Plant Natural Products E-Book

Herwig O. Gutzeit

• Herausgeber: Wiley-VCH
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 769

Veröffentlichungsjahr: 2014

Beschreibung

In contrast to existing books which either focus exclusively on the pharmacological properties of plant natural products or cover the secondary metabolism of plants as one section in general plant science book, this is the first to cover all aspects in one volume.

It has all the features of a modern textbook, including color figures, questions and answers and a complimentary website. In addition, the introductory chapters provide sufficient background knowledge in the chemistry and biochemistry of plant natural products and their biotechnological applications to allow its use as a true stand-alone text for student courses.

Leseprobe

CONTENTS

Cover

Related Titles

Title Page

Copyright

Preface

Chapter 1: Biosynthesis and Chemical Properties of Natural Substances in Plants

1.1 Selected Classes of Secondary Metabolites

1.2 Evolution of Natural Compounds in Plants

1.3 Biotechnological Applications

Chapter 2: Function of Natural Substances in Plants

2.1 Secondary Compounds in Primary Metabolism

2.2 Development

2.3 Abiotic Stress

2.4 Symbioses

2.5 Defense

2.6 Allelopathy

Chapter 3: Biological Effects on Microorganisms and Animals

3.1 Coevolution

3.2 Effects on Vertebrates: An Overview

Chapter 4: Metabolism and Toxicity of Natural Substances in Mammals

4.1 Metabolism of Natural Substances in Mammals

4.2 Toxicity

Chapter 5: Examples of Physiological Effects of Natural Substances Present in Food and in Medicinal Plants

5.1 Are Antioxidants Good for Health?

5.2 Endocrine Effects of Natural Substances

5.3 Interference with Neural Functions

Chapter 6: Nature's Drugstore for a Healthy Life

6.1 Biologically Active Food and Food Components

6.2 Natural Compounds in Pharmacological Research

6.3 The Problem of Complexity: Analysis and Consequences

Further Reading

Index

End User License Agreement

List of Tables

Table 1.1

Table 1.2

Table 1.3

Table 1.4

Table 1.5

Table 1.6

Table 1.7

Table 1.8

Table 1.9

Table 1.10

Table 2.1

Table 2.2

Table 2.3

Table 2.4

Table 2.5

Table 2.6

Table 2.7

Table 2.8

Table 2.9

Table 2.10

Table 2.11

Table 2.12

Table 2.13

Table 2.14

Table 2.15

Table 3.1

Table 3.2

Table 4.1

Table 4.2

Table 4.3

Table 4.4

Table 4.5

Table 4.6

Table 5.1

Table 5.2

Table 5.3

Table 6.1

List of Illustrations

Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4

Figure 1.5

Figure 1.6

Figure 1.7

Figure 1.8

Figure 1.9

Figure 1.10

Figure 1.11

Figure 1.12

Figure 1.13

Figure 1.14

Figure 1.15

Figure 1.16

Figure 1.17

Figure 1.18

Figure 1.19

Figure 1.20

Figure 1.21

Figure 1.22

Figure 1.23

Figure 1.24

Figure 1.25

Figure 1.26

Figure 1.27

Figure 1.28

Figure 1.29

Figure 1.30

Figure 1.31

Figure 1.32

Figure 1.33

Figure 1.34

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

Figure 2.6

Figure 2.7

Figure 2.8

Figure 2.9

Figure 2.10

Figure 2.11

Figure 2.12

Figure 2.13

Figure 2.14

Figure 2.15

Figure 2.16

Figure 2.17

Figure 2.18

Figure 2.19

Figure 2.20

Figure 2.21

Figure 2.22

Figure 2.23

Figure 2.24

Figure 2.25

Figure 2.26

Figure 2.27

Figure 2.28

Figure 2.29

Figure 2.30

Figure 2.31

Figure 2.32

Figure 2.33

Figure 2.34

Figure 2.35

Figure 2.36

Figure 2.37

Figure 2.38

Figure 2.39

Figure 2.40

Figure 2.41

Figure 2.42

Figure 2.43

Figure 2.44

Figure 3.1

Figure 3.2

Figure 3.3

Figure 3.4

Figure 3.5

Figure 3.6

Figure 3.7

Figure 3.8

Figure 3.9

Figure 3.10

Figure 3.11

Figure 3.12

Figure 3.13

Figure 3.14

Figure 3.15

Figure 3.16

Figure 3.17

Figure 3.18

Figure 3.19

Figure 3.20

Figure 3.21

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

Figure 4.7

Figure 4.8

Figure 4.9

Figure 4.10

Figure 4.11

Figure 4.12

Figure 4.13

Figure 4.14

Figure 4.15

Figure 4.16

Figure 4.17

Figure 4.18

Figure 4.19

Figure 4.20

Figure 5.1

Figure 5.2

Figure 5.3

Figure 5.4

Figure 5.5

Figure 5.6

Figure 5.7

Figure 5.8

Figure 5.9

Figure 5.10

Figure 5.11

Figure 5.12

Figure 5.13

Figure 5.14

Figure 5.15

Figure 5.16

Figure 5.17

Figure 5.18

Figure 5.19

Figure 5.20

Figure 5.21

Figure 5.22

Figure 5.23

Figure 5.24

Figure 5.25

Figure 5.26

Figure 5.27

Figure 5.28

Figure 5.29

Figure 5.30

Figure 5.31

Figure 5.32

Figure 5.33

Figure 5.34

Figure 5.35

Figure 5.36

Figure 5.37

Figure 5.38

Figure 5.39

Figure 5.40

Figure 5.41

Figure 6.1

Figure 6.2

Figure 6.3

Figure 6.4

Figure 6.5

Figure 6.6

Figure 6.7

Figure 6.8

Figure 6.9

Figure 6.10

Figure 6.11

Figure 6.12

Figure 6.13

Figure 6.14

Figure 6.15

Guide

Cover

Table of Contents

Preface

Chapter 1

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Related Titles

Dewick, P.M.

Medicinal Natural Products - A Biosynthetic Approach, 3rd Edition

2009

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Annual Plant Reviews Volume 40 - Biochemistry of Plant Secondary Metabolism, 2nd Edition

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Wink, M. (ed.)

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ISBN: 978-3-527-31650-2

Plant Natural Products

Synthesis, Biological Functions and Practical Applications

Herwig O. Gutzeit

Jutta Ludwig-Müller

Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty can be created or extended by sales representatives or written sales materials. The Advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.

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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany

Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley's global Scientific, Technical, and Medical business with Blackwell Publishing.

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

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Preface

All organisms synthesize biologically active organic compounds that confer a selective advantage for the respective species. Natural substances play a central role in the evolutionary struggle for survival and hence Nature has invented a plethora of organic substances with different activities that, for example, help to cope with stressful situations, permit communication with other organisms, or deter enemies. We feel that this important interdisciplinary topic should be represented in student courses of biochemistry, pharmacology, and, of course, biology. The book is based on a lecture course on the biology of natural substances, which is part of the curriculum for biology students at the TU Dresden.

The book covers a wide range of different aspects concerning the importance of secondary metabolites for the plants in their respective ecological niche. We discuss, for example, the biosynthesis of biologically active substances, their role in the development of plants, and their interaction with beneficial organisms as well as pathogens, and present examples of the communication between plants and animals feeding on the plants. The toxicity of some substances in vegetarian food is of concern and a toxicological assessment is essential. For this reason, we outline basic principles of toxicological tests with relevant natural compounds. The public discussion about health-promoting substances addresses important scientific questions that are not easy to answer. In the chosen examples of natural substances affecting human health, we summarize the results of intensive research and illustrate the complexity of the elicited reactions. Estrogenic compounds and hallucinogenic drugs exemplify the perils and promises of natural compounds. Are some compounds in our food disease-preventing? In the book, we discuss current attempts to answer this important question. Finally, the inspiration of Nature for medicinal chemistry will be briefly addressed.

The focus of the book is on plant secondary metabolites but the reader is occasionally reminded that animals and bacteria also contain most interesting compounds. In view of the vast published literature on the subject, we had to resist the temptation to address every interesting aspect concerning the biology of natural substances. Rather, we present current concepts of general biological mechanisms and illustrate the essential interdisciplinary approach in the functional analysis of natural substances. We hope that the reader will share our enthusiasm for the subject and will find this book stimulating and an incentive for further studies.

Dresden

Herwig O. Gutzeit

February 2014

Jutta Ludwig-Müller

1Biosynthesis and Chemical Properties of Natural Substances in Plants

The number of known so-called “secondary metabolites” (also referred to as “natural products”) that have been discovered to date is increasing at a constant rate. Yet, it is not only plants (as described in this book) that produce these bioactive compounds; rather, other organisms such as bacteria, fungi, sponges, as well as animals, are also capable of synthesizing a plethora of these metabolites.

Whilst some of these metabolites are discussed in Chapter 4 and 5, a large number remain undiscovered. Moreover, secondary metabolites often possess interesting pharmacological properties, and therefore their characterization is very important. It should not be forgotten that plants synthesize these compounds as part of their own survival strategies, typically as defense compounds or as signals for pollinators or symbionts. In addition, recent evidence has pointed to additional roles for secondary metabolites in plant development. Although the term “secondary metabolites” perhaps infers a less important role for these compounds than those involved in primary metabolism, this is not the case. In fact, many essential and nonessential compounds in this group are found in plants, and even so-called “nonessential materials” can play a role in a plant's responses against abiotic and biotic stress. In this situation, the deletion of a biosynthetic pathway would cause damage to the plant, even if the pathway was not needed under favorable conditions. Interest in the secondary metabolites of plants was further increased when more sensitive analytical instruments became available, as well as genome sequence data for many plant species. Together, these tools allow the details of biosynthetic pathways to be investigated, an example being biotechnological manipulation. Notably, attention also began to be focused on the evolutionary aspects of secondary metabolite synthesis.

In general, secondary metabolites occur as complex mixtures (Figure 1.1). Their biosynthesis can be influenced by a variety of factors during development, in addition to stress, which makes the determination of their complete pattern essentially impossible. Whilst secondary metabolites can occur in the tissues as active compounds, they can also be synthesized as inactive compounds that must be transformed into active products; such metabolites that pre-exist are known as “phytoanticipins.” Compounds that are biosynthesized under stress conditions are typically not detectable in unstressed tissues; when they are synthesized after the invasion of plants by various pests these metabolites are termed “phytoalexins.”

Figure 1.1Correlation between secondary metabolites, their occurrence, and various functions. Secondary metabolites occur always as complex mixtures, and each plant has a specific set of compounds. The composition varies over time, development, and in different tissues and organs. In addition, the metabolites can be inactive and must be metabolized to the active form. Others will be produced only in response to biotic stress factors and other stress situations. The latter have a function in deterring a variety of herbivores, but can also attract insects for pollination.

Secondary metabolites play a variety of roles in the interaction of plants with their environment, but they may also be involved in plant development. They can function in defense mechanisms against herbivores such as nematodes, insects or vertebrates, as well as against microbes and competing plants. Metabolites may also play important roles in the attraction of symbionts, such as insects for pollination or zoophagous insects in multitrophic (when more than two organisms are involved) defense mechanisms. Animals that are required for seed-dispersal purposes are attracted by color and aroma compounds; however, some specialist insects and vertebrates have developed mechanisms to cope with the toxic effects of these compounds, and may generate an ecological niche in recognizing a specific host plant for either feeding or oviposition. Abiotic signals also contribute to the specific patterns of secondary metabolites; for example, some compounds are good protectors against ultraviolet (UV) light and photo-oxidation (i.e., oxidation reactions induced by light).

In order to understand the complexity of these compounds it is essential to have a good knowledge of their biosynthesis and the regulation of genes that encode the enzymes involved in syntheses, under changing conditions. A brief description of the biosynthesis of selected compounds is provided in the following sections of this chapter, but for additional information relating to the enzymes and their biochemistry, the reader is referred to respective textbooks on plant physiology and plant biochemistry.

1.1 Selected Classes of Secondary Metabolites

1.1.1 Occurrence and Compartmentation

The patterns of secondary metabolites will differ depending on the species, the developmental stages, and the stress situations occurring in that species. The differences in pattern are the result of the metabolite biosynthesis, and the regulation of genes encoding the biosynthetic enzymes, transcription factors and transport proteins (see Sections 1.1.3 and 1.1.4). It is therefore difficult to generalize the metabolic patterns for a given plant species. The occurrence of some metabolites can be confined to a certain plant species, such as betalains in the Caryophyllales, glucosinolates in the Brassicales (with few exceptions), and polyacetylenes mainly in the Asteraceae and Apiaceae families. In the latter case, the compounds are important as bioactive substances in food crops (see Chapter 6) such as carrot (Daucus carota), celery (Apium graveolens) and fennel (Foeniculum vulgare). A compound from the Asteraceae Carlina acaulis has a strong antimicrobial activity and is also toxic for trypanosomes. Based on their chemical structures, the secondary metabolites can be placed into different classes (Table 1.1).

Table 1.1The major classes of secondary metabolites based on their chemical structures. Chemical structures are from The PubChem Project.

Class of compound

Example

Structure

N-containing

Alkaloids

Nicotine

Nonproteinogenic amino acid

Canavanine

Amines

Butylamine

Cyanogenic glycosides

Dhurrin

N- and S-containing

Glucosinolates

Glucobrassicin

Without N

Anthraquinones

Emodin

Flavonoids

Quercetin

Polyacetylenes

Falcarinol

Polyketides

Aloeresin

Phenylpropanoids

Rosmarinic acid

Terpenes

Mono-

Thymol

Sesqui-

Helenaline

Di-

Gingkolide

Tri-

Oleanolic acid

Tetra-

ß-Carotin

Poly-

Rubber

The synthesis of secondary metabolites can occur in all plant organs, including the roots, shoots, leaves, flowers, fruit, and seeds. Some metabolites are stored in specific compartments, which may be either whole organs or specialized cell types. Within these compartments the concentration of toxic secondary metabolites may be very high, so that they can exert an efficient defense against herbivores. For example, the glandular trichomes are often rich in toxic compounds, and some pathways – an example being the synthesis of the labdane diterpene Z-abienol in tobacco – occur exclusively in these trichomes. The glandular trichomes of peppermint leaves also contain large amounts of aromatic oils. The latex of Euphorbia is a good example of a toxic mixture of compounds being stored in specialized cells, the laticifers (Figure 1.2).

Figure 1.2Some examples of secondary metabolite-containing plant compartments or cells. (a) Resin duct; cross-section through the needle of a Pinus species; (b) Laticifers; longitudinal section through the shoot of an Euphorbia species; (c) Oil cells; cross-section through a magnolia (Magnolia sp.) leaf; (d) Trichome; section through a foxglove (Digitalis sp.) leaf with glandular trichome. Image (a) from Bernd Schulz and Jutta Ludwig-Müller, Technische Universität Dresden, Germany; images (b–d) from James D. Mauseth, University of Texas, USA.

Lipophilic compounds can be found in the membranes of organelles or in the endoplasmic reticulum (ER), although specific storage vesicles have been reported for some compounds, such as the alkaloid berberin. Hydrophilic compounds are stored preferentially in vacuoles in the inner parts of the organelles and in the cytosol, as well as in the extracellular space (apoplast). Biosynthesis can occur in the cytosol, organelles and the ER, whereas transcriptional control takes place in the nucleus.

Some metabolites occur only in specialized subcellular compartments. In such cases, the tissue- and cell-specific localizations will depend on the solubility of the compound, notably whether it is lipophilic or hydrophilic (Table 1.2). Most hydrophilic compounds accumulate in vacuoles, and are typically present as inactive precursor substances that must be activated either chemically and/or enzymatically; this is the case for cyanogenic glycosides and glucosinolates. Antimicrobial tannins, which exert their protective effects in the extracellular space, are located in the apoplast, while the laticifers contain both hydrophilic and lipophilic compounds. The metabolite patterns are species-specific, but all are toxic; if human skin comes into contact with a latex extract, a severe irritation results. Lipophilic storage compartments also contain antimicrobial and/or defense compounds. Metabolites located in the membranes of the plastids and mitochondria are necessary for primary metabolism, an example being electron-transport chains and light absorbance. Plants secrete volatile compounds either to attract insects for pollination purposes, or zoophagous insects to defend themselves against herbivorous insects (Figure 1.1; see also Chapter 2). Plants may also deliver nonvolatile compounds into the soil, thereby altering the rhizosphere (termed allelopathy; see Section 2.6) that leads to an inhibition of the growth of other, competing, plants.

Table 1.2Tissue-specific and subcellular compartmentation of hydrophilic and lipophilic compounds.

Characteristics

Storage compartment

Class of compounds

Tissue

Cell

Hydrophilic

Laticifer

Some alkaloids

Nonproteinogenic amino acids

Digitalis glycosides

Cyanogenic glycosides

Vacuole

Many alkaloids

Nonproteinogenic amino acids

Saponins

Glycosides

Flavonoids

Anthocyanidins

Betalains

Tannins

Cyanogenic glycosides

Glucosinolates

Amines

Apoplast

Tannins

Cytoplasm

Most hydrophilic compounds during their biosynthesis

Nucleus

Flavonoids

Lipophilic

Trichome

Terpenoids

Resin duct

Flavonoids

Terpenoids

Laticifer

Diterpenes

Quinones

Flavonoids

Polyterpenes

Oil cell

Anthraquinones

Terpenoids

Cuticula

Wax

Flavonoids

Terpenoids

Mitochondrion

Alkaloids (

Conium

)

Plastid

Terpenes

Alkaloids (coniin, quinolizidine, coffein)

Specialized vesicles

Alkaloids (protoberberin)

Plastid membrane

Ubiquinones

Tetraterpenes

Endoplasmic reticulum

Lipophilic substances during enzymatic hydroxylation steps

The process of compartmentation has three important implications:

The plant itself may not be in contact with the toxic compounds, because it is only after wounding or attack by other organisms that the metabolites will be released from the storage compartments.

The biosynthetic pathways are also compartmentalized, but this necessitates the involvement of transport processes for metabolite movement.

The target molecules for the secondary metabolites may be present in all compartments; however, the target is not necessarily colocalized with an active compound.

A few cellular targets will be discussed in the following section (see Figure 1.3), and further descriptions are provided in Chapters 5 and 6, with details of the compounds' biological activities. Many compounds that are used to protect plants against microbes and herbivores exert their biological activities outside the plant tissues; however, if these compounds were to exert any toxic effect on the plant, their compartmentation would provide protection against self-poisoning.

Figure 1.3Cellular and molecular targets for secondary metabolites in plants and other organisms. In the plant cell (green), secondary metabolites (SecMet) are synthesized and can exert their effects in different compartments, such as the electron-transport chain in organelles, transporters at the plasma membrane, as antioxidants, and as fortification elements in the cell walls. They are stored in vacuoles or cell wall compartments as coloring or defense compounds. In the cells of the target organisms (brown), into which the metabolites must be transported, the targets can be in all compartments. Target molecules encompass transporters and channels, enzymes and proteins, nucleic acids, the cytoskeleton, receptor molecules and signal transduction chains, as well as membrane lipids.

Membrane targets can be found at the plasma membrane, ER or Golgi apparatus, where the functions of molecules such as transporter and channels, and also of membrane receptors, can be altered. The electron-transport chains of both plastids and mitochondria may also serve as targets, and plant compounds may inhibit their function. In addition, the structural proteins of the cell wall and of enzymes may be affected, while the cytoskeleton, with its component proteins myosin and tubulin, may be a target for secondary metabolites and result in an altered cell cycle (see Figure 2.5). Within the nucleus and the organelles, desoxyribonucleic acid (DNA) is a target for interaction with secondary metabolites; it is well known that some molecules alkylate DNA, which in turn affects its replication, transcription, and repair mechanisms. It should be noted that, in order to be active in these compartments, the metabolites must be transported across the respective membranes (see Section 1.1.4).

Although the major role of these compounds seems to be the defense of plants against other organisms, humans are also affected by secondary metabolites (see Chapters 3.2 and 6). When defending plants against microbes, the toxic effect of the secondary metabolites on one or several target molecules constitutes a major role, although their “bitter-tasting” properties will generally deter attacks by herbivores. In these organisms, the targets may be located as indicated in Figure 1.3, with the exception of plastids, which only play a role as target in allelopathy. This might not be the case for parasitic plants, however, which are dependent on the host photosynthesis for their nutrient acquisition. Many secondary metabolites, such as sterols, can influence membrane fluidity (see Figure 2.28), while other compounds may interact with or intercalate in DNA, and are thus cytotoxic; mitosis may also be inhibited (see Figure 2.5).

Secondary metabolites can also inhibit the activity of various enzymes. Alkaloids and amines are often similar to the neurotransmitters of vertebrates, or to hormones involved in the development of insects, and thus mimic their effects. Cyanogenic glycosides release toxic cyanide, which inhibits cytochrome c oxidase in the mitochondrion and, as a consequence, energy production in the form of adenosine triphosphate (ATP). Isothiocyanates, which are released from glucosinolates, can influence membrane fluidity. Terpenes also interact with membranes, though some demonstrate similarities to biologically important sterols in fungi and animals. For example, the Na+-, K+-ATPase, which is important in animals, can be inhibited by several groups of metabolites, including the pyrrolizidine alkaloid monocrotalin, the digitalis glycosides, and/or polyketides such as anthraquinones. The latter also interfere with the formation of cyclic AMP (cAMP) by inhibiting adenylate cyclase, which itself influences many signal transduction pathways. Interestingly, plants contain cyclic guanosine monophosphate (cGMP) rather than cAMP. Flavonoids also have the ability to inhibit enzymes, but may cause additional mutagenic and toxic effects on DNA via alkylation.

1.1.2 Biosynthesis

The biosynthetic pathways derive from various precursors of primary metabolism (Figure 1.4). The precursor is defined as a molecule used by a biosynthetic enzyme as a substrate and converted to a product. The product can be an intermediate in the pathway, and in this case it is used as precursor for the next biosynthetic enzyme, or it is the final product of the reaction chain.

Figure 1.4General scheme of biosynthetic pathways and precursors for the major classes of secondary metabolites. Pathways deriving from aromatic amino acids are shown in green; pathways deriving from carbohydrates are shown in brown; pathways deriving from lipids are shown in yellow. Modified from Larcher, W. (1995) Physiological Plant Ecology, 3rd edn, Springer-Verlag.

In a complex reaction scheme, which has many junctions, an intermediate is simultaneously also a precursor for another part of the pathway. In Figure 1.4, shikimic acid would be an intermediate for the amino acid metabolism, and also a precursor for the biosynthesis of aromatic secondary metabolites. Similar precursors are used within one class of compounds for the biosynthesis, but the same precursors can be also used for a range of different metabolites.

The final products are derived from three major classes of compounds and marked in the same color in Figure 1.4. The major precursors are derived from protein (amino acids), carbohydrate (sugars) and lipid (fatty acid) metabolism. The biosynthetic pathway for aromatic amino acids is one of the major sources of aromatic compounds such as phenols, flavonoids and some alkaloids. Acetyl-CoA is a central metabolite formed by glycolysis and also via the β-oxidation of fatty acids, and is used in the tricarboxylic acid cycle in the synthesis of organic acids, which are also precursors for secondary metabolites. In addition, acetyl-CoA is involved in the synthesis of terpenes, which form a distinct class of metabolites. In the following sections some selected biosynthetic pathways will be discussed, but for more detail the reader is referred to textbooks on plant biochemistry. Knowledge of the biosynthetic pathways of natural compounds is essential for the targeted manipulation of these pathways in biotechnology. In addition to knowledge on the enzymes and genes involved in these biosyntheses, it is important to understand the transcriptional control of the genes. Therefore, a few examples will be provided where the transcriptional control of pathways has been well elucidated, notably of the glucosinolates and flavonoids (Section 1.1.3). Finally, details on biosynthetic pathways and corresponding genes will help to provide an understanding of how these secondary metabolites have evolved (Section 1.2).

When the basic skeleton of a secondary metabolite has been synthesized, it is the many enzymatic modifications (which are added later to the structure) that form the main reason for the large variations among these molecules. The chemical reactions involved include oxidations, hydroxylations, reductions, acylations, methylations, prenylations and glycosylations (see also Table 2.5).

1.1.2.1 Alkaloids

Within the group of alkaloids many compounds with stimulating, hallucinogenic and analgesic properties can be found (see Chapter 5). Many of these compounds are toxic, or they can be converted from nontoxic to toxic compounds. Depending on the chemical structure of an alkaloid, different precursors are required for the biosynthesis. Examples of the major groups of alkaloids, together with a typical chemical structure, a plant in which the compound is found and the main precursor(s) involved in the biosynthesis, are shown in Table 1.3.

Table 1.3The major classes of alkaloids and their precursors, according to their chemical structure. One example, with chemical structure, and one plant species where it occurs, is given for each class. Chemical structures are from The PubChem Project.

Basic structure

Precursor

Example of structure (Plant)

Formula basic structure

Acridine

Anthranilate

OH-N-Methylacridone (

Ruta graveolens

)

Benzophenanthrinidine

Tyrosine

Sanguinarine (

Eschscholzia californica

)

Quinazoline

Ornithine

Anthranilate

Peganine (

Peganum harmala

)

Quinoline

Tryptophan

Quinine (

Cinchona officinalis

)

Quinolizidine

Lysine

Lupanine (

Lupinus polyphyllus

)

Imidazole

Histidine

Pilocarpine (

Pilocarpus jaborandi

)

Indole

Tryptophan

Serotonin (

Musa x paradisiaca

)

Isoquinoline

Tyrosine

Morphine (

Papaver somniferum

)

Phenylalkylamine

Phenylalanine

Cathinone (

Catha edulis

)

Piperidine

Lysine

Coniine (

Conium maculatum

)

Purine

Aspartate

Glycine

Glutamate

Caffeine (

Coffea arabica

)

Pyridine

Aspartate

Ornithine

Nicotine (

Nicotiana tabacum

)

Pyrrolidine

Aspartate

Ornithine

Muscarine (

Amanita muscaria

)

Pyrrolizidine

Ornithine

Valine

Senecionine (

Senecio jacobaea

)

Terpene indole

Tryptophan

Isoprene

Physostigmine (

Physostigma venenosum

)

Tropane

Ornithine

Arginine

Cocaine (

Erythroxylon coca

)

Tropolone

Tyrosine

Phenylalanine

Colchicine (

Colchicum autumnale

)

The biosynthetic pathway for an individual compound is very complex. Many different precursors are involved for the various pathways, including not only aromatic amino acids such as tryptophan, tyrosine and phenylalanine but also aspartate, glutamine, lysine, glycine and valine for other alkaloids (Figures 1.5 and 1.6; Table 1.3). In addition, the nonproteinogenic amino acid ornithine is an important precursor for various alkaloids. Anthranilic acid, from which tryptophan is synthesized, is the precursor for acridine alkaloids, and thus the shikimate pathway (which is responsible for the biosynthesis of all aromatic amino acids) is involved. For several alkaloids two different precursors are needed for the biosynthetic pathways. In the case of terpene indole alkaloids (Figures 1.5 and 1.6; see also Figure 1.34), it is not only tryptophan that is involved as a precursor for the indole moiety, but also monoterpenes for the synthesis of side chains. Another example is the biosynthesis of the tropane alkaloids hyoscyamine and scopolamine, where ornithine and phenylalanine are required for the different parts of the molecule (Figure 1.5; Table 1.3).

Figure 1.5Overview on the biosynthesis of selected alkaloids. The shikimate pathway leads to the synthesis of many different alkaloids given in colors depending on their origin (red = anthranilate/tryptophan, green = tyrosine). In addition, ornithine and arginine via putrescine result in the synthesis of pyridine (dark blue), tropane (blue) and pyrrolizidine alkaloids (light blue). Phenylalanine together with ornithine is needed for the synthesis of a second group of tropane alkaloids (violet). Caffeine and related substances is derived from purine (brown). The class of compounds is given in brackets.

Figure 1.6Compartmentation of terpene indole alkaloid biosynthesis in Catharanthus roseus. The cellular compartments (plastids, cytosol and vacuole) participate in the biosynthetic steps. Enzymatic reactions with dashed arrows are hypothetical. Circles symbolize membrane-associated enzymes. Putative transporters are also indicated. The structure for the terpene indole alkaloid vindoline is shown. IPP: isopentenylpyrophosphate; G10H: geraniol-10-hydroxylase; NMT: S-adenosyl-methionine hydroxytabersonine-N-methyltransferase; DAT: acetyl-coenzyme-A deacetylvindoline 17-O-acetyltransferase; OHT: 2-oxoglutarate-dependent dioxygenase; POX: peroxidase; SSβG: strictosidine-β-glycosidase; SSS: strictosidine synthase. Modified from Verpoorte, R. (2000) Metabolic engineering of plant secondary metabolism. Kluwer Academic Publisher, pp. 1–29. Chemical structure is from The PubChem Project.

Although many alkaloids are of importance to humans (Chapter 5), their biosynthetic pathways are often not fully understood. However, it is important to know as much as possible about the precursors, enzymes and limiting steps in the pathway, if these secondary metabolites should be changed in abundance by breeding or biotechnological methods employed in plants (Section 1.3). It is important that changes in one pathway do not lead to limitation of a precursor for a second pathway, in case the two pathways compete for the same precursor. This can be observed especially for aromatic amino acids, which play many roles in different pathways in the plant; examples include their requirement in proteins, a role in the synthesis of cell wall components, and as precursors for the plant hormone indole-3-acetic acid (IAA). In secondary metabolite synthesis, amino acids are involved in the synthesis of alkaloids, of phenolic compounds, and also of pigments such as anthocyanins and betalains. This indicates that, even under natural conditions, alkaloid biosynthesis competes with many other pathways, because the other amino acids are also needed for protein synthesis.

The shikimate pathway contributes to the synthesis of acridone and indole alkaloids via anthranilic acid or tryptophan. Tyrosine is a precursor for a series of compounds, namely the protoberberine type, benzylisoquinoline, morphinane, and benzophenanthridine alkaloids; the intermediate is reticuline (Figure 1.5). Pyridine (e.g., nicotine) and tropane alkaloids (e.g., cocaine) are derived from ornithine, where the polyamine putrescine is a common precursor; however, the pathways diverge before the intermediate tropolone is formed. For a second class of tropane alkaloids, the amino acid phenylalanine is required in addition to ornithine for synthesis of the backbone of the molecule littorine, which is an intermediate on the route to hyoscyamine and scopolamine. Putrescine is converted to homospermidine, which is then metabolized to retronecine, the precursor for the group of pyrrolizidine alkaloids (Figure 1.5). The reaction converting homospermidine is catalyzed by homospermidine synthase, the first specific enzyme for this pathway. For an additional part of the molecule – the complex ester structure of mono- or dibasic acids – either leucine or isoleucine are mainly involved as precursors. Pyrrolizidine alkaloids are synthesized mainly in the roots and then transported to the upper parts of the plant. The problem here for humans and animals is the hepatotoxic potential of these compounds.

Although quinolizidine alkaloids are structurally very similar to pyrrolizidine alkaloids, they are synthesized from the proteinogenic amino acid lysine. An important intermediate in this pathway is cadaverine (another polyamine), the biosynthesis of which occurs in the chloroplasts of mesophyll cells. A third group with structural similarity to the above-mentioned compounds are the indolizidine alkaloids; these are found in the orchid genus Dendrobium, but also occur in the South American poison dart frog of the genus Dendrobates. Even though the synthesis of the two major compounds swainsonine and castanospermine derives from the amino acid lysine, a different intermediate is found, namely pipecolic acid. The second part of the ring structure is derived from acetate.

In the way that the same precursor is used for a range of metabolic pathways, intermediates can also serve in the synthesis of a variety of compounds (Figure 1.5). The intermediate reticuline serves as a precursor for different groups of alkaloids, such as isoquinoline-, benzophenanthridine- or protoberberine-type compounds; examples are morphines, sangurinarine, and berberine. Within these pathways there are alternatives to synthesize the same metabolite; for example, nicotine can be produced from either ornithine alone or by a fusion of ornithine with putrescine. Morphine (for structure, see Figure 5.30) can either be synthesized via thebaine, oripavin and morphinone (the latter is the direct precursor for morphine), or by the conversion of thebaine to neopinone, which is then converted via codeinone to the bioactive codeine, a known remedy against cough (see Section 4.1); ultimately, codeine is converted to morphine.

Amphetamine-like compounds such as cathinone from Catha edulis are also derived from an aromatic amino acid (phenylalanine in this case). The mild stimulating activity of the plant can be achieved by chewing leaves, whereby cathinone is absorbed via the mucosa of the mouth. The genus Ephedra produces ephedrine and pseudoephedrine, which are used in medicine to reduce nasal congestions (Section 5.3.7). The slightly stimulating activity is, again, due to the similarity with amphetamine. The biosynthesis might proceed via a route similar to phenylpropanoid synthesis, but with benzoic acid or benzoyl-CoA as intermediates.

Simple indole alkaloids such as serotonin most likely derive from tryptophan via tryptamine as intermediate. The complex structure of terpene indole alkaloids is also dependent on the terpene pathway for the side-chain moiety. The biosynthesis of terpene indole alkaloids is also an example of a highly compartmented pathway (Figure 1.6). On the one hand, the localization of enzymes is critical, but on the other hand the solubility of the compounds plays a role and these factors necessitate the compartmentalization of the compounds (Figure 1.3; Table 1.2), and therefore transporters are essential for the respective intermediates. Tryptophan is synthesized in the plastids, whereas tryptamine is formed in the cytosol. The latter must be transported between the two compartments, and the same is true for the monoterpene moiety, which is again synthesized in the plastids (see Figure 1.14). Tryptamine is further converted in the cytosol, whereas the first step to the terpene indole alkaloid takes place in the vacuole. Although further enzymatic modification steps are observed again in the cytosol, the later steps also occur in the vacuole (Figure 1.6).

1.1.2.2 Phenol Derivatives, Especially Flavonoids

As a representative group for bioactive phenols, the biosynthesis of flavonoids is described in more detail. The biosynthetic pathways derive from the shikimate pathway (Figure 1.7), which is shared by indoles, and by several alkaloids and betalains. The precursor for the cinnamic acid derivatives and flavonoids is phenylalanine, which is converted by the key enzyme for this part of the pathway, phenylalanine ammonia lyase (PAL), to cinnamic acid. Simple molecules (e.g., cinnamic acids) as well as very complicated molecules (e.g., rosmarinic acid) are synthesized via this pathway. Rosmarinic acid has a high antioxidative potential and also good aromatic qualities. Coumarin occurs in woodruff as an aroma and an intoxicating (poisonous) substance. The cinnamic acid derivatives also serve as precursors for polymers such as lignin, which is synthesized via cinnamaldehydes and the respective alcohols, termed monolignols. Flavonoids and stilbenes, such as resveratrol from grapes, are also formed via this pathway. Activation by coenzyme-A plays an important role in this biosynthesis network; the cinnamic acid is activated and acts then, as cinnamoyl-CoA, as a precursor for other pathways in the synthesis of flavonoids and coumarins.

Figure 1.7Schematic overview for the biosynthetic pathways of selected phenols from phenylalanine as precursor (bold). A key enzyme, phenylalanine ammonia lyase (PAL), is shown (red). Some example structures are depicted (blue). Chemical structures are from The PubChem Project.

Flavonoid biosynthesis has been quite well elucidated in the model plant Arabidopsis thaliana, in which only a small set of secondary metabolites are synthesized. Much information is also available from maize and legumes, the latter also contain isoflavonoids. The scheme for the biosynthetic steps leading to the major flavonoid compounds includes information derived from several plant species (Figure 1.8). The key enzyme for the flavonoid pathway is chalcone synthase, which catalyzes the reaction to naringenin chalcone, the first flavonoid in the pathway. Chalcone synthase mutants of Arabidopsis have a transparent seed coat (testa), and are therefore referred to as transparent testa (tt) mutants. However, other mutants in the pathway of, for example, the next enzyme encoding chalcone isomerase (which is responsible for the synthesis of naringenin), also show this phenotype, and consequently the mutations were numbered consecutively, starting with “1.” Mutations in the transcription factors (see Section 1.1.3) that control the synthesis of flavonoids have similar phenotypes.

Figure 1.8The main pathways for flavonoid synthesis derived from different plant species. Because of the complexity of the different biosynthetic steps, only the basic features of the pathways are illustrated. The names in brackets are examples for each class of compounds, which is then also presented in a chemical structure. The red arrows indicate changes in the individual flavonoid groups compared to naringenin. CHS: chalcone synthase; CHI: chalcone isomerase; IFS: isoflavonoid synthase; FNS: flavone synthase; F3H: flavanone-3-hydroxylase; FLS: flavonol synthase; DFR: dihdroflavonol reductase; ANS: anthocyanidin synthase; UGT: glycosyltransferase; ANR: anthocyanidin reductase. Chemical structures are from The PubChem Project.

Several biosynthetic pathways originate from naringenin, which lead to (i) the isoflavonoids of legumes, (ii) flavones, and (iii) flavonols, which constitute the largest group among the flavonoids (Table 1.4). The flavonols in the model organism for molecular biology, Arabidopsis thaliana, are kaemfperol and quercetin, and their metabolites (glycosides). Dihydroflavonols are intermediates for the formation of flavonols, but are also converted to the anthocyanin pigments. In addition, they give rise to the proanthocyanidins (also known as “condensed tannins”), which are found in the seed coats of many plants. The catechins are derived from intermediates of the anthocyanin pathway, the (leuco)anthocyanidins. The highly diverse structures are achieved by glycosylation reactions with various sugars, where mostly monoglycosides or diglycosides are formed (see Figure 2.13). The sugar moiety is added either as a monosaccharide or disaccharide to one position, though the sugars can be also added at different positions of the molecule. Glycosylations also render the molecules more hydrophilic, and this may change the storage compartment involved. Further details on the water-soluble anthocyan(in)s are provided in Chapter 2.

Table 1.4The major classes of flavonoids with the estimated number of compounds and some functions (see also Chapter 2).

Class

No. of known compounds

Biological function in plants

Anthocyanins

250

Red and blue pigments

a)

Aurones

20

Yellow pigments

Biflavonoids

65

Not known yet

Catechins

40

Antimicrobial

Chalcones

60

Yellow pigments

Dihydrochalcones

10

Feeding deterrents

Flavones

350

Light yellow pigments

Flavonols

350

Feeding deterrent

Isoflavonoids

15

Antimicrobial, attraction

Proanthocyanidins

50

Antimicrobial

a)

Pigmentation can have the function in flowers of pollination, but also in fruit of seed dispersal or the deterrence of herbivores in combination with bitter-tasting compounds. In leaves, the pigmentation can serve as a protection against high levels of light.

1.1.2.3 Betalains

Betalains fulfill the function of anthocyanins in the Caryophyllales as pigments and UV-protecting compounds. They have never been detected in combination with anthocyanins in any plant, and the reason for that remains a mystery. In addition to plants, betalains occur in some mushrooms, such as fly agaric (Amanita muscaria). Betalain-containing plant species cannot convert flavane-3-dioles to the respective anthocyanin, whereas flavonoids up to the step of leucoanthocyanidins occur in betalain-containing plants.

The group of N-containing betalains are derived from tyrosine, though an alternative pathway based on tyramine has been reported for some plant species. In contrast to anthocyanins and carotenoids, the biosynthetic pathway of betalains is only partially understood. Like anthocyanins, they also occur in a glycosylated form, but the color does not depend on the pH. Betalains are comprised of two major groups: the yellow betaxanthins and the red-pink betacyanins (Figure 1.9). The more than 50 betacyanins known to date are water-soluble and stored in vacuoles. The most important intermediates are DOPA (L-3,4-dihydroxyphenylalanine; see Figure 1.9) and betalamic acid. Next to betanidin, amararanthin is a prominent compound in Amaranthus species, while betalamic acid serves as intermediate for the betaxanthins and betanidins. The reaction is nonenzymatic in both cases; rather, a spontaneous addition occurs of either cyclo-DOPA to yield betanidin, or of an amino acid or amine, which results in the formation of a betaxanthin. The glycosylated betanin is formed by a glycosyltransferase reaction with either cyclo-DOPA or betanidin. The enzyme tyrosinase is known from the synthesis of melanin in animals, and in this case two reactions are being catalyzed by the one enzyme; but this enzyme has not been characterized in plants. In contrast, evidence has been advanced that for these two steps a different enzyme is required.

Figure 1.9Betalain biosynthesis from the aromatic amino acid tyrosine. Enzymatic steps are shown in red, spontaneous reactions in blue. The inferred enzyme tyrosinase is shown in light red. DOPA = (2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoic acid; DODA = DOPA 4,5-dioxygenase. Insert: Expression of the cytochrome P450 gene and the DODA gene in yeast (a. DODA expression + DOPA as substrate; b. DODA expression – DOPA; c. both genes + DOPA as substrate; d. both genes – DOPA). Photographs from Hatlestad, G.J. et al. (2012) Nat. Genet., 44, 81. Reprinted with permission from Nature Publishing Group. Chemical structures are from The PubChem Project.

A novel gene involved in the biosynthesis of betacyanins in Beta vulgaris has been isolated with the help of yellow mutants encoding a novel cytochrome P450 protein that is involved in the conversion of DOPA to DOPA-quinone, and most likely again by the spontaneous cyclization to cyclo-DOPA. The biosynthesis of betalamic acid is dependent on a DOPA 4,5-dioxygenase (DODA), which results in the formation of both betacyanins and betaxanthins. Functional proof of this was derived from the silencing of red wild-type B. vulgaris plants using both genes alternatively. Whereas loss of the cytochrome P450 gene resulted in yellow-pigmented plants, loss of the DODA gene yielded white (unpigmented) plants; this showed that in beet, betalamic acid is indeed the precursor for both the yellow and red pigments (Figure 1.9). Complementation of the yellow beet mutant with the cytochrome P450 gene led to red tissues. The expression of these genes in yeast, with subsequent addition of the substrate DOPA, resulted in a red color in the case of both enzymes, in a yellow color when only the DODA gene was expressed, and in no color when the DOPA substrate was omitted. The results of this experiment indicated that both genes are essential for the red betacyanin formation, whereas the DODA gene alone is sufficient to yield the yellow betaxanthins.

1.1.2.4 Cyanogenic Glycosides, Glucosinolates, and other Indole Derivatives

While alkaloids, phenols, flavonoids and terpenes are present in most plant families – albeit with their plant-specific patterns – glucosinolates occur mainly within the crucifer family (Brassicaceae). This family includes many crop plants, vegetables and spices are found, such as cabbages, radish, mustard, or oilseed rape (canola) (Figure 1.10); the model plant A. thaliana is also a member of the Brassicaceae. Although glucosinolates and cyanogenic glycosides share similar intermediates in their biosynthetic pathways, it has been hypothesized that both have evolved separately (Section 1.2), and both classes of metabolites have been identified only in a few plant species, among them in papaya (Carica papaya).

Figure 1.10Some examples of crop, vegetable and spice plants belonging to the Brassicaceae. Only the species, not cultivars, are given. Source: Fotolia.com (Chinese cabbage, © Bernd, S.; oilseed rape, © Schliemer; radish, © photocrew; cauliflower, © dimakp; Brussels sprout, © narumol pug; cress, © eyetronic; mustard, © Andrey Starostin; rucola, © hvoya; horseradish, © Hetizia; broccoli, kohlrabi, © Natika).

The cyanogenic glycosides and glucosinolates are both present as glycosides in the vacuoles; however, they are inactive and must first be metabolized to form the active compounds, either via an enzymatic reaction or spontaneously. This process occurs only following damage of the tissue due to colonization by microbes, or by the feeding of herbivores, and consequently the glycosides and glucosinolates are considered as preformed defense compounds (see Section 2.5). Although the glucosinolates are localized in the vacuole, the degrading enzyme – β-thioglycosidase (myrosinase) – is compartmentalized in the cytosol. Following tissue disruption, however, both components of the system come together, whereupon the glucosinolates are metabolized to their toxic active components that are referred to as “mustard oils”; hence, the whole system is known as “The Mustard Oil Bomb.” As these compounds might also be toxic towards the plant cell, compartmentation is essential in order to maintain healthy tissues. The application of immunohistochemical techniques has shown that myrosinases also occur in the vacuoles, albeit in specialized “myrosin cells,” named after the enzyme myrosinase. In contrast, the glucosinolates are localized in so-called S-cells (Figure 1.11), so-named due to their high sulfur (S) content. This specific cell-type localization of the glucosinolate–myrosinase system has been identified in the inflorenscences of A. thaliana and Brassica napus (oilseed rape).

Figure 1.11Cellular compartmentation of the glucosinolate–myrosinase system in inflorescences of Arabidopsis thaliana. A light microscopy cross-section shows the different cell types labeled in different colors: glucosinolate-containing S-cells (green), the myrosin cells (M) containing the β-thioglycosidase myrosinase (red and orange), the epidermal cells (E), which contain epithiospecifier proteins (yellow), the cortex (C) (dark blue), the endodermis (En; cyan), phloem (P; pink) and xylem (X; violet). From Kissen, R. et al. (2009) Phytochem. Rev., 8, 69 (see Further Reading). Reprinted with permission from Springer.

Among the Brassica species used as food or spices, compartmentation is especially important because it gives rise to the special taste of these crops. Some of the metabolites also have anticancerogenic potential, so that their formation is desirable (Chapter 6). Other cell types contain the so-called “specifier proteins”; these are important when determining the metabolic fate of the glucosinolate as either volatile (iso)thiocyanates, or as less-toxic nitriles that can be further converted to carboxylic acids (Figure 1.12).

Figure 1.12Biosynthetic pathways (light gray) and metabolism (gray) of glucosinolates and cyanogenic glycosides in comparison. For the glucosinolates the general scheme is shown for the different amino acids as precursors (a) and for cyanogenic glycosides the main pathway is shown (b). In both cases the metabolism leads to toxic compounds. For cyanogenic glycosides the deglycosylation yields cyanide, whereas for glucosinolates the metabolism is more complicated involving so-called specifier proteins. MAM = methylthioalkylmalate synthase; FMO = flavin-dependent monooxygenase; CytP450 = cytochrome P450-dependent monooxygenase; “?” indicates a reaction step for which no enzyme has yet been identified; ESP = epithiospecifier protein; NSP = nitrile-specifier protein; TFP = thiocyanate-forming protein.

The precursors required for the biosynthesis of all these compounds are amino acids (Figure 1.12). The glucosinolates can be divided into three large groups, depending on the respective precursor amino acid: (i) aliphatic glucosinolates deriving from methionine; (ii) aromatic glucosinolates deriving from phenylalanine and tyrosine; and (iii) indolic glucosinolates originating from tryptophan. Not all brassicaceous plants contain all groups of glucosinolates. For example, the model plant Arabidopsis contains aliphatic and indole glucosinolates, but no aromatic representatives. On the other hand, this is the major glucosinolate group in the garden cress (Lepidium sativum) and in nasturtium (Tropaeolum majus) which, in addition, contains indole glucosinolates.

The major variation in the aliphatic glucosinolates is derived from enzymatic chain elongation reactions of the amino acid methionine. These chain-elongated precursors are then used for the formation of the next intermediate aldoxime. This enzymatic step, catalyzed by cytochrome P450-dependent monooxygenases, is quite specific for the amino acid substrate. In contrast, the later steps – that is, the addition of a sulfur group and glycosylation – are not very specific, and the respective enzymes accept a variety of different glucosinolate structures. This implies that only a modification of the specific step could lead to novel compounds in other plant species, because they can transform the new substance by their set of enzymes to the final glucosinolate. This has been achieved with Arabidopsis, where the biosynthesis of benzylglucosinolate was engineered by transformation with the gene encoding the protein for the aldoxime synthetic reaction. The late modification steps, such as hydroxylation or methylation, are again specific for the individual glucosinolates. The range of possible enzymatic reactions can differ greatly between the aliphatics on the one hand and indole glucosinolates on the other hand (Figure 1.12). Finally, unusual modification reactions have been reported for glucosinolates, such as benzoylation and sinapoylation. In these cases, a benzyl or sinapoyl moiety is attached following the esterification of hydroxylated glucosinolates to the core structure.

Closely related to the indole glucosinolate pathway is the biosynthesis of other indolic compounds, for example the indole-type phytoalexins in Brassica species. In Arabidopsis there is one phytoalexin (camalexin) with an indole moiety present, whereas other Brassica species contain several indole phytoalexins, such as cyclobrassinin, brassilexin, rutalexin, or spirobrassinin (see Section 2.5). The biosynthetic pathway of camalexin is a branch of the indole-3-acetaldoxime pathway leading to indole glucosinolates (see Figure 1.19), whereas the biosynthesis of the Brassica indole phytoalexins derives from either tryptophan, indole or indole-3-glycerolphosphate, thus representing an earlier branch point in the metabolic network of indole metabolites.

Apart from the indole glucosinolates and indole phytoalexins of the Brassicaceae, other indole derivatives – which are also considered as defense compounds – are synthesized in different plant species. Among these are the terpene indole alkaloids (Figure 1.6) and a volatile compound in maize (DIMBOA; 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one). A detailed description of the biosynthetic pathway of this compound is provided in Section 1.2 (see Figure 1.25). Briefly, the precursor is also indole-3-glycerol phosphate, which is converted in two enzymatic steps to DIBOA (2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one). DIBOA is glycosylated by a uridine diphosphate (UDP)-dependent glycosyltransferase; the glycosylated DIBOA is then converted to DIMBOA-glycoside, which is stored in the vacuoles. As in the case of glucosinolates, the glycosylated form is not toxic and must be deglycosylated to yield the toxic metabolite (the chemical structure is shown in Figures 2.30 and 3.12).

The cyanogenic glycosides are also synthesized from an amino acid (see Figure 1.12) although, in contrast to the glucosinolates, they do not occur predominantly in one plant family. The best-understood pathway is that yielding dhurrin, the cyanogenic glycoside of millet (Sorghum bicolor). In general, the amino acid is converted to an aldoxime, which is reminiscent of a biosynthetic step in glucosinolate synthesis. The aldoxime is further converted via a nitrile as an intermediate to the cyanogenic glycoside. The nitrile can also be converted to an aldehyde and cyanide. Detoxification of cyanide is therefore an essential part of the metabolic pathways leading to these compounds. In contrast to the formation of aldoxime in the glucosinolate pathway, the enzyme catalyzing the first step in cyanogenic glycoside synthesis is dependent on NADPH. Metabolic channeling was also shown to occur for the plasma membrane-bound enzymes, which means that the metabolites are not released from the enzymes after each step of the reaction. Nonetheless, this is a very efficient procedure in terms of the availability of precursors in low concentrations.

The degradation of glucosinolates and cyanogenic glycosides to toxic metabolites closely resembles one another in the first steps (see Figure 1.12). Metabolic conversion is especially vital for glucosinolates to become bioactive, and their fate is mainly determined by protein factors, which influence the outcome of the reaction. While the β-thioglucosidase myrosinase catalyzes the deglycosylation, the so-called “specifier proteins” determine the next reaction product. Isothiocyanates may be formed spontaneously. An epithiospecifier protein is involved in the formation of an epithionitrile, a nitrile specifier protein in the formation of a nitrile, which is less toxic. These reaction products can be further metabolized to the corresponding carboxylic acid by nitrilases (see Figures 1.12 and 1.24). Finally, a thiocyanate-forming protein gives rise to the toxic thiocyanate. If none of these specifier proteins is present, then the isothiocyanate is mostly released, because of its volatility. Cyanogenic glycosides are also deglycosylated, which results in an aglycone that is further converted to cyanohydrin and the toxic cyanide (HCN) by the enzyme hydroxynitrile lyase. The cyanide group can be transferred enzymatically to the amino acid cysteine, such that β-cyanoalanine is the final product. The latter may then be converted by a specialized nitrilase to aspartate, or by a nitrile hydratase to asparagine (see Figure 1.24). These reactions are important to the plant, because of the extreme toxicity of the cyanide.

1.1.2.5 Terpenes

Among the terpenes some intoxicating or hallucinogenic compounds can be found; examples are the cannabinoids (see Section 5.3.2), which are synthesized predominantly via the methylerythritol–phosphate (MEP) pathway in the plastids. Terpenes can be classified as either essential or nonessential compounds (Figure 1.13). Essential terpenes include the carotenoids, which play an important role in photosynthesis as components of light-harvesting complexes, and also as protective compounds against high light intensities, but also antioxidative compounds such as tocopherol. Many plant hormones also belong to the terpenoid family, but as these are essential signaling molecules for growth and development and occur in rather small amounts, they are not usually viewed as secondary metabolites. Among nonessential terpenes antimicrobial substances such as the monoterpenes can be found. The term nonessential means that the plant is at an advantage if it can produce these substances under stress conditions; however, under normal conditions nonessential terpenes will not result in a phenotype or prove to be fatal if the biosynthetic pathway is mutated.

Figure 1.13Classification of terpenes into essential and nonessential compounds for the plant. Examples of compounds and their biological function are given. Within the respective chemical scaffold, substances deriving from odd numbers of carbon atoms are shown in green; those deriving from even numbers of carbon atoms are shown in blue.

The biosynthesis of terpenes can proceed via two different pathways: (i) the mevalonate (MVA) pathway; or (ii) the methylerythritol-phosphate (MEP) pathway (Figure 1.14