Phytochemistry Reviews (v.7, #3)

Preface by Kirsi-Marja Oksman-Caldentey (385-386).

A short history of plant biotechnology by Indra K. Vasil (387-394).
The foundations of modern plant biotechnology can be traced back to the Cell Theory of Schleiden (Arch Anat Physiol Wiss Med (J Müller) 1838:137–176, 1838) and Schwann (Mikroscopische Untersuchungen über die Übereinstimmung in der Struktur und dem Wachstum des Tiere und Pflanzen. W Engelmann: Leipzig No 176, 1839), which recognized the cell as the primary unit of all living organisms. The concept of cellular totipotency, which was inherent in the Cell Theory and forms the basis of plant biotechnology, was further elaborated by Haberlandt (Sitzungsber K Preuss Akad Wiss Wien, Math-Naturwiss 111:69–92, 1902), who predicted the production of somatic embryos from vegetative cells. This brief historical account traces the development of technologies for the culture, regeneration and transformation of plants that led to the production of transgenic crops which have become central to the many applications of plant biotechnology, and celebrates the pioneering men and women whose trend-setting contributions made it all possible.
Keywords: Cell theory; Genetic transformation; Plant regeneration; Totipotency; Transgenic crops

Plant metabolites have been the successful source of drugs and provided considerable value not only to the pharmaceutical industry but also to human health problems. Although pharmaceutical companies significantly decreased their activities in natural product discovery during the past few decades, various multidisciplinary approaches have been made to create new opportunities for finding innovative plant derived pharmaceuticals in post-genome era. Strategies to integrate the knowledge on medicinal plants into rational drug screening, the unique biodiversity of plant metabolites into random drug screening, and the chemical diversity of plant metabolites into combinatorial chemistry have been reviewed with concrete examples. Innovative biotechnologies in plant cell and tissue cultures, and the latest achievements in metabolic engineering and genetic modification should significantly improve the production sustainability and efficiency of plant-derived pharmaceuticals.
Keywords: Biodiversity; Biotechnology; Chemical diversity; Metabolic engineering; Plant metabolites

Plant foods contain substantial amounts of phenolic compounds. Dietary interventions with phenolic supplementation show that phenolic compounds are transformed into phenolic acids or lactone structures by intestinal microbiota. The colon is the main site of microbial fermentation. The metabolites circulate in plasma and are excreted via urine. The entero-hepatic circulation ensures that their residence time in plasma is extended compared to that of their parent compounds. Thus these metabolites may exert systemic effects, which however have not been studied adequately. In particular the health implications of microbial metabolites of flavonoids, mostly phenolic acids, are unknown. This review aims to elucidate the microbial metabolism of most of the phenolic classes: flavonoids, isoflavonoids, lignans, phenolic acids and tannins. Some examples of biological activity studies of flavonoid and lignan metabolites are given. Biological significance of enterolactone, a mammalian plant lignan metabolite, has been studied quite extensively, but convincing evidence of the health benefits of the diverse pool of microbial metabolites is still scarce. Hopefully, novel tools in systems biology and the constant search for biomarkers will elucidate the role of the phenolic metabolome in health and in the prevention of chronic diseases. In conclusion, the colon is not only an excretion route, but also an active site of metabolism and deserves further attention from the scientific community.
Keywords: Flavonoids; Phytoestrogens; Tannins; Phenolic acids; Lactones

Phytoestrogens as natural prodrugs in cancer prevention: a novel concept by Randolph R. J. Arroo; Vasilis Androutsopoulos; Asma Patel; Somchaiya Surichan; Nicola Wilsher; Gerry A. Potter (431-443).
It has been generally accepted that regular consumption of fresh fruits and vegetables is linked with a relatively low incidence of cancers (e.g. breast, cervix, and colon). A number of plant-derived compounds have been identified that are considered to play a role in cancer prevention. However, at present there is no satisfactory explanation for the cancer preventative properties of the above-mentioned compound groups. The current review is an effort to develop a consistent and unambiguous model that explains how some plant-derived compounds can prevent tumour development. The model is based on recent discoveries in the fields of genomics and drug-metabolism; notably, the discovery that CYP1 genes are highly expressed in developing tumour cells but not in the surrounding tissue, and that a variety of plant-derived compounds are substrates for the CYP1 enzymes. Our hypothesis is that some dietary compounds act as prodrugs, i.e. compounds with little or no biological effect as such, but become pharmaceutically effective when activated. More specifically, we state that the abovementioned prodrugs are only activated in CYP1-expressing cells—i.e. cells in the early stages of tumour development—to be converted into compounds which inhibit cell growth. Thus, the prodrugs selectively kill precancerous cells early in tumour development. The review focuses on the identification of naturally-occurring prodrugs that are activated by the tumour-specific CYP1 enzymes and aims to assess their role in cancer prevention.
Keywords: CYP1; Drug metabolism; Flavones; Natural products; Prevention

Biosynthesis and genetic engineering of proanthocyanidins and (iso)flavonoids by Li Tian; Yongzhen Pang; Richard A. Dixon (445-465).
Plant natural products have been used since ancient times as medicines and herbal remedies. Over the past two decades, the results of population and intervention studies, or assays in animal or cell model systems, have revealed positive health beneficial effects for various classes of phytochemicals, particularly polyphenols. The results of such studies have ignited an interest in being able to manipulate the levels of such bioactive compounds in plants using biotechnological approaches. Although still in its infancy, this technology promises to deliver health benefits to humans and animals through direct consumption of genetically-modified or -enhanced dietary plant materials. We here review the strategies currently being used for engineering two classes of nutraceuticals, the proanthocyanidins and the isoflavones, in transgenic plants. We also provide an overview of recent advances in our understanding of the biosynthesis of these classes of compounds.
Keywords: Isoflavone; Proanthocyanidin; Genetic engineering; Nutraceutical

Mechanisms of resistance to self-produced toxic secondary metabolites in plants by Supaart Sirikantaramas; Mami Yamazaki; Kazuki Saito (467-477).
Plants produce a variety of secondary metabolites to protect themselves from pathogens and herbivores and/or to influence the growth of neighbouring plants. Some of these metabolites are toxic to the producing cells when their target sites are present in the producing organisms. Therefore, a specific self-resistance mechanism must exist in these plants. Self-resistance mechanisms, including extracellular excretion, vacuolar sequestration, vesicle transport, extracellular biosynthesis, and accumulation of the metabolite in a non-toxic form, have been proposed thus far. Recently, a new mechanism involving mutation of the target protein of the toxic metabolite has been elucidated. We review here the mechanisms that plants use to prevent self-toxicity from the following representative compounds: cannabinoids, flavonoids, diterpene sclareol, alkaloids, benzoxazinones, phenylpropanoids, cyanogenic glycosides, and glucosinolates.
Keywords: Secondary metabolite; Toxicity; Self-resistance; Detoxification

As the final downstream product of the genome, the plant metabolome is a highly complex, dynamic assortment of primary and secondary compounds. Although technological platforms to study genomes, transcriptomes and even proteomes are presently available, methods to pursue genuine metabolomics have not yet been developed due to the extensive chemical diversity of plant primary and secondary metabolites. No single analytical method can accurately survey the entire metabolome. However, recent technical, chemometric and bioinformatic advances promise to enhance our global understanding of plant metabolism. Separation-based mass spectrometry (MS) approaches, such as gas (GC) or liquid chromatography (LC)-MS, are relatively inexpensive, highly sensitive and provide excellent identifying capacity. However, Fourier transform-ion cyclotron resonance (FT-ICR)-MS is better suited for rapid, high-throughput applications and is currently the most sensitive method available. Unlike MS-based analyses, nuclear magnetic resonance (NMR) spectroscopy provides a large amount of information regarding molecular structure, and novel software innovations have facilitated the unequivocal identification and absolute quantification of compounds within composite samples. Due to the size and complexity of metabolomics datasets, numerous chemometric methods are used to extract and display systematic variation. Coupled with pattern recognition techniques and plant-specific metabolite databases, broad-scope metabolite analyses have emerged as functional genomics tools for novel gene discovery and functional characterization. In this review, key metabolomics technologies are compared and the applications of FT-ICR-MS and NMR to the study of benzylisoquinoline alkaloid metabolism in opium poppy are discussed.
Keywords: Chemometrics; Mass spectrometry; Metabolite analysis; Nuclear magnetic resonance; Opium poppy

Phenylanthraquinones belong to the quite rare class of fully unsymmetric biaryls, consisting of two different molecular portions, an anthraquinone part, chrysophanol, and a phenyl part, 2,4-dihydroxy-6-methoxyacetophenone, linked together by phenol-oxidative coupling. The biosynthesis of these two moieties, from eight and four acetate units, respectively, bears some unique features: Chrysophanol is the first example of an acetogenic natural product that is, in an organism-specific manner, formed via more than one folding mode: In eukaryotes, like, e.g., in fungi, in higher plants, and in insects, it is formed via folding mode F, while in prokaryotes it originates through mode S. It has, more recently, even been found to be synthesized by a third pathway, which we have named mode S′. It is thus the first example of biosynthetic convergence in polyketide biosynthesis. The monocyclic “southern” portion of the molecule, which is much simpler (arising from only four acetate units and without decarboxylation), unexpectedly does not show the anticipated randomization of the C2-labeling in the aromatic ring, but has clearly localized C2 units, excluding any symmetric intermediate like, e.g., 2,4,6-trihydroxyacetophenone. This is confirmed by competitive feeding experiments with specifically 13C2-labeled acetophenones, showing the O-methylation to be the decisive symmetry-preventing step, which hints at a close collaboration of the participating enzymes. The results make knipholone an instructive example of structure, function, and evolution of polyketide synthases and O-methyltransferases, and their cooperation.
Keywords: Polyketides; Chrysophanol; Knipholone; Biosynthesis; Feeding experiments; Polyketide synthase; O-Methyltransferase

Secondary transport as an efficient membrane transport mechanism for plant secondary metabolites by Kazufumi Yazaki; Akifumi Sugiyama; Masahiko Morita; Nobukazu Shitan (513-524).
Plants produce a large number of secondary metabolites, such as alkaloids, terpenoids, and phenolic compounds. Secondary metabolites have various functions including protection against pathogens and UV light in plants, and have been used as natural medicines for humans utilizing their diverse biological activities. Many of these natural compounds are accumulated in a particular compartment such as vacuoles, and some are even translocated from source cells to sink organs via long distance transport. Both primary and secondary transporters are involved in such compartmentation and translocation, and many transporter genes, especially genes belonging to the multidrug and toxin extrusion type transporter family, which consists of 56 members in Arabidopsis, have been identified as responsible for the membrane transport of secondary metabolites. Better understandings of these transporters as well as the biosynthetic genes of secondary metabolites will be important for metabolic engineering aiming to increase the production of commercially valuable secondary metabolites in plant cells.
Keywords: ABC protein; MATE; Membrane transport; Secondary transport; Translocation

Metabolomics: back to basics by R. Verpoorte; Y. H. Choi; N. R. Mustafa; H. K. Kim (525-537).
Metabolomics has developed into a major tool in functional genomics and plant systems biology. The various methods used for metabolomic analysis will be discussed from the analytical methods back to the preanalytical phase and the biological experiment. Particularly aspects of the preanalytical phase of the analysis is dealt with, including the risks of artefact formation with the various commonly used solvents. Metabolomics is like a snap shot, and conclusions from dynamic systems must be drawn with great care as demonstrated with a biosynthetic study of salicylate in Catharanthus roseus cell cultures.
Keywords: Metabolomics; NMR spectrometry; Sample preparation; Extraction; Artefacts; Catharanthus roseus

Production of recombinant allergens in plants by Georg Schmidt; Gabriele Gadermaier; Heidi Pertl; Marc Siegert; Kirsi-Marja Oksman-Caldentey; Anneli Ritala; Martin Himly; Gerhard Obermeyer; Fatima Ferreira (539-552).
A large percentage of allergenic proteins are of plant origin. Hence, plant-based expression systems are considered ideal for the recombinant production of certain allergens. First attempts to establish production of plant-derived allergens in plants focused on transient expression in Nicotiana benthamiana infected with recombinant viral vectors. Accordingly, allergens from birch and mugwort pollen, as well as from apple have been expressed in plants. Production of house dust mite allergens has been achieved by Agrobacterium-mediated transformation of tobacco plants. Beside the use of plants as production systems, other approaches have focused on the development of edible vaccines expressing allergens or epitopes thereof, which bypasses the need of allergen purification. The potential of this approach has been convincingly demonstrated for transgenic rice seeds expressing seven dominant human T cell epitopes derived from Japanese cedar pollen allergens. Parallel to efforts in developing recombinant-based diagnostic and therapeutic reagents, different gene-silencing approaches have been used to decrease the expression of allergenic proteins in allergen sources. In this way hypoallergenic ryegrass, soybean, rice, apple, and tomato were developed.
Keywords: Allergy; Expression system; Green biotechnology; Molecular farming; Recombinant protein

A variety of plant species have been genetically modified to accumulate vaccine antigens for human and animal health and the first vaccine candidates are approaching the market. The regulatory burden for animal vaccines is less than that for human use and this has attracted the attention of researchers and companies, and investment in plant-made vaccines for animal infectious disease control is increasing. The dosage cost of vaccines for animal infectious diseases must be kept to a minimum, especially for non-lethal diseases that diminish animal welfare and growth, so efficient and economic production, storage and delivery are critical for commercialization. It has become clear that transgenic plants are an economic and efficient alternative to fermentation for large-scale production of vaccine antigens. The oral delivery of plant-made vaccines is particularly attractive since the expensive purification step can be avoided further reducing the cost per dose. This review covers the current status of plant-produced vaccines for the prevention of disease in animals and focuses on barriers to the development of such products and methods to overcome them.
Keywords: Animal health; Molecular farming; Oral vaccine; Plant-produced vaccine

Molecular pharming in cereal crops by Koreen Ramessar; Teresa Capell; Paul Christou (579-592).
There are many different agricultural expression systems that can be used for the large-scale production of recombinant proteins, but field-grown cereal crops are among the most attractive because recombinant proteins can be targeted to accumulate in the seed, and specifically in the endosperm, which has evolved naturally as a protein storage tissue. Within the developing endosperm, proteins are supplied with molecular chaperones and disulfide isomerases to facilitate folding and assembly, while the mature tissue is desiccated to prevent proteolytic degradation. Proteins expressed in cereal seeds can therefore remain stable for years in ambient conditions. Recent basic research has revealed a surprising diversity of protein targeting mechanisms in the endosperm, which can help to control post-translational modification and accumulation. Applied research and commercial development has seen several pharmaceutical proteins produced in cereals reach late stage preclinical development and the first clinical trials, with a number of companies now dedicated to developing cereal-based production platforms. In this review we discuss the basic science of molecular pharming in cereals, some of the lead product candidates, and challenges that remain to be addressed including the emerging regulatory framework for plant-made pharmaceuticals.
Keywords: Transgenic plant; Cereal; Maize; Rice; Barley; Wheat; Recombinant protein; Plant made pharmaceutical; Expression; Regulation; Biosafety

Plant cell suspension cultures and hairy roots are potential sources of secondary metabolites and recombinant proteins. In contrast to traditionally grown “whole wild plants” or “whole transgenic plants”, their production in bioreactors guarantees defined controlled process conditions and therefore minimizes or even prevents variations in product yield and quality, which simplifies process validation and product registration. Moreover, bioreactors and their configuration significantly affect cultivation results by accomplishing and controlling the optimum environment for effective cell growth and production of bioactive substances. This review highlights the main design criteria of the most widely used bioreactor types, both for plant cell suspension cultures and for hairy roots, and outlines suitable low-cost disposable bioreactors which have found increasing acceptance over the last 10 years.
Keywords: Disposable bioreactors; Hairy roots; Plant cell suspension cultures; Secondary metabolites and recombinant proteins; Traditional bioreactors

Potential of plant cells in culture for cosmetic application by Cornelia Schürch; Peter Blum; Fred Zülli (599-605).
Plants and plant derived ingredients are common and of major importance in the fields of pharmacy, food and cosmetics. The cosmetic industry is a fast moving market. Products have short life-cycles and the industry has to come up with innovative products constantly. Most cosmetic products and their applications are defined by active ingredients. These active ingredients may derive from either synthetic sources or from plant sources. Beside this, no other origin like human or animal are accepted or allowed in cosmetics nor are genetically modified plant sources. The whole cosmetic research and development society is therefore desperately seeking for new innovative plant ingredients for cosmetic application. Unfortunately, new plant derived ingredients are limited because several plants of cosmetic interest are not to be used due to following facts: the plants contain toxic metabolites, the plants grow too slow and a seasonal harvesting is not possible, the concentration of plant constituents differ from harvest to harvest or the plant is endangered and not allowed to harvest. With the plant cell culture technology we bring complete new aspects in the development of novel cosmetic plant derived actives. Due to all these findings, we decided to risk the step into plant cell culture derived cosmetic active ingredient production. This article describes the successful establishment of an apple suspension culture producing a high yield of biomass, cultured in disposable, middle-scale bioreactors. The use of a bioactive extract out of these cells for cosmetic application and the efficacy of this extract on mammalian stem cells is also outlined in this article. To obtain a suitable cosmetic product we used the high pressure homogenization technique to decompose the plant cells and release all the beneficial constituents while encapsulating these components at the same time in liquid Nanoparticles. With the plant cell culture technology we bring complete new aspects in the development of novel cosmetic plants derived actives.
Keywords: Active ingredient; Large scale production; Suspension culture

Improvement of plastic-based disposable bioreactors for plant science needs by J. P. Ducos; B. Terrier; D. Courtois; V. Pétiard (607-613).
The present article describes two new applications of plastic-based cell culture systems in the plant biotechnology domain. Different types of bioreactors are used at Nestlé R&D Center-Tours for large scale culture of plants cells to produce metabolites or recombinant proteins and for mass propagation of selected plant varieties by somatic embryogenesis. Particularly, recent studies are directed to cut down the production costs of these two processes by developing disposable cell culture systems. For large scale culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 l working volumes, validated with several plant species (“Wave and Undertow” and “Slug Bubble” bioreactors). Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has been recently set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 2.5–3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-l glass bioreactors. An improved process has been developed using a 10-l disposable bioreactor consisting in a bag containing a rigid plastic box (“Box-in-Bag” bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design.
Keywords: Coffee; Disposable; Plant cell culture; Somatic embryogenesis; Temporary immersion

Secondary metabolism in cannabis by Isvett Josefina Flores-Sanchez; Robert Verpoorte (615-639).
Cannabis sativa L. is an annual dioecious plant from Central Asia. Cannabinoids, flavonoids, stilbenoids, terpenoids, alkaloids and lignans are some of the secondary metabolites present in C. sativa. Earlier reviews were focused on isolation and identification of more than 480 chemical compounds; this review deals with the biosynthesis of the secondary metabolites present in this plant. Cannabinoid biosynthesis and some closely related pathways that involve the same precursors are disscused.
Keywords: Alkaloids; Cannabinoid biosynthesis; Flavones and flavonols; Lignan group; Stilbenes