GUS expression in immature seeds. The results from both T_o and T₁ tobacco plants indicated thatGlbl-driven GUS expression in tobacco is embryo specific.     

In planta analysis of the Agrobacterium tumefaciens T-cyt gene promoter: identification of an upstream region essential for promoter activity in leaf,stem and root cells of transgenic tobacco

The promoter region of the Agrobacterium tumefaciens T-cyt gene was fused to a -glucuronidase (gusA) reporter gene and introduced into tobacco plants. Detection of gusA expression in transgenic F1 progeny revealed that the T-cyt promoter is active in many, if not all, cell types in leaves, stems and roots of fully developed plants. Developmental stage-dependent promoter activity was observed in seedlings. Analysis of 5-deleted promoter fragments showed that sequences located between positions–185 and –139 with respect to the T-cyt translational start codon are essential for T-cyt promoter activity in transfected tobacco protoplasts as well as in transformed tobacco plants.     

The indole alkaloid tryptamine produced in transgenic Petunia hybrida

Tryptophan decarboxylase (TDC) from Catharanthus roseus (periwinkle) converts tryptophan to the indole-alkaloid tryptamine, an anti-insect compound. This TDC cDNA was transformed and expressed in transgenic Petunia hybrida under the control of the strong and constitutive 35S promoter from cauliflower mosaic virus. Kanamycin screening and Southern hybridization with the TDC cDNA confirmed plant transformation. Northern analysis indicated greater TDC mRNA accumulation in transgenic plants compared to non-transformed plants. Additionally, eight-fold more tryptamine accumulated in leaves of kanamycin resistant transgenic plants compared to non-transformed plants. Flower petals from the transgenic plants contained lower tryptamine levels than their leaves. Because tryptamine titers were higher in transformed plants compared to controls, over-expression of the TDC enzyme may partially overcome endogenous tryptamine catabolism and/or other negative biosynthetic regulation. Future alteration of tryptamine breakdown in Petunia may further increase total endogenous tryptamine concentrations, potentially discouraging insect reproduction on these transgenic plants.     

Overexpression of the feedback-insensitive anthranilate synthase gene in tobacco causes tryptophan accumulation

A total of 35 independent transgenic tobacco plants were produced using the Agrobacterium tumefaciens-leaf segment co-cultivation method followed by selection with kanamycin for the nptII gene. The vector also carried the tobacco feedback-insensitive anthranilate synthase gene (ASA2). Many of the lines showed increased ASA2 mRNA levels but only three contained increased free tryptophan (Trp) and many lines contained lower Trp than the untransformed control. The line with the highest Trp level (threefold that of the untransformed control) contained increased anthranilate synthase activity (AS) both in leaves and a cell suspension culture derived from the plant while the feedback insensitivity was most evident in the suspension culture. Other kinetic data also indicated that the ASA2 encoded AS -subunit was more abundant in the tissue culture than in leaves. Progeny seedlings from this line were resistant to certain toxic Trp analogs, especially -methyltryptophan (MT) and less so to the most commonly used analog, 5-methyltryptophan. Shoots formed more readily from leaves of two of the transgenic lines than from leaves of the untransformed control on MT, indicating that it might be possible to use ASA2 as a selectable marker gene and MT as the selection agent.     

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by wounding and methyl jasmonate

HMGR (3-hydroxy-3-methylglutaryl-coenzyme A reductase; E.C.1.1.1.34) supplies mevalonate for the synthesis of many plant primary and secondary metabolites, including the terpenoid component of indole alkaloids. Suspension cultures of Camptotheca acuminata and Catharanthus roseus, two species valued for their anticancer indole alkaloids, were treated with the elicitation signal transducer methyl jasmonate (MeJA). RNA gel blot analysis from MeJA treated cultures showed a transient suppression of HMGR mRNA, followed by an induction in HMGR message. Leaf disks from transgenic tobacco plants containing a chimeric hmgl::GUS construct were also treated with MeJA and showed a dose dependent suppression of wound-inducible GUS activity. The suppression of the wound response by MeJA was limited to the first 4 h post-wounding, after which time MeJA application had no effect. The results are discussed in relation to the differential regulation of HMGR isogenes in higher plants.Abbreviations GUS -glucuronidase - hmg gene of hmgr - HMGR 3-hydroxy-3-methylglutaryl-coenzyme A reductase - JA jasmonic acid - MeJA methyl jasmonate - MUG methylumbelliferyl--d-glucuronide - TDC tryptophan decarboxylase - SDS sodium dodecyl sulfate - SS strictosidine synthase     

Expression of a Synthetic Porcine α-Lactalbumin Gene in the Kernels of Transgenic Maize

The main nutritional limitation of maize used for feed is the content of protein that is digestible, bioavailable and contains an amino acid balance that matches the requirements of animals. In contrast, milk protein has good digestibility, bioavailability and amino acid balance. As an initial effort to create maize optimized as a source of swine nutrition, a codon-adjusted version of a gene encoding the milk protein porcine -lactalbumin was synthesized. Maize expression vectors containing this gene under the control of the Ubi-1 promoter and nos 3 terminator were constructed. These vectors were used to transform maize callus lines that were regenerated into fertile plants. The -lactalbumin transgenes were transmitted through meiosis to the sexual progeny of the regenerated plants. Porcine -lactalbumin was detected in callus and kernels from transgenic maize lines that were transformed by two constructs containing the 27-kDa maize gamma-zein signal sequence at the 5 end of the synthetic porcine -lactalbumin coding sequence. One of these constructs contained an ER retention signal and the other did not. Expression was not observed in kernels or callus from transgenic maize lines that were transformed by a construct that does not contain an exogenous protein-targeting signal. This suggests that the signal peptide might play an important role in porcine -lactalbumin accumulation in transgenic maize kernels.     

The sequence surrounding the translation initiation codon of the pea plastocyanin gene increases translational efficiency of a reporter gene

The 5-upstream region of the pea plastocyanin gene (petE) directed 5–10-fold higher levels of -glucuronidase (GUS) activity than the cauliflower mosaic virus 35S promoter in transgenic tobacco plants, although the levels of GUS mRNA were similar. The sequence (AAAAAUGG) around the translation initiation codon of petE enhanced translation of the GUS mRNA 10-fold compared to translation from the GUS translation initiation codon in transgenic tobacco plants and transfected protoplasts.     

Repeated sequences from theArabidopsis thaliana genome function as enhancers in transgenic tobacco

Sixteen segments ofArabidopsis thaliana DNA that function as enhancers in transgenic tobacco plants were isolated using the pROA97 enhancer cloning vehicle and library transformation ofNicotiana tabacum. The sequences were compared for AT content, homology, repeated motifs, and expression pattern in transgenicN. tabacum. The sequences were average with respect to the AT content ofA. thaliana DNA. They could be placed into seven homology groups. Five of the sequences are single-copy sequences. The remaining eleven sequences represent two homology groups. Homology Group I contains seven sequences with minor differences. Homology Group II contains four sequences with minor differences. Two repeated motifs were identified (5-CCTCT-3 and 5-AAGGAT-3). Both repeated motifs are found in other plant enhancers, and in the promoter region of the cauliflower mosaic virus 35S gene. In the 35S gene TATA region, the motifs can form two alternative stem-loop structures. The TATATAA sequence is located in the loop region of both stem-loop structures.     

A novel seed protein gene from Vicia faba is developmentally regulated in transgenic tobacco and Arabidopsis plants

Summary We have isolated a novel gene, denoted USP, from Vicia faba var. minor, which corresponds to the most abundant mRNA present in cotyledons during early seed development; however, the corresponding protein does not accumulate in cotyledons. The characterized USP gene with its two introns is 1 of about 15 members of a gene family. A fragment comprising 637 bp of 5 flanking sequence and the total 5 untranslated region was shown to be sufficient to drive the mainly seed-specific expression of two reporter genes, coding for neomycin phosphotransferase 11 and -glucuronidase, in transgenic Arabidopsis thaliana and Nicotiana tabacum plants. We showed that the USP promoter becomes active in transgenic tobacco seeds in both the embryo and the endosperm, whereas its activity in Arabidopsis is detectable only in the embryo. Moreover, we demonstrated a transient activity pattern of the USP promoter in root tips of both transgenic host species.     

Expression of S-locus glycoprotein genes from Brassica oleracea and B. campestris in transgenic plants of self-compatible B. napus cv Westar

Summary Self-compatible Brassica napus var Westar was transformed with SLG, the S-locus-derived gene that encodes S-locus-specific glycoproteins (SLSG). Four allelic variants of SLG isolated from self-incompatible B. oleracea and B. campestris strains homozygous for different S alleles were used. We show that the transgenic plants synthesized SLSG with the same apparent charge, molecular weight, and antigenic properties as that produced by the corresponding self-incompatible strains from which the cloned SLG genes were isolated. In addition, transgene-encoded SLSG was detected specifically in the papillar cells of the stigma, and was correctly targeted to the papillar cell wall. However, SLSG was produced at reduced levels in transgenic plants relative to self-incompatible strains. The introduction of the SLG genes did not confer a self-incompatibility phenotype on the Westar cultivar.