Studies of the molecular genetics, mechanisms of activation and functional connections between general phenylpropanoid metabolism and certain branch pathways in parsley, bean, potato plants and cell cultures

Physiology and Molecular Biology of Phenylpropanoid Metabolism

Antibiotic phenols have been found in all plants investigated to date. Some occur constitutively and are thought to function as preformed inhibitors associated with nonhost resistance (84, 94, 128, 134). Others, which are the subject of this review, are formed in response to the ingress of pathogens, and their appearance is considered as part of an active defense response. Since the first suggestions that phenolic intermediates have a role in the active expres­ sion of resistance, an underlying problem in ascertaining that such secondary metabolites are of primary (rather than secondary) importance has been the localization and timing of the host response. In this review we address the problem of response localization and localization of phenolics relative to the sequential development of stages of disease that lead ultimately to resistance expression. Initial demonstrations that phenols are significant components of the host

Phenolic Compounds and Their Role in Disease Resistance

Pharmaceutically significant secondary metabolites or phytopharmaceuticals include alkaloids, glycosides, flavonoids, volatile oils, tannins, resins etc. Currently, most of these secondary metabolites are isolated from wild or cultivated plants because their chemical synthesis is either extremely difficult or economically infeasible. Biotechnological production in plant cell cultures is an attractive alternative, but to date this has had only limited commercial success because of a lack of understanding of how these metabolites are synthesized. Plants and/or plant cells in vitro, show physiological and morphological responses to microbial, physical or chemical factors which are known as ‘elicitors’. Elicitation is a process of induced or enhanced synthesis of secondary metabolites by the plants to ensure their survival, persistence and competitiveness. Here, we discuss the classification of elicitors, their mechanism of action, and applications for the production of phyto-pharmaceuticals from medicinal plants.

Plant cell elicitation for production of secondary metabolites: A review

Index-Flowering plants; antifungal agents; constitutive compounds; phytoalexins; second- ary metabolites. Abstract-Recent work on the characterization of antifungal metabolites in higher plants is reviewed. Interesting new structures are discussed and the distribution of those substances in different plant families is outlined. Distinction is made between constitutive antifungal agents and phytoalexins, which are specifically formed in response to fungal inoculation. The literature survey covers the 12 years since 1982. INTRODUCTION A fungal spore landing on the leaf surface of a plant has to combat a complex series of defensive barriers set up by the plant before it can germinate, grow into the plant tissues and survive. The arsenal of weapons against the fungus includes physical barriers (e.g. a thick cuticle) and chemical ones, i.e. the presence or accumulation of anti- fungal metabolites. These can be preformed in the plant, the so called ‘constitutive antifungal substances’, or they are induced after infection involving de

Review article no. 92 a survey of antifungal compounds from higher plants, 1982-1993

The synthesis of secondary metabolites in response to stress conditions has been implicated as a major defence response of higher plants1. Cell suspension cultures of parsley (Petroselinum hortense) have been studied extensively as a model system and shown to respond to UV irradiation and treatment with fungal elicitor by the synthesis of flavonoids and furanocoumarins, respectively2,3. The induced synthesis of these compounds might afford the plant protection against the respective environmental hazards, because flavonoids strongly absorb UV light and furanocoumarins possess fungitoxic properties4. The production of each class of compound is preceded by the induction of groups of coordinately regulated enzymes2,5–8, based on rapid changes in amounts and activities of the corresponding messenger RNAs9,10. We now report that these changes are due to transient increases in the transcription rates of the respective genes, which indicates that gene activation has an important role in UV and disease resistance in higher plants.

Transcription of plant defence genes in response to UV light or fungal elicitor

Dark-grown cell suspension cultures of parsley, Petroselinum hortense, produce furanocoumarins after treatment with elicitor preparations of either Phytophthora megasperma f.sp. glycinea (Pmg elicitor) or Alternaria carthami Chowdhury (Ac elicitor). The linear furanocoumarins, psoralen and xanthotoxin, and the benzodipyrandione, graveolone, are the major products synthesized in response to Pmg elicitor, besides small amounts of the furanocomarin bergapten. Treatment with Ac elicitor induces predominantly the formation of bergapten and the furanocoumarin isopimpinellin, as well as small amounts of graveolone. While Pmg elicitor leads to cell death within a few days, cell mass increased for at least 6 days after treatment with Ac elicitor. Brefeldin A, a phytotoxin produced by A. carthami, inhibits growth of parsley cell suspension cultures considerably at a concentration of 0.01 mM and growth of the cells ceased at a concentration of 0.1 mM toxin. Concomitantly, furanocoumarin biosynthesis was suppressed in our system by a concentration of brefeldin A within 0.01–0.1 mM.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1983.tb07277.x

Differential response of cultured parsley cells to elicitors from two non-pathogenic strains of fungi. 1. Identification of induced products as coumarin derivatives.

Parsley cell cultures produce linear furanocoumarins and the linear benzodipyrandione, graveolone, in response to treatment with an elicitor from either Phytophthora megasperma or Alternaria carthami. Activities of enzymes involved in general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate: CoA ligase, as well as of an enzyme involved specifically in furanocoumarin biosynthesis, dimethylallyl diphosphate: umbelliferone dimethylallyltransferase, were monitored over several days after treatment with A. carthami elicitor. In addition, the activities of chalcone synthase, an enzyme involved in flavonoid formation, and of glucose-6-phosphate: NADP 1-oxidoreductase were also monitored. The lyase and the ligase activities increased steadily for 48 h and the dimethylallyltransferase activity for 54 h, while the synthase activity was not altered and the oxidoreductase activity decreased gradually. In some experiments, phenylalanine ammonia-lyase activity reached a maximum value of 250 mukat/kg, twice the maximal activity observed previously in parsley cells after treatment with either ultraviolet light or an elicitor preparation from P. megasperma. In crude extracts, phenylalanine ammonia-lyase activity was shown to be inhibited by unidentified small-molecular-weight compounds which were formed in proportion to the elicitor treatment. While phenylalanine ammonia-lyase and dimethylallyl diphosphate: umbelliferone dimethylallyltransferase are known to be required for furanocoumarin biosynthesis, the involvement of 4-coumarate: CoA ligase is as yet unclear. The concomitant increase and decrease of the ligase activity with the activities of the lyase and the dimethylallyltransferase, as well as its similar response to elicitor concentrations, suggest that CoA esters of cinnamic acids play a role in the biosynthesis of furanocoumarins.

Differential response of cultured parsley cells to elicitors from two non-pathogenic strains of fungi. 2. Effects on enzyme activities.

Induction and Suppression of Phytoalexin Biosynthesis in Cultured Cells of Safflower, Carthamus tinctorius L., by Metabolites of Alternaria carthami Chowdhury

Three phytotoxins and two of their non-toxic derivatives were purified from cultures of several Alternaria carthami isolates, the causal agent of a leaf spot disease in safflower, Carthamus tinctorius. These toxins induced leaf spots in safflower similar to the symptoms seen after infection with the fungus. Considerable amounts of the phytotoxins brefeldin A and zinniol were identified from all isolates grown in synthetic culture, while only some isolates formed two derivatives related structurally to zinniol. Only one isolate produced small amounts of 7-dehydrobrefeldin A in addition. Of the toxins, brefeldin A was shown to be the most crucial for the expression of disease symptoms. When brefeldin A was fed toroots of safflower seedlings, toxin accumulated in the leaves and induced wilting within 24h, but the roots appeared unaffected for several days. No immediate effect of this toxin was observed in the electrolyte leakage bioassay using safflower leaf discs but after 5 h treatment, concentration dependent ion losses occurred followed by destruction of the tissue. A microscopic examination of safflower leaf protoplasts, after treatment for several hours with brefeldin A revealed no observable change. Furthermore, the toxin did not significantly alter the membrane potential of suspension cultured safflower cells. Thus, a primary action of brefeldin A on the plasmalemma of safflower is excluded.

Phytotoxins from Alternaria carthami chowdhury: structural identification and physiological significance

A radioimmunoassay for the phytotoxin brefeldin A has been developed employing [7-3H] brefeldin A and an antiserum raised against 7-dehydrobrefeldin A conjugated to bovine serum albumine. The antisera allowed the determination of as little as 1 pmol of toxin and had a similar affinity for both 7-epi-brefeldin A and 7-dehydrobrefeldin A. Several other compounds, including some with structures similar to brefeldin A and certain toxins from other Alternaria species, were bound by the antiserum but at least 6000-fold less strongly than brefeldin A. The radioimmunoassay was used to determine the amounts of brefeldin A in leaf tissues adjacent to and distant from the inoculation site in safflower leaves inoculated with the pathogen Alternaria carthami or with the non-pathogens Ascochyta imperfecta and Eupenicillium brefeldianum. High concentrations of brefeldin A, up to approximately 3 m M , accumulated in leaf tissues over a period of 17 days after inoculation with A. carthami. The other two fungi, although able to produce large quantities of brefeldin A in stationary culture, did not accumulate it in inoculated leaves and the small amount of toxin present with the inoculum disappeared with time. Alternaria carthami, on the other hand, failed to accumulate the toxin when inoculated onto leaves of Zinnia elegans, Helianthus annus, Calendula officinalis or Lactuca sativa. These results support our earlier hypothesis that efficient production of brefeldin A is a factor in the mechanism of infection of safflower tissue by the pathogen A. carthami.

Klaus Günther Tietjen

Papers

Differential response of cultured parsley cells to elicitors from two non-pathogenic strains of fungi. 1. Identification of induced products as coumarin derivatives.

Differential response of cultured parsley cells to elicitors from two non-pathogenic strains of fungi. 2. Effects on enzyme activities.

Induction and Suppression of Phytoalexin Biosynthesis in Cultured Cells of Safflower, Carthamus tinctorius L., by Metabolites of Alternaria carthami Chowdhury

Phytotoxins from Alternaria carthami chowdhury: structural identification and physiological significance

Determination of toxin distribution in Alternaria leaf spot diseased tissue by radioimmunoassay