AmABCB1, an alkaloid transporter from seeds of Argemone mexicana L (Papaveraceae).

MAIN CONCLUSION: An ABCB-type transporter for sanguinarine, a benzophenanthridine alkaloid, was isolated from Argemone mexicana seeds. An ABCB-type transporter, AmABCB1, was identified in a transcriptome from unfolding seedlings of A. mexicana by its amino acid sequence identity to previously characterized alkaloid transporters from Coptis japonica and Thalictrum minus. Expression analysis revealed mature seeds as its main location; meanwhile, in vitro assays in yeast cells showed that AmABCB1 had uptake and efflux activities for sanguinarine and berberine, respectively.

Salicylic-acid elicited phospholipase D responses in Capsicum chinense cell cultures.

The plant response to different stress types can occur through stimulus recognition and the subsequent signal transduction through second messengers that send information to the regulation of metabolism and the expression of defense genes. The phospholipidic signaling pathway forms part of the plant response to several phytoregulators, such as salicylic acid (SA), which has been widely used to stimulate secondary metabolite production in cell cultures. In this work, we studied the effects of SA treatment on [(32)-P]Pi phospholipid turnover and phospholipase D (PLD) activity using cultured Capsicum chinense cells. In cultured cells, the PIP2 turnover showed changes after SA treatment, while the most abundant phospholipids (PLs), such as phosphatidylcholine (PC), did not show changes during the temporal course. SA treatment significantly increased phosphatidic acid (PA) turnover over time compared to control cells. The PA accumulation in cells treated with 1-butanol showed a decrease in messengers; at the same time, there was a 1.5-fold increase in phosphatidylbutanol. These results suggest that the participation of the PLD pathway is a source of PA production, and the activation of this mechanism may be important in the cell responses to SA treatment.

Light activation of vindoline biosynthesis does not require cytomorphogenesis in Catharanthus roseus seedlings.

Upon illumination, the cotyledons of Catharanthus roseus seedlings readily synthesise vindoline from late biosynthetic intermediates, which accumulate in etiolated seedlings. The cellular localisation of tryptophan decarboxylase (TDC) and desacetoxyvindoline 4-hydroxylase (D4H), which catalyse the first and penultimate reactions of vindoline biosynthesis, was identified by immunocytochemistry in developing seedlings. The expression of TDC was restricted to the upper epidermis of cotyledons, whereas that of D4H was confined to laticifer cells. Light exposure of etiolated seedlings significantly induced D4H enzyme activity without changing the steady-state levels of D4H immunoreactive protein or modifying the cellular distribution of D4H expression in dark-grown seedlings. These results suggest that the early and late stages of vindoline biosynthesis occupy different cellular compartments, even in the early phases of etiolated seedling development. The role of light in activating the late stages of vindoline biosynthesis does not, therefore, seem to be related to the formation of the laticifer and idioblast cell types. It is concluded that light is not required for formation of these cell types, whereas regulatory factors, restricted to idioblasts and laticifers, may respond to light to activate localised expression of the late stages of vindoline biosynthesis.

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.

Molecular cloning and characterization of desacetoxyvindoline-4-hydroxylase, a 2-oxoglutarate dependent-dioxygenase involved in the biosynthesis of vindoline in Catharanthus roseus (L.) G. Don.

A 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11) which catalyzes the 4-hydroxylation of desacetoxyvindoline was purified to homogeneity. Three oligopeptides isolated from a tryptic digest of the purified protein were microsequenced and one oligopeptide showed significant homology to hyoscyamine 6 beta-hydroxylase from Hyoscyamus niger. A 36-mer degenerate oligonucleotide based on this peptide sequence was used to screen a Catharanthus roseus cDNA library and three clones, cD4H-1 to -3, were isolated. Although none of the three clones were full-length, the open reading frame on each clone encoded a putative protein containing the sequence of all three peptides. Primer extension analysis suggested that cD4H-3, the longest cDNA clone, was missing 156 bp at the 5' end of the clone and sequencing of the genomic clone, gD4H-8, confirmed these results. Southern blot analysis suggested that d4h is present as a single-copy gene in C. roseus which is a diploid plant, and the significant differences in the sequence of the 3'-UTR between cD4H-1 and -3 suggest that they represent dimorphic alleles of the same hydroxylase. The identity of the clone was further confirmed when extracts of transformed Escherichia coli expressed D4H enzyme activity. The D4H clone encoded a putative protein of 401 amino acids with a calculated molecular mass of 45.5 kDa and the amino acid sequence showed a high degree of similarity with those of a growing family of 2-oxoglutarate-dependent dioxygenases of plant and fungal origin. The similarity was not restricted to the dioxygenase protein sequences but was also extended to the gene structure and organization since the 205 and 1720 bp introns of d4h were inserted around the same highly conserved amino acid consensus sequences as those for e8 protein, hyoscyamine-6 beta-hydroxylase and ethylene-forming enzyme. These results provide further support that a common ancestral gene is responsible for the appearance of this family of dioxygenases. Hydroxylase assays and RNA blot hybridization studies showed that enzyme activity followed closely the levels of d4h transcripts, occurring predominantly in young leaves and in much lower levels in stems and fruits. In contrast, etiolated seedlings which contained considerable levels of d4h transcripts had almost undetectable hydroxylase activity, whereas exposure of seedlings to light resulted in a rapid increase of enzyme activity without a significant further increase in d4h transcripts over those detected in dark-grown seedlings. These results suggest that the activating effect of light may occur at a point downstream of transcription which remains to be elucidated.