Indian Journal of Experimental Biology Vol. 38, November 2000, pp. 1086-1091

Review Article

Polyamines and alkaloids

Bharati Ghosh Department of Botany, Bose Institute, Calcutta 700 009,

Naturally occurring alkaloids are nitrogenous compounds th at constitute th e pharmacogenically acti ve basic principles of flowering . Alkaloids are classified into several biogenically related groups . Tobacco alkaloids are metabolised from polyamines and diamines putrescine and cadaverine . N-methyl transferase is the tirst enzyme in alk aloid biosynthetic pathway which drives the flow of nitrogen away from polyamine biosynthesis to alkaloid biosynthesis. Arginine decarboxylase has been suggested to be primarily responsible for providing putrescine for nicotine synthesis. Tryptophan is the precursor of indole alkaloids . However, th e biosynthetic pathway of tropane and isoquinoline alkaloids are not clear. Genes for several key biosynthetic enzymes like arginine decarboxylase, ornithine decarboxylase, putrescine N-methyl transferase and spermidine synth ase, hyoscyamine 6 p hydroxylase,tryptophan decarboxylase etc have been cloned from different plant species. These genes arc regulated by plant hormones, li ght, different kinds of stress an e li cito rs like jasmonates and their strong expression is primarily in the cultured roots. In view of thi s, th e axenic hairy root cultures induced by Agrobacterium rhizoge11 es have been utilised to synthesise secondary metabolites. The current development in the knowledge of alk aloid biosynthesis, particularly molecular analysis, has been discussed in this review that may help to open up new avenues of investigation for th e researchers.

Plants produce a wide range of natural products instrumentation, precursors labeled with stab le derived from secondary metabolic pathways which isotopes, analysis of products by nuclear magnetic are useful for pharmaceutical,agrochemical, fragrance resonance spectroscopy and the use of plant cell and food industries .These naturally occurring culture as experimental system has helped in secondary metabolites are alkaloids which contain understanding the alkaloid biosynthetic pathway in 2 secondary, tertiary or quaternary nitroge n atoms in different plants . Alkaloids are classified into several their molecules. These nitrogenous compounds biogen ically related groups but the enzymes and constitute the pharmacogenically active 'basic genes have been characterised on ly in following principles' of flowering plants, and have traditionall y groups 26 been of interest due to their various physiological activities in animals and human . Alkaloids are • Nicotine and Tropane alkaloids important for defense of the plant against pathogenic • Indole alkaloids organisms and herbivores, or protoxins for in sects • Isoquinoline alkaloids which further modify the alkaloids and incorporate 1 them into their own defense/secretions • Alkaloids This review describes the bi osynthesis and have been isolated from diverse organi sms . However, molecular aspects of tobacco alkal oids in detail and little information is available regarding the synthesis the rest of the groups in brief. of complex structure of alkaloids which contain multiple asymmetric centers. Nevertheless the Polyamines as precursors of tobacco alkaloids pharmacological effects of these compounds are well The ubiquitous polycationic polyamines, a class of known. Nowadays the sophi sti cated analytical aliphatic amines, are found in almost every cell compartment including the cell wall. They are deeply Abbreviations used: ODC, Ornithine decarboxylase; ADC, Arginine decarboxylase; SPDS, Spermidine synthase; SPMS, involved in a series of cellular events such as Spermine synthase; SAMDC, S-adenosylmcthi onine replicat ion , cell division ,protein synthesis' ;md plant 4 5 decarboxylase; PMT, Putrescine N-methyl transferase; DAO, growth and developmental processes · but the exact di amine oxidase: H6H, Hyoscyamine 6P hydroxylase; PAs, 5 function of polyamines is not known . In plants, Polyamines; PUT, Putrescine; Spd, Spermidine; Spm, Spermine; SAM, S-adenosyl methionine; dSAM , decarboxylated SAM; diamine putresci ne (Put) can be sy nthesized by two MeJA, Methyl esters ofjasmonates independent pathways which involve ornithine GHOSH: POLYAMINES & PLANT ALKALOIDS 1087 decarboxylase (ODC) and argmme decarboxylase SAM to Put. Substrate specifi city studies and (ADC) and these two pathways regulate th e growth, inhibition kineti cs by amines have predicted th at development and stress responses of plants.4 .5 SPDS 11 and partially purified PMT 12 may have Spermidine sy nthase (SPDS) transfers the similar active sites for enzyme catalysis. Previously aminopropyl moiety of decarboxylated S- conflicting hypotheses have been proposed for the adenosylmethionine (dSAM) to Put producing source of Put in nicotine synthes is in tobacco. It was Spermidine(S pd). Spermine (Spm) is similarly beli eved th at the ODC pathway is a major source of synth esized by another enzyme Spermine synthase Put as accumulated ni cotine showed a good (SPMS). dSAM is formed from SAM by SAM correlati on with the increase in ODC activity in roots 13 decarbo xy lase (SAMDC). of decapitated tobacco seedlings . In later A wide va ri ety of secondary metabolites of experiments ADC pathway has been suggested to be biological interest can be metabolized from Put primarily responsi ble for providing Put for ni cotine through largely unelucidated pathways. The synthes is, as nicotine production in tobacco callus is pyrrolidine rings of tobacco alkaloids (ni cotine, effectively inhibited by DFMA, a specific inhibitor of nornicot ine), tropane alkaloids (hyoscyamine, ADC, and incorporat ion of 14C into ni cotine was more hyosc ine, metelod ine), pyrrolizidine (retronecine) are efficient from the uniformly labeled Arg than th at 6 14 Put derivatives . Cadaverine and Spd are precursors from the uniformly labeled Orn • of quinolizidine whil e lunarine alkaloids are derived The bi osynthetic path way of tropane alkaloids is from Pue. not yet clear. Recently activities of two enzymes of Nicotine, the biologically active component of biosynthesis, PMT and hyoscyamine tobacco , is found in the genu s and 6~ hydroxy lase (H6H), 2-0xoglutarate-dependent other genu s of th ough it occurs in many enzyme, (EC 1.14. I I .I I), have been shown to be hi gh 7 other pl including lycopods and horsetail s . It is in cultured roots but very low in cultured cell s and the predominant alkaloid in mo st commercial cultured shoots. H6H catal yse the first oxidati ve culti vars of tobacco, hi ghl y toxic and se rves as a very reacti on in the bi osyntheti c pathway leading from effective in secticide and fumigant. The tropane hyoscyamin e to scopol amine and requires alkaloid 15 alkaloids th at include the medica ll y important substrate 2-oxoglutarate, ferrous ion, ascorbate etc . • anti cholinergic compounds, hyoscyamine and H6H not onl y hyd roxylates various hyoscyamine , have been used traditionall y for their derivatives but also epoxidi zes 6,7- medicinal, hallucinogeni c and poisonous properties. dehydrohyoscyami ne, a sy nth etic alk aloid to 16 In addition to the Solanaceae family , tropane scopolamine • H6H has been purified to alkaloid s are also found in the Convo lvulaceae, homogenei ty from cu ltured roots of Hyoscyamus 8 17 Cruciferae and Rhizophoraceae . Both these niger L . Four monoclonal antibodies were rai sed alkaloid s are sy nthesized in tobacco roots from Arg against purified H6H and Western blot analysis and/or Orn by th e ac ti on of ADC and ODC whi ch showed that H6H is abundant in plant roots, but involves the symmetric diamine Puts th at is converted absent in , stem, calyx, cultured cell s and cultured to N-methyl Put with the help of Put N-methyl roots. Immunohistoc hemi cal studies using transferase (PMT, EC 2. 1.1 .53). This is the first monoclonal antibody and immunogo ld -silver committed step in alkaloid biosynth esis whi ch drives enhancement studi es detected H6H onl y in the the flov,: of ni trogen away from PA biosynthesis to pericycle cell s of the you ng root in several 18 1 alkaloid biosynthesis. N-methyl Put can also be scopolami ne-produci ng plants . Hartmann produced through di rect meth ylation of Put or demonstrated a close relati onship between the decarboxylation of !-methyl Orn . N- met hyl Put is biosynthesis of PAs and senceionine N-oxide, the then oxidatively deaminated by diamine ox idase major pyrrolizidine alk aloids produced in root (DAO, EC 1.4.3.6) and cyc li sed spontaneously to th e cu ltures of Solanum vulgaris. 9 10 1-met hyi-A-pyrro linum via 4-met hylaminob utano1 · . It is then condensed with ni cotinic ac id or its Molecular cloning of polyamine and alkaloid 10 derivati ves by an unknown enzyme to form ni cotine . biosynthetic genes SPDS transfers the aminopropyl moiety of dSAM to The eDNA clone representing ODC from Datura 8 Put whereas PMT transfers the methy l moiety of has been obtained recentl / . ADC gene ha s bee n 1088 INDIAN 1 EXP BIOL, NOVEMBER 2000

19 20 2 1 b .d . ?? c I oned f rom oat , tomato , pea , Ara t opsl.!;-- and which was expressed as a galactosidase fusion protein 23 31 soyabean . The cloning of eDNA encoding the in E. coli • enzyme nicotiana PMTs is generated by subtrac ti on 24 hybridi zation , the deduced aminoacid sequence of Regulation of alkaloid biosynthetic gene expression PMT is hi ghly homol ogous to the sequence of SPDS Like other pl genes, expression of the genes from human (73 % identical), mouse (70% identi cal) encoding biosyntheti c enzymes fo r alkaloids are 5 and E.coli (58 % identicali . Nicotiana PMT is more regulated spatially and temporally by plant hormones, homologous to the peptide fragments (73% identity li ght and stress.2 Ni cotine is synthesised in response and 88% homology in 66 amin o acid res idu es) to wounding. PAs are not only essenti al for cell encoded by a parti al eDNA sequence of ri ce SPDS divi sion ,but also play an import:m t role in the 5 (GenBank accession No. D 1540 I). The Hyoscyamus development and stress res ponses in plants • Salinity niger PMT eDNA was I ,350-bp long and encoded of stress causes a rapid increase in the level of Put in 338 amino acids. The belladonna PMT sensitive rice seedlings 32 through activation of ADC cDNAs was I ,305 bp lon g and encoded of 336 amino and treatment of MeJA also increase accumul ati on of 33 aci ds. AbPMT eDNA was more simili ar in nucl eot ide Put in rice leaves . The levels of Put, Spd and Spm sequence to HnPMT eDNA (86% identity) .AbPMTs did not change significantly after treatment of tobacco and HnPMTs lacked the N-terminal tandem repeat ce ll s with MeJA (meth yl esters of jas monates) but it 26 arrays previously found in Ni coti ana PMTs . Very caused accumulation of N-methyl Put and the recentl y four cDNAs for SPDS were iso lated from ni cotine n MeJA treatment induces accumul ation of Nicotiana sylvestris, and not only mRNAs fo r ODC and SAMs but also mR NA 27 Arabidopsis thaliana • The plant SPDS has for PMT. The ex pression of genes for ADC, an mol ecular mass of about 34 kDa , posses th e cofac tor alternative enzy me for the synthesis of Put, and binding motifs and are more homologous in amino SAMDC in vo lved in the sy nthesis of higher PAs (Spd acid sequ ence to tobacco PMT th an to SPDS from and Spm) from Put, were not affected by MeJA 34 . The mammals and E. coli. The SPDS gene is ex pressed in eli citors MeJA induces expression of genes cod ing root, stem and leaf in N. sylvestris whereas th e PMT for enzy mes at various steps in the synthes is of 33 ge ne is ex pressed onl y in root. From the ph ylogeneti c ni cotine by multiple regul atory mechanisms . tree constructed from a matrix of sequence Negative effects of auxin on alkaloid biosy nthesis simil ariti es (U PGMA Tree Programme in Gene ha ve been known for a long time. It was shown Works) it ca n be shown that PMT has evo lved from recently that auxin down regulates several genes 3 SPDS in tobacco during the diversifi cation of involved in alkalo id bi osynth es is .J. Auxin may be 28 Solanaceae . transported from the shoo t to the root where it suppresses the genes for ni cotine biosynthes is. The eDNA clones encoding H6H were obtained Ex pression of ni cotine converter gene which converts from mRNA of the cultured roots of Hyoscyamus 9 ni cotine to norni cotine is induced with ethephon or nige/ . Nucleotide sequ ence analysis of the cloned 25 eDNA revealed an open readi ng fra me th at encodes ethrel . 344 aminoacids.RNA hybridi zat ion showed th at mRNA of the hydrolase is abundant in cultured roots Biotechnological manipulation of th e alkaloid and present in plant roots but abse nt in leaves, stems biosynthetic genes 29 and cultured cells of Hyoscyamus niger . Expression By the application of in vitro cu lture techniques, of H6H eDNA clone in E. coli has demonstrated that efforts have been made to produce plant metaboli sm 35 H6H catalyses both reactions and th at the hydrolase . The axeni c hairy root cultures ind uced by infecti on ac ti vi ty is about 40 fold stronger th an epox id ase with Agrobacterium rhizogenes have the ability to 0 ac ti vi t/ . The ami no ac id sequence of H6H in sy nthes ise second ary metabolites such as tropane 35 Hyoscyamus niger is homol ogous to eth ylene forming alkaloids, tanin, and coumari ns . Hi gher enzyme (ACC ox idase). Tropine redu ctase (EC accumulation of atropin e and scopolamine was 1.1.1.236) is another enzyme of bi osyntheti c pathway reported in hairy root cultures of Atropa baetica 36 of tropane alkaloids which conve rt s the 3-keto group infected with_Agrobacterium rhizogenes . of tropinone to the 3 hyd rox yl of trop in e. The eDNA The synthes is of alk aloid is contro ll ed at multiple 2 of thi s enzy me was is olated from Datura stramonium steps by several enzy mes . The overex pression of GHOSH: POLYAM!NES & PLANT ALKALOIDS 1089 only one of the enzyme may not be enough in LA Burley 21 resulted in isolation of two eDNA achieving significant amount of end product of the clones whose expression in the roots were repressed pathway. Metabolic engineering of the plants that proportionally to the nicotine content (N. Hibi, serve as commercial sources of nicotine or unpublished information). Exogenous supply of auxin scopolamine could enhance classical breeding in the down regulated both genes in cultured tobacco roots effort to develop plants with optimal alkaloid patterns which suggested that NIC I and NIC2 are regulatory that serve as improved sources of pharmaceuticals. gene for nicotine biosynthesis (T. Hashimoto, The exploitation of the biotechnological potential of unpublished information) the alkaloid biosynthesis has only just begun. Feeding low levels of Put (I to 5 mM) can have a stimulatory Indole alkaloids effect of nicotine by transformed root culture of The indole alkaloids comprise about 1800 members 34 . of alkaloids of which the antimalaria drug quinine Expression of yeast ODC gene into Nicotiana from Cinchona .officina/is, the antineoplastic drug rustica roots resulted an increased accumulation of camptothecin from Cinchona .acuminata, the Put and nicotine37 which first shows plant secondary homeopathic drug strychnin from Strychnos product can be elevated by means of genetic nuxvomica and the chemotherapeutic agents manipulation. The 5' flanking region of the vincristine and vinblastine from Catharanthus roseus AbPMTgene was transferred to are isolated from plant materials 2 and further analysis showed that root is the main organ of PMT expression. Hyoscyamus niger H6H is T1yptophan as precursors of Indole alkaloids expressed specially in root pericycle cells but absent Tryptophan decarboxylase catalyses the 17 in leaf, stem, calyx, cultured cells • The H6H eDNA decarboxylation of L-tryptophan to tryptamine which of Hyoscyamus niger was introduced into Atropa can serve as substrate for strictosidine synthase . belladona; in which the enzyme was strongly and Strictosidine synthase catalyses tryptamine and 2 25 constitutively expressed in all parts of the plant 25 secologanine to form the indole alkaloids ' like ajmalicine,cantharanthine,vindoline etc. The eDNA clone encoding tryptophan Mutational analysis decarboxylase was isolated from Catharanthus roseus Well defined mutants defective 111 alkaloid and amino acid sequence show similiarities with an biosynthetic steps are valuable tools for the aromatic L-aminoacid decarboxylase from elucidation and regulation of biosynthesis of 39 Drosophila melangoster .The eDNA encoding secondary metabolites. In the cured leaves of tobacco Strictodine synthase was isolated from Rauvolfia cultivars, nicotine is the predominant alkaloid though 40 1 serpentina and Catharanthus roseus .4 . The gene for certain cultivars posses a gene that demethylates 42 strictosidine synthase,str 1 ,has been isolated from nicotine to nornicotine during curing. The nicotine Rauvolfia serpentine and the transcript accumulates converter gene was considered to be a means of primarily in the roots and mature leaves. lowering the nicotine content of tobacco but this approach was discarded due to poor smoking quality lsoquinoline alkaloids 28 of tobacco products with high nornicotine contents . The active members of this large and diverse class In 1930, a genetically stable breeding line LA-Burley of alkaloids are emetine (an antiamoebic), colchicine 21 was established by series of back crossing with (a microtubular disrupter and gout suppressant), Cuban cigar tobacco cultivars Burley 21. LA-Burley berberine (an antimicrobial against eye and intestinal 21 and parental strain Burley are isogenic and have infections), morphine (a narcotic ),codeine low nicotine biosynthesis loci NIC I and NIC2 which (a narcotic analgesic and antitussive and sanguinarine regulates nicotine level in tobacco. Two mutants nic I (an antimicrobal used in oral hygeine) which are 2 and nic2 with low nicotine levels accumulate largely isolated from plants . Due to the complexity proportionally more PAs, mostly Put and Spd in of structure only berberine is isolated and synthesised leaves and culture roots than the wild type probably from cell suspension of Coptisjaponica43 because of the block of PMT (T. Hashimoto and S. The complete picture of aligning 13 enzymes Higashiguchi,unpublished information). Differential comprising methyl transferase and oxidases for 2 screening between the cultured roots of Burley 21 and berberine synthesis is not yet clear . 1090 INDIAN J EXP BIOL, NOVEMBER 2000

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