Melatonin mediates selenium-induced tolerance to cadmium stress in tomato plants.

Both selenium (Se) and melatonin reduce cadmium (Cd) uptake and mitigate Cd toxicity in plants. However, the relationship between Se and melatonin in Cd detoxification remains unclear. In this study, we investigated the influence of three forms of Se (selenocysteine, sodium selenite, and sodium selenate) on the biosynthesis of melatonin and the tolerance against Cd in tomato plants. Pretreatment with different forms of Se significantly induced the biosynthesis of melatonin and its precursors (tryptophan, tryptamine, and serotonin); selenocysteine had the most marked effect on melatonin biosynthesis. Furthermore, Se and melatonin supplements significantly increased plant Cd tolerance as evidenced by decreased growth inhibition, photoinhibition, and electrolyte leakage (EL). Se-induced Cd tolerance was compromised in melatonin-deficient plants following tryptophan decarboxylase (TDC) gene silencing. Se treatment increased the levels of glutathione (GSH) and phytochelatins (PCs), as well as the expression of GSH and PC biosynthetic genes in nonsilenced plants, but the effects of Se were compromised in TDC-silenced plants under Cd stress. In addition, Se and melatonin supplements reduced Cd content in leaves of nonsilenced plants, but Se-induced reduction in Cd content was compromised in leaves of TDC-silenced plants. Taken together, our results indicate that melatonin is involved in Se-induced Cd tolerance via the regulation of Cd detoxification.

Non-dopaminergic neurons expressing dopamine synthesis enzymes: differentiation and functional significance.

The development and functional significance of neurons in the arcuate nucleus expressing tyrosine hydroxylase and/or aromatic L-amino acid decarboxylase were studied in rat fetuses, neonates, and adults using immunocytochemical (single and double immunolabeling of tyrosine hydroxylase and aromatic L-amino acid decarboxylase) methods with a confocal microscope and computerized image analysis, HPLC with electrochemical detection, and radioimmunological analysis. Single-enzyme neurons containing tyrosine hydroxylase were first seen on day 18 of embryonic development in the ventrolateral part of the arcuate nucleus. Neurons expressing only aromatic L-amino acid decarboxylase or both enzymes of the dopamine synthesis pathway were first seen on day 20 of embryonic development, in the dorsomedial part of the nucleus. On days 20-21 of embryonic development, dopaminergic (containing both enzymes) neurons amounted to less than 1% of all neurons expressing tyrosine hydroxylase and/or aromatic L-amino acid decarboxylase. Nonetheless, in the ex vivo arcuate nucleus and in primary neuron cultures from this structure, there were relatively high leveLs of dopamine and L-dihydroxyphenylalanine (L-DOPA), and these substances were secreted spontaneously and in response to stimulation. In addition. dopamine levels in the arcuate nucleus in fetuses were sufficient to support the inhibitory regulation of prolactin secretion by the hypophysis, which is typical of adult animals. During development, the proportion of dopaminergic neurons increased, reaching 38% in adult rats. Specialized contacts between single-enzyme tyrosine hydroxylase-containing and aromatic L-amino acid decarboxylase-containing neurons were present by day 21 of embryonic development; these were probably involved in transporting L-DOPA from the former neurons to the latter. It was also demonstrated that the axons of single-enzyme decarboxylase-containing neurons projected into the median eminence, supporting the secretion of dopamine into the hypophyseal portal circulation. Thus, dopamine is probably synthesized in the arcuate nucleus not only by dopaminergic neurons, but also by neurons expressing only tyrosine hydroxylase or aromatic L-amino acid decarboxylase.

Effect of precursor feeding on alkaloid accumulation by a tryptophan decarboxylase over-expressing transgenic cell line T22 of Catharanthus roseus.

To obtain more insight into the regulation of terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus (L.) G. Don cell cultures and particularly to identify possible rate limiting steps, a transgenic cell line over-expressing tryptophan decarboxylase (Tdc), and thus having a high level of tryptamine, was fed with various amounts of precursors (tryptophan, tryptamine, loganin and secologanin) in different time schedules and analyzed for TIA production. When these precursors were added to this culture it was found that the optimal time for supplying the precursors was at inoculation of the cells into the production medium. Alkaloid accumulation by line T22 was enhanced by addition of loganin or secologanin; however, the secologanin feeding was less effective. Tryptamine or tryptophan alone had no effect on TIA accumulation. The over-expression of Tdc causes this cell line to produce quite large quantities of alkaloids after feeding loganin or secologanin. However, in combination with tryptophan or tryptamine, feeding of these precursors resulted in an even further increase of alkaloid accumulation and under optimal conditions line T22 accumulated around 1200 micromol l(-1) of TIAs whereas the control cultures accumulated less than 10 micromol l(-1) TIAs.

Jasmonate modulates development- and light-regulated alkaloid biosynthesis in Catharanthus roseus.

Methyl jasmonate, a chemical inducer of secondary metabolism, has been shown to promote vindoline biosynthesis in developing seedlings, as a result of induction of tryptophan decarboxylase (TDC) and desacetylvindoline 4-hydroxylase (D4H). The present studies suggest that jasmonate-based induction of TDC and D4H activities involves modulation of transcriptional, post-transcriptional and post-translational controls. The effects of jasmonate on both enzymes were transient with maximum TDC activity appearing 12 h earlier than that of D4H. Jasmonate treatment of etiolated seedlings neither enhanced TDC activity nor could it replace the light requirement for D4H induction. Jasmonate, therefore, appears to modulate events which are already triggered by developmental and environmental specific controls. Salicylic acid, another chemical inducer of secondary metabolism, was ineffective in activating either TDC or D4H under the experimental conditions used.

Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus.

Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg.L-1 of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment.

D2 dopamine receptor antisense increases the activity and mRNA of tyrosine hydroxylase and aromatic L-amino acid decarboxylase in mouse brain.

A D2 dopamine receptor antisense oligodeoxynucleotide was administered intracerebrovetricularly to mice twice on the first day and then once daily for 2 days. The animals were killed 2 h after the last injection, and tyrosine hydroxylase and aromatic L-amino acid decarboxylase activities assayed in the corpus striatum, olfactory tubercle and frontal cortex. Tyrosine hydroxylase activity increased in corpus striatum but not in the olfactory tubercle or in the frontal cortex, while the activity of aromatic L-amino acid decarboxylase increased in all three brain regions. The treatment with the antisense oligomer also elevated the mRNA levels for the two enzymes in the midbrain. In contrast, repeated injection of a vehicle or a random oligomer was without effect on enzyme activity or mRNA D2 antisense oligodeoxynucleotides appear to be selective tools to investigate the role of D2 dopamine receptors in brain.

Changes in activity and mRNA for rat tryptophan hydroxylase and aromatic L-amino acid decarboxylase of brain serotonergic cell bodies and terminals following neonatal 5,7-dihydroxytryptamine.

In the present study, we examined time-dependent changes in activity, mRNA and immunoreactivity of the serotonin biosynthetic enzymes, tryptophan hydroxylase (TPH) and aromatic L-amino acid decarboxylase (AADC) in dorsal raphe nucleus (DRN), caudal brainstem and hypothalamus, following intracisternal injection of 5,7-dihydroxytryptamine (5,7-DHT) in neonatal rats. TPH activity in central serotonergic cell bodies and terminals was reduced to 20-30% of control levels at 1-8 weeks after neonatal, low-dose 5,7-DHT injection (24 micrograms free base). In contrast, AADC activity was either not changed or decreased to 40% of control levels, depending on the region. In situ hybridization and immunocytochemical staining indicated that 5,7-DHT caused a marked reduction in TPH and AADC message levels as well as the number of 5-HT and AADC-immunoreactive cells within the DRN as early as 1 week after 5,7-DHT. Even 15 weeks after drug administration recovery did not occur. This apparent neuronal loss was region-specific suggesting that some serotonergic neurons are more resistant to neonatal 5,7-DHT treatment than others. Taken together, these studies indicate that neonatal treatment with 5,7-DHT produces a marked and permanent (up to 15 weeks) reduction in the number of central serotonergic neurons.

Dopamine increases the expression of tyrosine hydroxylase and aromatic amino acid decarboxylase in primary cultures of fetal neurons.

Previous studies, aimed at identifying which diffusible signals may influence the differentiation of embryonic neurons towards the monoaminergic phenotypes during brain development, have shown that serotonin itself could promote the 'serotoninergic-like properties' of hypothalamic cells from mouse embryos. We presently reinvestigated such 'autocrine/paracrine' regulatory mechanisms by exposing dissociated cell cultures from embryonic rat hypothalamus and brain stem to dopamine--or related agonists--in an attempt to influence their differentiation towards the catecholaminergic phenotype. Chronic treatment of cells by dopamine or apomorphine (a mixed D1/D2 agonist), but not selective D1 and D2 agonists, significantly increased the number of cells that expressed tyrosine hydroxylase (TH: as assessed with a specific anti-TH antiserum) and the activity of aromatic L-amino acid decarboxylase (AADC) in the cultures. Furthermore, apomorphine treatment also decreased the levels of cholecystokinin-like material in primary cultures from the brainstem (but not the hypothalamus) where both dopamine and cholecystokinin are--partly--colocalized in mesencephalic dopaminergic neurons. The maximal effects of both dopamine and apomorphine on TH expression and AADC activity occurred earlier in the brainstem (on cells from 14- to 15-day-old embryos) than in the hypothalamus (on cells from 15- to 16-day-old embryos), in line with the well established caudo-rostral maturation of the rat brain. Furthermore both the expression and the dopamine-induced modulation of AADC activity and TH immunoreactivity appeared to occur independently of each other. Present and previous data are in agreement with a possible autocrine/paracrine action of dopamine and serotonin during brain development.(ABSTRACT TRUNCATED AT 250 WORDS)

Elicitor-mediated induction of tryptophan decarboxylase and strictosidine synthase activities in cell suspension cultures of Catharanthus roseus.

Treatment of one cell line (No. 615) of Catharanthus roseus c.v. Little Delicata with an elicitor preparation of autoclaved and homogenized Pythium aphanidermatum culture resulted in rapid accumulation of indole alkaloids. Alkaloid formation was preceded by rapid transient increases in the extractable activities of the enzymes tryptophan decarboxylase and strictosidine synthase. The induction of these two enzyme activities occurred when cells were transferred to alkaloid production medium or treatment with fungal elicitors. Treatment of this cell line with translational or transcriptional inhibitors prevented the Pythium-induced increases of enzyme activity as well as alkaloid accumulation. When cells were transferred to alkaloid production medium the induction of strictosidine synthase activity preceded that of tryptophan decarboxylase by many hours even when cells were also treated with Pythium elicitor. Results suggested that tryptophan decarboxylase induction proceeds only when endogenous tryptamine levels were decreased by two-third. The internal cellular level of tryptamine, therefore, could regulate expression of tryptophan decarboxylase, whereas induction of strictosidine synthase or of another enzyme in the biosynthetic pathway could control channeling of tryptamine into alkaloids. The results demonstrate that fungal elicitors can be used to facilitate studies of the factors which regulate expression of indole alkaloid pathway enzymes and their ultimate pathway products.