Nailish Samanani

Cell Type–Specific Localization of Transcripts Encoding Nine Consecutive Enzymes Involved in Protoberberine Alkaloid Biosynthesis

Molecular clones encoding nine consecutive biosynthetic enzymes that catalyze the conversion of l-dopa to the protoberberine alkaloid (S)-canadine were isolated from meadow rue (Thalictrum flavum ssp glaucum). The predicted proteins showed extensive sequence identity with corresponding enzymes involved in the biosynthesis of related benzylisoquinoline alkaloids in other species, such as opium poppy (Papaver somniferum). RNA gel blot hybridization analysis showed that gene transcripts for each enzyme were most abundant in rhizomes but were also detected at lower levels in roots and other organs. In situ RNA hybridization analysis revealed the cell type–specific expression of protoberberine alkaloid biosynthetic genes in roots and rhizomes. In roots, gene transcripts for all nine enzymes were localized to immature endodermis, pericycle, and, in some cases, adjacent cortical cells. In rhizomes, gene transcripts encoding all nine enzymes were restricted to the protoderm of leaf primordia. The localization of biosynthetic gene transcripts was in contrast with the tissue-specific accumulation of protoberberine alkaloids. In roots, protoberberine alkaloids were restricted to mature endodermal cells upon the initiation of secondary growth and were distributed throughout the pith and cortex in rhizomes. Thus, the cell type–specific localization of protoberberine alkaloid biosynthesis and accumulation are temporally and spatially separated in T. flavum roots and rhizomes, respectively. Despite the close phylogeny between corresponding biosynthetic enzymes, distinct and different cell types are involved in the biosynthesis and accumulation of benzylisoquinoline alkaloids in T. flavum and P. somniferum. Our results suggest that the evolution of alkaloid metabolism involves not only the recruitment of new biosynthetic enzymes, but also the migration of established pathways between cell types.

Opium poppy: blueprint for an alkaloid factory

Opium poppy (Papaver somniferum) produces a large number of benzylisoquinoline alkaloids, including the narcotic analgesics morphine and codeine, and has emerged as one of the most versatile model systems to study alkaloid metabolism in plants. As summarized in this review, we have taken a holistic strategy—involving biochemical, cellular, molecular genetic, genomic, and metabolomic approaches—to draft a blueprint of the fundamental biological platforms required for an opium poppy cell to function as an alkaloid factory. The capacity to synthesize and store alkaloids requires the cooperation of three phloem cell types—companion cells, sieve elements, and laticifers—in the plant, but also occurs in dedifferentiated cell cultures. We have assembled an opium poppy expressed sequence tag (EST) database based on the attempted sequencing of more than 30,000 cDNAs from elicitor-treated cell culture, stem, and root libraries. Approximately 23,000 of the elicitor-induced cell culture and stem ESTs are represented on a DNA microarray, which has been used to examine changes in transcript profile in cultured cells in response to elicitor treatment, and in plants with different alkaloid profiles. Fourier transform-ion cyclotron resonance mass spectrometry and proton nuclear magnetic resonance mass spectroscopy are being used to detect corresponding differences in metabolite profiles. Several new genes involved in the biosynthesis and regulation of alkaloid pathways in opium poppy have been identified using genomic tools. A biological blueprint for alkaloid production coupled with the emergence of reliable transformation protocols has created an unprecedented opportunity to alter the chemical profile of the world’s most valuable medicinal plant.

Cell type-specific protoberberine alkaloid accumulation inThalictrum flavum

Summary Berberine is a common benzylisoquinoline alkaloid with potent antimicrobial properties, which suggest it functions to protect some plants from pathogen challenge. Berberine was identified as the major alkaloid in meadow rue (Thalictrum flavum ssp. glaucum), a medicinal member of the Ranunculaceae, and was detected in seeds and all organs of the plant. The high level of berberine in roots, rhizomes, and older petioles is mainly responsible for the intense yellow color of these organs. In rhizomes, protoberberine alkaloids were detected throughout the pith and, to a lesser extent, the cortex, but were absent from the vascular tissues. Similarly, protoberberine alkaloids were detected in the rib parenchyma of older petioles. In roots, alkaloid accumulation was detected only in the endodermis at the onset of secondary growth. Rather than being sloughed off, the endodermis was found to undergo extensive anticlinal division leading to an expanding cellular cylinder that ultimately displaced all external tissues. Endodermal-specific protoberberine alkaloid accumulation continued throughout root development, but was extended to include 3 to 4 layers of smaller pericycle cells in the oldest roots near the base of the stem. The cell type-specific accumulation of antimicrobial alkaloids and the unusual development of the endodermis and pericycle in T. flavum roots support the putative role of berberine in plant defense.

Chapter Three – Compartmentalization of Plant Secondary Metabolism

Bifunctional or multifunctional enzymes targeted to alternative subcellular compartments may interact with different substrates to produce unique products. This may partially explain the observed diversity of plant secondary products. Broad enzyme specificities have been observed for O -methyltransferases, glucosyltransferases, P450-dependent monooxygenases, polyketide synthases, and monoterpene synthases. Flavonoid biosynthesis in plants was thought previously to occur exclusively in the cytoplasm although flavonoids could accumulate in distinct subcellular compartments in different tissues. However, at least two of the enzymes of flavonoid biosynthesis occur in the nuclei of Arabidopsis cells, where the flavonoids also accumulate. Although much progress has recently been made toward the deciphering of the compartmentalization of secondary product metabolism, a comprehensive understanding of the spatial relationships among transcripts, enzymes, and biosynthetic products requires further research in several important areas.

Chapter seven Multiple levels of control in the regulation of alkaloid biosynthesis

Summary Alkaloid biosynthetic pathways are under strict regulation in plants. Until now, our limited knowledge of the fundamental mechanisms involved in the control of alkaloid metabolism has severely restricted our ability to harness the vast biotechnological potential of these important secondary pathways. For example, the use of plant cell cultures for the commercial production of pharmaceutical alkaloids has not become a reality despite decades of empirical research. The application of traditional and modern biochemical, molecular, and cellular techniques has revealed important clues about the reasons why C. roseus cultures accumulate tabersonine and catharanthine, but not vindoline or vinblastine, and why opium poppy cultures produce sanguinarine, but not codeine or morphine. The inability of dedifferentiated cells to accumulate certain metabolites was interpreted as evidence that the operation of many alkaloid pathways is tightly coupled to the development of specific tissues. Recent studies have shown that alkaloid pathways are regulated at multiple levels, including cell type-specific gene expression, induction by light and elicitors, enzymatic controls, and other poorly understood metabolic mechanisms. Our ability to exploit the biosynthetic capacity of plants through, for example, metabolic engineering will require a thorough understanding of the mechanisms that allow a cell to produce specific alkaloids. Advances in genomics will provide a more rapid and efficient means to identify new biosynthetic and regulatory genes involved in alkaloid pathways. The apparently unique aspects of alkaloid biosynthesis also provide intriguing targets for plant cell biology research, in general. Novel insights obtained using a combination of traditional and modern technologies, including biochemistry, molecular biology, cell biology, and genetic engineering, highlight the importance of a multifaceted approach in studying the regulation of alkaloid biosynthesis in plants.