NPC Natural Product Communications Plant Secondary Metabolism: Diversity, Function and its Evolution 2008 Vol. 3 No. 8 1205 - 1216 Michael Wink Institute of Pharmacy and Molecular Biotechnology, INF 364, 69120 Heidelberg, Germany wink@uni-hd.de Received: April 2nd, 2008; Accepted: May 23rd, 2008 A typical character of plants is the production and storage of usually complex mixtures of secondary metabolites (SM). The main function of secondary metabolites is defense against herbivores and microbes; some SM are signal compounds to attract pollinating and seed dispersing animals or play a role in the symbiotic relationships with plants and microbes. The distribution of SM in the plant kingdom shows an interesting pattern. A specific SM is often confined to a particular systematic unit, but isolated occurrences can occur in widely unrelated taxonomic groups. This review tries to explain the patchy occurrence of SM in plants. It could be due to convergent evolution, but evidence is provided that the genes that encode the biosynthesis of SM appear to have a much wider distribution than the actual secondary metabolite. It seems to be rather a matter of differential gene regulation whether a pathway is active and expressed in a given taxonomic unit or not. It is speculated that the genes of some pathways derived from an early horizontal gene transfer from bacteria, which later became mitochondria and chloroplasts. These genes/pathways should be present in most if not all land plants. About 80% of plants live in close symbiotic relationships with symbiotic fungi (ectomycorrhiza, endophytes). Recent evidence is presented that these fungi can either directly produce SM, which were formerly considered as plant SM or that these fungi have transferred the corresponding pathway gene to the host plant. The fungal contribution could also explain part of the patchy occurrence patterns of several secondary metabolites. Keywords: evolution, chemotaxonomy, horizontal gene transfer, molecular phylogeny. Diversity and function of plant secondary metabolites A typical character of plants is the production and storage of usually complex mixtures of secondary metabolites (SM). The constituents of these mixtures often belong to several classes of SM, for example terpenoids are often accompanied by phenolics. Usually we find a limited number of major SM and several minor components, which are often biosynthetically related to the main constituents [1-7]. For more than 60 years phytochemists have separated complex mixtures of SM by prep.TLC, LC, and HPLC and have isolated more than 100,000 phytochemicals. Their structures have been determined by mass spectrometry, NMR spectroscopy and X-ray crystallography. It has been suggested that only 20 to 30% of the known 350,000 plant species have been examined phytochemically in some detail; therefore, the real number of SM present in the plant kingdom very likely exceeds 200,000 compounds. The structurally diverse SM can be grouped according to their biosynthetic pathways and structures into two large groups: SM with nitrogen in their structures and those without (Table 1). Within both complexes we can define further units, which can be split into even finer subgroups [1,4-7, 8-10]. Whereas phytochemists were and are always enthusiastic about the high structural diversity of SM, biologists tried to understand why and how plants synthesize, transport and store all these different SM. As compared to the numbers of known SM, only limited numbers of SM have been studied in detail in terms of physiology, biochemistry and ecology. Nevertheless, it is safe to assume, that SM are not functionless waste products (as suggested earlier in the 20th century) [4], but important for the plants in an ecological context [8, 12-14]. Since early days when land plants evolved in the Devonian period, they had to cope with animals, 1206 Natural Product Communications Vol. 3 (8) 2008 Table 1: Approximate numbers of known secondary metabolites from higher plants [11] Type of secondary metabolite Nitrogen-containing SM Alkaloids Estimated numbers* 21,000 Non-protein amino acids (NPAAs) 700 Amines 100 Cyanogenic glycosides Glucosinolates Alkylamides Lectins, peptides, polypeptides 60 100 150 Wink overcome the defense barriers. Some insects even use the defense compounds of their host plants to protect themselves against predators [8,15-19]. Herbivores have developed a number of enzymatic means to detoxify, inactivate or excrete toxic plant compounds (liver enzymes, ABC transporters). Also the microorganisms that are present in the rumen or intestines of herbivores can play a role in inactivating plant defense chemicals. 2,000 SM without nitrogen Monoterpenes (C10) ** 2,500 Sesquiterpenes C15)** 5,000 Diterpenes (C20)** 2,500 Triterpenes, steroids, saponins (C30, C27)** Tetraterpenes (C40)** 5,000 500 Flavonoids, tannins 5,000 Phenylpropanoids, lignin, coumarins, lignans 2,000 Polyacetylenes, fatty acids, waxes 1,500 Polyketides 750 Carbohydrates, simple acids 400 *Approximate number of known structures; **total number of all terpenoides exceeds 22,000 at present. which tried to feed on plants (herbivores) and with infectious microorganisms (bacteria, fungi). It is obvious but still important to note that plants cannot run away when challenged by a herbivore nor do they have an elaborate immune system to fight off a microbial infection. As a common defense measure, plants and other sessile organisms evolved bioactive natural products, which repel, deter or poison herbivores and which can inhibit growth and development of bacteria, fungi and even viruses. Some of the defense compounds are constitutive, while others can be induced under stress conditions: several SM (so-called phytoalexins) and defenserelated proteins are synthesized de novo when a plant is challenged by bacteria, fungi or viruses. Because plants have to compete with other plants for light, water and nutrients, SM often also serve as mediators in plant-plant interactions (so-called allelopathy). During evolution, SM were apparently optimized in such a way that they did not only exhibit defensive but also additional non-defense functions: Some SM have additional physiological and ecological functions (for example, as nitrogen storage compounds; UV protectants) or serve as signal compounds to attract pollinating or seed dispersing animals and can mediate the interactions between symbiotic bacteria and their plant hosts (for example, Rhizobia). Because no defense system is absolute, a number of specialists have evolved, which have The ecological functions of SM become apparent if plants are grown in the wild with all their enemies and competitors around. Plant breeders have selected varieties without toxins or which taste better as food plants. Consequently, food crops have often lost their natural resistance to herbivores and/or pathogens, so that we have to use synthetic pesticides to protect them. Using alkaloid-rich and alkaloid-free lupins, we have shown that the alkaloids are important for lupin plants to protect themselves effectively against a wide diversity of herbivores [13,14,19-21]. Origins of plant secondary metabolism Early in the 20th century it was argued that secondary metabolites arise either spontaneously or with the aid of non-specific enzymes. There is good evidence today that biosynthetic enzymes are highly specific in most instances and that most have been selected towards this special task (although they often derive from common progenitors with a function in primary metabolism). As a consequence of specific enzymatic synthesis, final products nearly always have a distinct stereochemistry. Only the enzymes that are involved in the degradation of SMs, such as glucosidases, esterases and other hydrolases, are less substratespecific. Despite the enormous diversity of secondary metabolites (Table 1), the number of corresponding basic biosynthetic pathways is restricted and distinct [7,21,22]. Precursors usually derive from basic metabolic pathways, such as glycolysis, Krebs cycle and the shikimate pathway. For pathways leading to cyanogenic glycosides, glucosinolates, some alkaloids and non-protein amino acids (NPAAs), amines, flavonoids and several terpenes, the enzymes that catalyze individual steps have been identified. In pathways leading to isoquinoline, indole, pyrrolidine, pyrrolizidine and tropane alkaloids, flavonoids, coumarins, NPAAs, mono-, sesqui- and triterpenes, some of the genes that encode biosynthetic enzymes have already been isolated and characterized. Diversity, function and evolution of plant secondary metabolism Natural Product Communications Vol. 3 (8) 2008 1207 Among the unsolved problems of secondary metabolism is the question of when, where and how the genes evolved that encode enzymes of SM biosynthesis, as well as those of transport, storage and turnover. Alkaloids Terpenes Flavonoids & other phenolics Chlorophyta Charophyta Marchantiophyta Anthoceratophyta Bryophyta Lycopodiophyta Theoretically the following scenarios can be considered: Secondary metabolism could be a young phenomenon and modern plants in the periphery of the tree of plants have developed their pathways independently. Alternatively, secondary metabolism is an old key innovation, which was developed early in the evolution of land plants and was inherited by present-day plants. This would mean that the corresponding genes should be detectable in at least most of modern plants. Plants could have developed the genes of SM from their own genes of primary metabolism. Starting with duplication of a gene, the new gene became mutated, exhibited new metabolic functions, and was established by natural selection. Plants might also have inherited some of the genes/pathways in early evolution by horizontal gene transfer from their bacterial symbionts, which later developed into modern mitochondria and plastids. It is well-established that bacteria (especially Actinomyces and Streptomyces) and cyanobacteria produce a wide diversity of secondary metabolites, showing similar structures as plant SM (for example, some anthraquinones, terpenoids and alkaloids). About 80% of modern plants live in symbiosis with fungi (endophytes, ectomycorrhiza); these fungi could directly have supplied its host with SM or might have transferred the corresponding genes to the host’s genome (i.e. by horizontal gene transfer). Inference from molecular phylogenies During the last 20 years botanists have reconstructed a tree of life for the plant kingdom using nucleotide sequences of plastid and nuclear marker genes [22-24]. The resulting molecular phylogenies can be used to map the distribution of SM in the plant kingdom [11]. Suggesting that all extant taxa, ranging from spore to seed producing plants, accumulate a certain class of SM makes it very likely that all individuals possess the genetic information for its synthesis and storage. Sphenophyta Filicophyta Angiospermae Pinophyta Gnetophyta Ginkgophyta Cycadophyta Figure 1: Molecular phylogeny of land plants. Clades that produce alkaloids are marked in red; those with terpenoids in green and those with phenolics in black. This in turn would suggest that the corresponding pathway must have evolved during early evolution. In Figure 1 a molecular phylogeny of major lineages of land plants is shown. All lineages are apparently able to synthesize flavonoids and other phenolics deriving from phenylalanine/tyrosine. This observation suggests that the genes of flavonoid biosynthesis must be present from early land plants to angiosperms. This is apparently true, since the corresponding key enzymes, such as phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) have been detected in spore forming plants and seed plants (Figure 2 A,B). Terpenoids, such as mono-, sesqui-, tri-, and tetraterpenes, and steroids are also present from mosses to higher plants, suggesting that the pathways leading to the active isoprene and the following combinations and cyclisation of C-5 units were present already in the ancestors of land plants (Figure 1). Alkaloids have a more patchy distribution and special alkaloidal types are usually specific for certain taxon groups; therefore, they were often used as chemotaxonomic markers [9,25,26]. Alkaloids, as such, can be detected in lycopods, horsetails, and some gymnosperms, but are predominant in angiosperms (Figure 1). Because many alkaloids are neurotoxins [27], which interfere with targets of neuronal signal transduction, we may speculate that the diversification of alkaloids in angiosperms coevolved with the rapid diversification of insect and vertebrate herbivores during the Cretaceous and Tertiary periods. The distribution of alkaloids offers a good example for understanding the dynamics of SM with restricted 1208 Natural Product Communications Vol. 3 (8) 2008 Wink Daucus carota Petroselinum crispum Lactuca sativa 30 100 Helianthus annuus Catharanthus roseus Digitalis lanata 23 27 Agastache rugosa 100 Nicotiana tabacum 8 Solanum tuberosum 100 Lithospermum erythrorhizon Rubus idaeus 36 62 Glycine max 55 100 Astragalus membranaceus Pisum sativum Prunus avium 13 48 Citrus limon 21 Quercus suber 37 Coffea canephora 99 38 Arabidopsis thaliana 100 Isatis tinctoria 100 Beta vulgaris Stellaria longipes 46 100 Allium cepa Oryza sativa 100 Triticum aestivum 99 Zea mays 70 100 Saccharum officinarum Pinus pinaster 100 Selaginella kraussiana 42 lycopods Lycopodium tristachyum 89 horsetail Equisetum arvense ferns Blechnum spicant 100 Aspergillus fumigatus Rhodotorula mucilaginosa Ustilago maydis Amanita muscaria 100 Coprinopsis cinerea 100 Anabaena variabilis 100 Nostoc punctiforme Streptomyces maritimus Photorhabdus luminescens 100 73 PAL O OH NH 2 O OH dicots monocots 98 100 74 fungi bacteria 87 0.1 2B OH 4-Coumaroyl-CoA + CHS 3 Malonyl-CoA HO OH OH O Solanum lycopersicum Nicotiana tabacum Pyrus communis Vitis vinifera Petroselinum hortense Daucus carota Arabidopsis thaliana Barbarea vulgaris 100 Allium cepa 100 Medicago sativa Physcomitrella patens Phaeosphaeria nodorum Aspergillus oryzae 100 Coccidioides immitis Magnaporthe grisea 100 Neurospora crassa Cytophaga hutchinsonii Arthrobacter Rhodobacterales bacterium Prochlorococcus marinus Synechococcus sp. W H 7805 100 Pseudomonas fluorescens 100 Streptomyces griseus 100 Stigmatella aurantiaca Anaeromyxobacter dehalogenans Deinococcus radiodurans 99 Rhodospirillum centenum Bacillus subtilis 99 87 Bacillus licheniformis Geobacillus kaustophilus 99 Bacillus halodurans 97 Bacillus clausii 62 plants fungi cyanobacteria 51 93 bacteria 0.1 Figure 2: Molecular phylogeny of PAL (A) and CHS (B) inferred from derived amino acid sequences of the corresponding genes (after [46]). Diversity, function and evolution of plant secondary metabolism occurrences. In Figure 3, a more detailed analysis of the distribution of two major classes of alkaloids in the angiosperms is illustrated as an example. The tree is based on several molecular markers and the basis for a reorganization of angiosperm phylogeny [24]. Quinolizidine alkaloids, such as lupanine and cytisine, are mainly found within the genistoid legumes (order Fabales); isolated occurrences have been reported from other plant families, which are not directly related to Fabaceae [28,29]. A similar picture can be seen for pyrrolizidine alkaloids (PAs), such as senecionine and symphytine, which are the main SM in Boraginaceae and in part of the Asteraceae (Senecioninae). PAs also occur in several unrelated monocot and dicot families. The patchy occurrence of QA and PA within the angiosperms continues if we look into finer details in the Fabaceae (Figure 4). Clades with PA production (genera Crotalaria and partially Lotononis) cluster within QA producing clades. In the genus Lotononis two clades are apparent [30], one producing QA the other PA. How can we interpret the patchy distribution of PA and QA in angiosperms and in the Fabaceae? Their occurrence in unrelated groups could be due to convergence; in this case we would expect an independent set of biosynthetic genes. In the case of the Genisteae s.L. the PA producing taxa are imbedded in clades with QA, which are ancestral. This observation unambiguously implies that the PA taxa must have inherited the genes of QA biosynthesis, but that the QA pathway is downregulated in PA producing clades. The PA pathway could be a new invention or caused by common PA genes or by horizontal gene transfer (see below). As a consequence, we could assume that the genes for QA and PA biosynthesis evolved at a much earlier stage in angiosperm phylogeny and that all descendants have a set of the corresponding genes. Whether QA or PA production is actually taking place would then depend on a differential expression of the corresponding genes (which in general is a common theme in plants). Because the genes of QA biosynthesis are not known we have presently no possibility to test our assumption. The QA/PA example is also representative of many other SM (cardiac glycosides, iridoid glycosides, sesquiterpene lactones, diterpenoids, many alkaloids) with more specific and restricted distribution patterns: In addition to one or more centers of SM Natural Product Communications Vol. 3 (8) 2008 1209 production, we can often detect the same trait in other unrelated members of the plant kingdom. This observation indicates that chemotaxonomy, which relies on the similarity of SM profiles, must be regarded with some caution. If we were to assemble all plants with similar SM in one clade, we should not obtain a meaningful phylogeny in most instances [11,25]. Because reliable molecular phylogenies are available by now for many plant groups, chemotaxonomic groupings need to be reanalyzed and reinterpreted. Inference from genome data mining A large number of bacterial, animal and plant genomes have been partly or completely sequenced, which provides us with the chance to test our assumptions explained in the last paragraphs by a data mining approach (as shown for PAL and CHS in Figure 2). Here we will concentrate on two genes of indole and isoquinoline alkaloid biosynthesis: Strictosidine synthase (STS) catalyzes the fusion of strictosidine and tryptamine, a key step in the biosynthesis of monoterpene indole alkaloids [26]. These alkaloids are abundant in four plant families – the Apocynaceae, Loganiaceae, Gelsemiaceae, and Rubiaceae. Therefore, we would expect that STS should be present in these families but not beyond. As can be seen from Figure 5, which is based on derived amino acid sequences, related genes/proteins occur in several other plants (among them Arabidopsis thaliana), which do not produce such alkaloids. This finding indicates that plants must have evolved a precursor of the STS gene much earlier during their phylogeny. Interestingly, related genes or STS precursor genes, which share many common amino acid sites, can be detected among animals and, more importantly, among bacteria. This observation could suggest that the progenitor of the STS gene evolved in protobacteria and was imported into plants via endosymbiotic protobacteria, which later became mitochondria. The modern mitochondrial genomes are quite small, suggesting that most of the original genes have been transferred to the nucleus of an ancestral eucaryotic or plant cell. Thus plant STS genes would be the result of an ancient horizontal gene transfer. As a second enzyme we have analysed the berberine bridge enzyme (BBE); it catalyses a specialized step in protoberberine alkaloid biosynthesis (Kutchan, 1210 Natural Product Communications Vol. 3 (8) 2008 Figure 3: Phylogeny of angiosperms and the distribution of quinolizidine (QA) and pyrrolizidine alkaloids (PA) (after [46]) Wink Diversity, function and evolution of plant secondary metabolism Natural Product Communications Vol. 3 (8) 2008 1211 NJ rbcL Argyrolobium lanceolatum 1 Argyrolobium lunaris Argyrolobium lotoides Goodia lotifolia Argyrolobium polyphyllum Argyrolobium marginatum Polhillia obsoleta 36 Polhillia brevicalyx 30 Polhillia canensis 31 Calicotome villosa I Cytisus hirsutus LG19 Chamaecytisus purpureus Cytisus villosus Cytisus scoparius Laburnum anagyroides Genista florida Genista januensis Genista tinctoria Petteria ramentacea Teline monspessulana AS67 Teline osyroides Ulex europaeus Ulex parviflorus Retama monosperma Retama raetam Retama sphaerocarpa Genista germanica Genista radiata Spartium junceum I Lupinus albus I Lupinus angustifolius I Lupinus arboreus Lupinus arcticus Lupinus polyphyllus Lupinus atlanticus Echinospartum horridum C38 Polhillia pallens 10 Dichilus pilosus 24 Dichilus strictus 26 Dichilus reflexus 25 Dichilus lebeckioides 40 Dichilus gracilis 6 Melolobium candicans 18a Melolobium adenodes 12a Aspalathus pendula 39 Aspalathus linearis 42 Aspalathus nivea 12 Aspalathus cephalotes Lebeckia inflata 13 Lebeckia pauciflora 28 Lebeckia bowieana 80 Lebeckia bowieana 127 Lebeckia pluckenetiana 29 Wiborgia tetraptera 46 Wiborgia obcordata 47 Wiborgia fusca 11 Lebeckia carnosa 7 Lebeckia meyeriana 33 Lebeckia meyeriana 118 Rafnia perfoliata 20a Rafnia amplexicaulis W40 Lebeckia meyeriana 11a Lebeckia wrighttii 81 Lebeckia wrighttii 59 Lebeckia wrighttii 128 Lebeckia wrighttii 129 Lebeckia simsiana 98 Lebeckia ambigna 99 Lebeckia leipoldtia 58 Lebeckia leipoldtia 130 Lotononis curvicarpa 14 Lotononis calycina 9 Lotononis foliosa 67 Lotononis eriantha 109 Lotononis adpresse 93 Lotononis decumbens 73 Lotononis platicarpa 53 Lotononis platicarpa 96 Lotononis bentamiana 8 Lotononis digitata 77 Lotononis magnifica 112 Lotononis magnifica 92 Lotononis mollis 52 Lotononis mollis 63 Lotononis plicata 66 Lotononis polycephala 114 Lotononis digitata 65 Lotononis globulosa 69 Lotononis densa ssp leu 15 Lotononis exstipulata 75 Lotononis filiformis 68 Lotononis elongata 108 Lotononis pulchella 51 Lotononis carnosa 104 Lotononis pulchella 115 Lotononis meyeri 123 Lotononis stricta 117 Lotononis meyeri 113 Lotononis eriocarpa 105 Lotononis lotononoides 111 Lotononis prostrata 18 Lotononis involucrata 70 Lotononis leptoloba 71 Lotononis maximiliani 91 Lotononis maximiliani 126 Lotononis pungens 124 Lotononis brevicaulis 94 Lotononis sericophylla 19 Lotononis divaricata 121 Lotononis sericophilla 122 Lotononis sericophilla 125 Lotononis umbellata 55 Lotononis falcata 16 Lotononis laxa 106 Lotononis macrosepala 76 Lotononis laxa 72 Lotononis crumania 54 Lotononis sabulosa 95 Lotononis parviflora 17 Lotononis fruticoides 107 Lotononis lenticula 110 Lotononis sparsiflora 116 Lebeckia cytisioides 27 Lebeckia cytisioides 34a Lebeckia sericea 102 Lebeckia multiflora 34 Lebeckia multiflora 119 Lebeckia macrocantha 103 Lebeckia pungens 101 Lebeckia melilotoides 100 Pearsonia aristata 20 Pearsonia aristata 17a Pearsonia grandifolia 21 Crotolaria distans 48 Crotalaria cunninghami AS26 Crotalaria laburnifolia 131 Crotalaria capensis Crotalaria pumila F27 Crotalariea eremeae AS52 Crotalaria pallida Bolusia amboensis 23 Bolusia amboensis 22 Bolusia capensis 35 Lotononis hirsuta 16a Lotononis hirsuta 64 Lotononis hirsuta 97 Bolusanthus speciosus 26a Virgilia oroboides 22a Virgilia divaricata 8a Virgilia divaricata Podalyria calyptrata 31a Liparia splendens Cyclopia genistoides Calpurnia sericea W19 Sophora davidii Sophora jaubertii Sophora flavescens Anagyris foetida Piptanthus nepalensis Baptisia leucantha F5 Thermopsis lupinoides F79 Baptisia tinctoria Thermopsis rhombifolia LG42 Maackia amurensis Hovea elliptica Lotus corniculatus 84 Lotus corniculatus 87 Chordospartium stevensonii F20 Swainsonia lessertifora AS65 Vicia ervilia AS11 Erythrina lysistimon 15a Erythrina caffra LG Erythrina falcata F35 Psoralea aphylla 28a Vigna unguiculata Daviesia brevifolia AS28 Daviesia ulicifolia AS61 Goodia lotifolia Belair AS47 Platylobium obtusangelicum AS62 Ormosia formosa 78 Ormosia hengchuniana 79 Ormosia formosana M20 Castanospermum australe Sophora secundiflora I Dichrostachis cinerea 7a Acagia saligna 37a Schotia afra 4a H N N H QAs Lebeckia Lotononis OH Lotononis Crotalaria Bolusia Lot. hirsuta Polygala amara O O O O N PAs Polygala chamaebuxus I 0.001 substitutions/site Figure 4: A molecular phylogeny of part of the Fabaceae, based on nucleotide sequences of the rbcL gene (after [30,46]) and distribution of QA and PA producing taxa. 1212 Natural Product Communications Vol. 3 (8) 2008 NH2 N H H NH O O O N H Glc O O Wink O O STS H3CO Glc O H3CO N HO HO OH O OCH3 N OH BBE OCH3 STS Arabidopsis thaliana NP 563818 100 Arabidopsis thaliana AAF22901 76 Oryza sativa AAR87254 Lycopersicon esculentum 93 Brassica napus Zea mays 100 100 Oryza sativa NP 001049635 100 Triticum aestivum 87 Medicago truncatula Ophiorrhiza pumila 96 Rauvolfia serpentina 100 100 Rauvolfia mannii 100 Drosophila melanogaster AAC47118 100 Drosophila pseudoobscura Anopheles gambiae Xenopus tropicalis 98 Danio rerio 86 Gallus gallus 100 Rattus norvegicus 100 Bos taurus 100 Canis familiaris 87 Homo sapiens BSCv 96 100 Pan troglodytes 100 Mesorhizobium loti Bradyrhizobium japonicum 100 Pseudomonas mendocina 100 Pseudomonas aeruginosa 94 Marinobacter aquaeolei Solibacter usitatus 81 Alcanivorax borkumensis 51 Deinococcus geothermalis 84 plants 100 BBE 54 97 99 86 animals bacteria 100 100 100 100 0.1 76 86 81 91 100 90 Helianthus annuus Lactuca sativa Medicago truncatula Vigna unguiculata Cannabis sativa Daucus carota Arabidopsis thaliana Glycine max Cynodon dactylon Phleum pratense 100 Lolium perenne Triticum aestivum 100 Hordeum vulgare Secale cereale Oryza sativa Berberis stolonifera Thalictrum flavum Eschscholzia californica Papaver somniferum 100 Bacillus thuringiensis Bacillus cereus Clostridium beijerincki Streptomyces avermitilis Ustilago maydis Aspergillus fumigatus 100 Aspergillus oryzae Phaeosphaeria nodorum Neurospora crassa Magnaporthe grisea plants bacteria fungi Figure 6: Phylogeny of berberine bridge enzyme (BBE) inferred from derived amino acid sequences (after [46]). Taxa that produce protoberberine alkaloids are marked by an arrow and printed in bold. 0.1 Figure 5: Phylogeny of strictosidine synthase (STS) inferred from derived amino acid sequences (after [46]). Taxa that produce monoterpene alkaloids are marked by an arrow and printed in bold. 1995). These alkaloids mainly occur in Ranunculaceae, Papaveraceae, Menispermaceae, Berberidaceae, Annonaceae and several other plant families. As can be seen from Figure 6, BBE genes and proteins are, however, widely present in higher plants, independent of whether a taxon actually makes these alkaloids or not, indicating common ancestry. Interestingly, BBE or similar proteins, which share a number of common conserved sites could also be found in fungi and bacteria. This finding implies again that the progenitor of the BBE gene evolved in protobacteria and was imported into plants and fungi via the endosymbiosis, which had led to mitochondria. Thus plant BBE would also be the result of an ancient horizontal gene transfer, similar to the situation in STS. These few examples support the assumption of a common distribution of SM genes in plants based on ancestral horizontal gene transfer. We do not know however, whether the genes are functional in species that do not express the corresponding pathway. These genes could be either silent copies or inactive pseudogenes; this possibility could be tested by expressing the genes in a heterologous system and then to evaluate their biological activity. A number of SM (for example, many terpenoids, quinolizidine alkaloids, the piperidine alkaloid coniine) are produced completely or partly in chloroplasts and/or mitochondria [21,31-33]. The corresponding genes are mostly nuclear. It is tempting to speculate that these localisations are indirect indicators of a former bacterial origin of the corresponding pathways. The PAL, CHS, STS and BBE examples indicate that plants have probably obtained the corresponding gene copies through early horizontal gene transfer from either protobacteria or cyanobacteria that gave rise to mitochondria and chloroplasts, respectively. It is likely that plants have also contributed some of their own genes to further modify the base structure of alkaloids, terpenoids and flavonoids that would produce the variation observed within most classes of SM. Diversity, function and evolution of plant secondary metabolism Contribution from endophytes and ectomycorrhizal fungi Mycorrhizal associations have apparently existed since the advent of vascular plants about 400 million years ago. It has been estimated that 80% of plants live in permanent contact with fungi [34-36]. Many of them are ecto- and endomycorrhiza that live in close contact with roots. The fungi substantially enlarge the root surface area and thus increase the possibility to catch more water and nutrients. The role and benefits of endophytes is less obvious. Some of these fungi apparently are also able to produce SM and contribute to the chemical defense of their host plants. An interesting example of a symbiotic fungus-plant interaction can be seen in ergot alkaloids. These alkaloids are mainly produced by saprophytic fungi: Claviceps purpurea, C. microcephala, C. paspali (Clavicipitaceae) and more than 40 further members of this genus live as symbionts on grasses (tribes Festucaceae, Hordeae, Avenae, Agrosteae). Rye is especially affected among cereals. Claviceps is not a parasite, but obviously a symbiotic organism. It takes nutrients from its host but provides chemical defense against herbivores as compensation (the ergot alkaloids are powerful neurotoxins). Field experiments have shown that such a fungal infection is an ecological advantage for grasses in the wild. Related symbiotic fungi such as Epichloe, Balansia and Myriogenspora (Clavicipitaceae) infect grasses and also produce alkaloids. Ergot alkaloids (such as agroclavine, chanoclavine, ergine, ergosine, and ergometrine) are also common SM of some genera of the Convolvulaceae (including Argyreia, Ipomoea, Turbina corymbosa, Stictocardia tiliafolia). It has been shown recently, that the ergot alkaloid formation in the Convolvulaceae is due to an endophytic clavicipateceous fungus that lives together with certain species in this plant family [37]. In this case, the isolated occurrence of ergot alkaloids in Convolvulaceae is due to a symbiotic relationship. Certain simple pyrrolizidine alkaloids, such as loline, were detected in the grass (Lolium pratense, ex. Festuca pratensis) and in a root hemiparasitic plant (Rhinanthus serotinus). The alkaloids derived from a symbiotic endophytic fungus (Neotyphodium uncinatum; Clavicipitaceae), which lives on the grass [38]. Similar to the situation in Claviceps, the fungus provides defense compounds, which help the grass and its hemiparasite to ward off herbivores. Natural Product Communications Vol. 3 (8) 2008 1213 The legume genera Astragalus and Oxytropis are famous for the production of toxic indolizidine alkaloids, such as swainsonine, causing locoweed poisoning in livestock. These alkaloids are apparently produced by an endophyte (Embellisia spp.; Pleosporaceae) [39]. The maytansinoid ansa antibiotics are produced by Actinosynnema pretiosum (Actinomycetes), but also in a number of angiosperms, including Celastraceae, Rhamnaceae, and Euphorbiaceae. It has been suggested that the occurrence of these SM depends on infection by this actinomycete [40]. Naphthodianthrones, such as hypericin are wellknown constituents of St. John’s wort (Hypericum perforatum; Clusiaceae). Kusari et al. [41] have isolated an endophytic fungus from H. perforatum that produces hypericin in culture. Also emodin, a precursor of hypericin is produced by both endophyte and the plant. Fungi are known producers of anthraquinones; in lichens, which are symbioses between an alga and a fungus, anthraquinones represent common defense metabolites, being probably produced by the fungal partner. Since anthraquinone producing plants are isolated over the plant kingdom (Asphodelaceae, Fabaceae, Rhamnaceae, Polygonaceae) it would be interesting to search for a former or extant endophyte association. Camptothecin (structurally a quinoline alkaloid, but derived from the tryptamine/secologanin pathway) occurs in unrelated families such as Camptotheca acuminata (Cornaceae), Nothapodytes foetida, Pyrenacantha klaineana, Merrilliodendron megacrapum (Icacinaceae), Ophiorrhiza pumila, O. mungos (Rubiaceae), Ervatamia heyneana (Apocynaceae) and Mostuea brunonis (Gelsemiaceae). It has been shown recently, that camptothecin can be produced by endophytes from Nothapodytes foetida [42]. It is tempting to speculate that the patchy distribution was originally caused by endophytes, which have either infected the respective plants or which have transferred their genes. The anticancer drug taxol is produced by endophytes from Taxus brevifolia [43]. An isolated occurrence of the taxan alkaloids has been reported from Corylus avellana (Betulaceae), which is not associated with an endophyte (although the pathway genes might haven imported by an endophyte at an earlier stage) [44]. Podophyllotoxin is produced by endophytes of 1214 Natural Product Communications Vol. 3 (8) 2008 EARLY EVOLUTION Wink protobacteria cyanobacteria mt Symbiotic fungi cp DNA DNA DNA -endophytes -ectomycorrhiza Genes Duplication Mutation Selection Specialization genome SM mRNA protein SM SM SM SM Figure 7: Hypothetical scheme for the evolution of secondary metabolism in plants. Podophyllum peltatum [45]. Although the endophytes, which were isolated from these plants, are capable of the biosynthesis of hypericin, taxol, camptothecin and podophyllotoxin in vitro, it is less likely that they alone perform the productions in the plant. It has been speculated that a horizontal gene transfer has taken place at some stage, thus importing the respective pathways from the fungi into the host plant [41]. It is a challenging question to determine the degree and contribution of endophytic and ectophytic SM pathways to the SM profiles of plants. If it were to be a more common phenomenon than usually assumed, it would offer an additional explanation for the patchy distribution of certain SM in the plant kingdom. It has been speculated that horizontal gene transfer could also be taking place when viruses or insects invade plants: Viruses could integrate some SM genes of a certain host plant into their genomes; if they are transferred to a different host plant species, they might transfer the corresponding genes. Insects often harbour endosymbiotic bacteria, which may carry genes of SM biosynthesis. Such bacteria could be transferred between species by sucking insects. Conclusions and Outlook: One of the main questions discussed in this communication concerns the origin and evolution of plant secondary metabolism. We have started with the observation that some SM (such as phenolics and terpenoids) are produced by nearly all vascular plants, whereas others, especially those with, for example, alkaloids, cardiac glycosides, and anthraquinones, show a more restricted but usually patchy distribution. The patchy distribution could be due to convergence, which is certainly the case in several instances (Figure 7). It could also be due to a wider distribution of SM pathway genes in the plant kingdom, which are silent or inactivated in most places, but become activated under certain conditions or in particular clades. Evidence is provided from the distribution of a few key genes/proteins that SM pathways might have been introduced into plants from SM producing bacteria via an early horizontal gene transfer; it is established that protobacteria became mitochondria and cyanobacteria plastids. Another external source for plant SM could be ectomycorrhizal fungi and endophytes, which either directly produce a particular SM or indirectly by transferring the pathway genes from fungi to plants. Because fungal infections do not necessarily follow plant phylogeny, they could cause (at least partly) the patchy distribution seen in some SM groups. 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