ORCAnization of jasmonate-responsive gene expression in alkaloid metabolism

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ORCAnization of jasmonateresponsive gene expression in alkaloid metabolism Johan Memelink, Rob Verpoorte and Jan W. Kijne Jasmonic acid is an important plant stress signalling molecule. It induces the biosynthesis of defence proteins and protective secondary metabolites. In alkaloid metabolism, jasmonate acts by coordinate activation of the expression of multiple biosynthesis genes. In terpenoid indole alkaloid metabolism and primary precursor pathways, jasmonate induces gene expression and metabolism via ORCAs, which are members of the AP2/ERF-domain family of plant transcription factors. Other jasmonate-regulated (secondary) metabolic pathways might also be controlled by ORCA-like AP2/ERF-domain transcription factors. If so, such regulators could be used to improve plant fitness or metabolite productivity of plants or cell cultures.

alkaloid science is the isolation of regulatory transcription factors for these genes. Alkaloid biosynthesis in plants is tightly controlled during development and in response to stress and pathogens. Developmental control of alkaloid biosynthesis has recently been reviewed3, therefore, here we will focus on the effects of the plant stress hormone jasmonic acid on the transcriptional regulation of alkaloid biosynthesis. Jasmonic acid

Johan Memelink* Jan W. Kijne Institute of Molecular Plant Sciences, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands. *e-mail: memelink@ rulbim.leidenuniv.nl Rob Verpoorte Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands.

The plant alkaloids caffeine and nicotine are common elements of daily life. Alkaloids are just one example of the large, diverse array of plant compounds known as secondary metabolites. Once considered as inessential byproducts of primary metabolism, it has now become clear that secondary metabolites serve key functions during a plant’s life cycle in the natural environment. Most classes of secondary metabolites are only found in specific families or genera, and each plant species contains a distinct profile of secondary metabolites, establishing a unique metabolic fingerprint. At present, >16 000 alkaloid structures are known. Alkaloids are low molecular weight nitrogencontaining organic compounds, usually with a heterocyclic structure. Traditionally, alkaloids have been of interest because of their biological activities1. About 25% of contemporary medicines are derived from plants, and many of these are alkaloids. In addition, alkaloids have served and continue to serve as leads for synthetic drugs. More recently, it was discovered that alkaloids have important ecological functions in, for example, plant defence against pathogens and herbivores2. About 20% of plant species accumulate alkaloids, which are mostly derived from the amino acids Phe, Tyr, Trp, Lys and Orn. Some have a mixed biosynthetic origin, such as the monoterpenoid indole alkaloids (TIAs), which are derived from Trp and terpenoid precursors. The basic biochemistry of several alkaloid biosynthesis pathways has been worked out over the past few decades. More recently, several biosynthesis genes have been cloned that encode enzymes from a few pathways1, which has allowed the study of pathway regulation at the transcriptional level. One of the latest trends in

Jasmonic acid and its volatile derivative methyljasmonate (MeJA), collectively called jasmonates, are plant stress hormones that act as regulators of defence responses4. The induction of secondary metabolite accumulation is an important stress response that depends on jasmonates as a regulatory signal5. Jasmonates are fatty acid derivatives with a 12-carbon backbone that is synthesized from 18-carbon intermediates via the socalled octadecanoid (ODA) pathway6. A lipase generates α-linolenic acid, which is the first precursor in the ODA pathway. α-Linolenic acid is then converted by a lipoxygenase, an allene oxide synthase and an allene oxide cyclase into the intermediate 12oxo-phytodienoic acid (12-oxo-PDA). This compound is converted into jasmonic acid through the action of a reductase and three rounds of β-oxidation. 12-OxoPDA might also play a major role as a stress signal. Structural analogues of 12-oxo-PDA that cannot be converted into jasmonic acid are effective in induction of alkaloid accumulation7. Jasmonic acid is essential for elicitor signal transduction

Addition of elicitors is a common method of enhancing secondary metabolism in plant cell cultures for metabolic, enzymatic or regulatory studies. In its broadest definition, an elicitor is any compound or mixture of compounds that induces a plant defence reaction. Most elicitors used in plant research originate from microorganisms but others are derived from the plant cell wall8. In addition, a variety of abiotic elicitors has been used, such as heavy metals. Elicitors are often applied in the form of crude mixtures, such as a fungal cell wall extract. In a few cases, elicitors have been purified to homogeneity.

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Table 1. Examples of plant secondary metabolites induced by jasmonates Metabolite class and subclass

Plant species

Polyamines

Hyoscyamus muticus Coumaroyl-conjugated polyamines

Hordeum vulgare Anthraquinones

Rubia tinctorum Naphthoquinones

Lithospermum erythrorhizon Gum (polysaccharide)

Prunus persica Tulipa gesneriana Terpenoids

Taxus spp.

Diterpenes (taxol)

Lactuca sativa

Sesquiterpenes

Phaseolus lunatus Zea mays Scutellaria baicalensis

Triterpenes Alkaloids Acridone

Ruta chalepensis

Nicotine

Nicotiana spp.

Tropane

Datura stramonium Eschscholzia californica

Benzylisoquinoline

Papaver somniferum Thalictrum tuberosum Catharanthus roseus

Terpenoid indole

Cinchona ledgeriana Rauvolfia spp. Phenylpropanoids

Coleus blumei

Rosmarinic acid

Lithospermum erythrorhizon Coumarins

Nicotiana tabacum

Furanocoumarins

Petroselinum crispum

Flavonoids

Arabidopsis thaliana Crotalaria cobalticola Glycine max Oryza sativa Petroselinum crispum Prunus persica Tulipa gesneriana

Most elicitors are considered to be general or nonrace-specific signals, to distinguish them from the elicitors encoded by microbial avirulence (avr) genes. These are recognized by plant resistance (R) gene products in race-specific resistance interactions. The specific plant receptors for avr gene products consist of R proteins and/or proteins associated with them9. It has now become clear that general elicitors might also be recognized by specific receptors via mechanisms that are not essentially different from R–avr-geneproduct interactions. The general bacterial elicitor flagellin is recognized by the Arabidopsis protein http://plants.trends.com

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FLS2, which shares structural similarity to several R proteins in that it has a leucine-rich repeat domain and a serine/threonine kinase domain10. In alkaloid research, an extract from baker’s yeast is commonly used as an elicitor. Yeast extract contains several components that can elicit plant defence responses, including chitin, N-acetylglucosamine oligomers, β-glucan, glycopeptides and ergosterol8. In addition, a low molecular weight component, which is probably a small peptide, induces TIA biosynthesis gene expression in cells of Catharanthus roseus (Madagascar periwinkle)11. Intensive research efforts, including pharmacological studies, have uncovered components of the elicitor signal transduction pathway connecting elicitor perception to induction of defence genes12. In several elicitor responses, including secondary metabolite production, (Me)jasmonic acid and some of its octadecanoid precursors play key roles as intermediate signals5,11,12. In different plant species, elicitors were shown to induce accumulation of endogenous jasmonic acid, and (Me)jasmonic acid itself increased secondary metabolite production (Table 1). In addition, blocking jasmonic acid biosynthesis abolished elicitor-induced metabolite accumulation and the expression of biosynthesis genes11,12. How elicitors affect jasmonic acid biosynthesis and how the jasmonic acid signal is transduced to affect gene expression is largely unknown. Many elicitors cause an increase in cytoplasmic calcium concentration, and calcium channel blockers inhibit downstream responses12. Elicitor-induced jasmonic acid biosynthesis in periwinkle cells also depends on an increase in cytoplasmic calcium concentration (Fig. 1; J. Memelink et al., unpublished). Several studies have indicated that elicitor-induced jasmonate accumulation is sensitive to protein kinase inhibitors7,11. A particular class of protein kinases, the mitogen-activated protein kinases were shown to be involved in wound- and elicitor-responsive jasmonic acid biosynthesis13. Also, downstream of the ODA pathway, one or more protein kinases are involved in transduction of the jasmonic acid signal leading to expression of metabolic pathway genes11. In Arabidopsis, many jasmonate responses depend on COI1, a protein of unknown function with leucinerich repeats and an F-box motif14. Based on its similarity to other F-box proteins with known activities, it has been suggested that COI1 might target a repressor of jasmonate responses for protein degradation via the ubiquitin–proteasome pathway. Figure 1 summarizes different components of the elicitor signal transduction pathway leading to expression of TIA biosynthesis genes in periwinkle cells (Ref. 11; J. Memelink et al., unpublished). Jasmonates affect gene transcription in secondary metabolism

The effects of jasmonates on secondary metabolism have been studied in detail for alkaloid biosynthesis.

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Ca2+ Plasma membrane

Receptor

Lipase

Protein kinase

Ca2+

Octadecanoid pathway Jasmonate

? Jasmonic acid receptor Protein kinase Nucleus CrBPF-1

ORCA

BA

JERE

TATA

Str

Activation of gene expression TRENDS in Plant Science

Fig. 1. Model for elicitor signal transduction leading to Str gene expression. The model shows the positions of CrBPF-1 in a signal transduction pathway independent of jasmonic acid, and of ORCAs in a jasmonic acid-dependent elicitor signal transduction pathway. Protein phosphorylation and calcium influx are required for elicitor-induced jasmonate biosynthesis, as well as for the induction of CrBPF-1, Orca2 and Orca3 gene expression. Orca gene expression and ORCA protein activation additionally depend on jasmonate biosynthesis. The existence of a lipase and of receptors for elicitor and jasmonic acid is postulated. The positions of the TATA box, the BA region and the jasmonate- and elicitor-responsive element (JERE) within the Str promoter are indicated. Abbreviations: Ca2+, calcium ion; CrBPF-1, Catharanthus roseus box P-binding factor 1 homologue; ORCA, octadecanoid-responsive Catharanthus AP2-domain protein; Str, strictosidine synthase gene.

For the three pathways (Fig. 2) leading to nicotine and tropane alkaloids, benzylisoquinoline alkaloids and terpenoid indole alkaloids, the genes for several biosynthetic enzymes have been cloned and the effects of jasmonates on gene transcription investigated. Detailed studies of promoter elements and transcription factors involved in jasmonateresponsive expression have been performed with genes involved in TIA biosynthesis. Nicotine and tropane alkaloid biosynthesis

Tropane alkaloids are mainly found in the Solanaceae and include the anticholinergic drugs hyoscyamine and scopolamine. Cocaine is a tropane alkaloid found outside the Solanaceae in members of the genus Erythroxylum. The alkaloid nicotine, present in the Solanaceous genus Nicotiana, is derived from the same precursor as the tropane ring, the N-methylpyrrolinium cation, which is itself derived from putrescine (Fig. 2a). Genes have been cloned from different plant species, including genes for http://plants.trends.com

putrescine-N-methyl transferase (PMT), which catalyses the first committed step in nicotine and tropane alkaloid biosynthesis (Fig. 2a). In Nicotiana species, nicotine biosynthesis2 and PMT gene expression15 are induced after jasmonate treatment. PMT gene expression is induced coordinately with the expression of genes for other enzymes in nicotine biosynthesis, including ornithine decarboxylase15 (Fig. 2a). Promoter regions of ~250 base pairs from three PMT genes from Nicotiana sylvestris could confer jasmonic acidresponsive expression on a GUS reporter gene in transgenic hairy roots16, showing that the jasmonic acid signal converges on relatively small promoter regions to confer transcriptional responses. For tropane alkaloid biosynthesis, stimulatory effects of jasmonate are more ambiguous. In root cultures of Datura stramonium (thornapple), MeJA treatment increased tropane alkaloid accumulation17. In root cultures of Hyoscyamus muticus (Egyptian henbane), MeJA induced the accumulation of methylputrescine but yielded only a modest increase in tropane alkaloid levels18. In roots of Atropa belladonna (deadly nightshade), MeJA did not induce expression of PMT genes19. The evaluation of jasmonate effects on tropane alkaloid biosynthesis awaits more systematic studies using larger sets of Solanaceae species and biosynthesis genes. It has been suggested19 that hyoscyamine and scopolamine might not function as defence compounds and so their synthesis might not respond to jasmonic acid. Benzylisoquinoline alkaloid biosynthesis

Benzylisoquinoline alkaloids compose a large, diverse class and are found in a range of plant families. The first biosynthetic step is decarboxylation of tyrosine by tyrosine decarboxylase (TYDC) to form tyramine. Coupling of two tyramine derivatives yields (S)norcoclaurine, which is the precursor of several thousand benzylisoquinoline alkaloids (Fig. 2b). Physiologically active members include the antimicrobial compounds berberine [produced by members of various families including Berberis vulgaris (barberry; Berberidaceae family)] and sanguinarine [produced by various members of the Papaveraceae including Sanguinaria canadensis (bloodroot)] and the narcotics morphine and codeine [produced by Papaver somniferum (opium poppy)]. Jasmonates were found to have strong inducing effects on the biosynthesis of benzophenanthridine alkaloids, a subclass of benzylisoquinoline alkaloids, in Eschscholzia californica (California poppy; Papaveraceae family)5. Several biosynthetic enzymes have been cloned from different plant species. The genes for TYDC, berberine bridge enzyme (BBE; catalyses the first step in the branch pathway leading to berberine and benzophenanthridine alkaloids) and CYP80B1 [(S)-N-methylcoclaurine 3′-hydroxylase] have been cloned from opium poppy; the genes for BBE and CYP80B1 have also been cloned from

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(a)

(b)

Tyrosine TYDC

Ornithine

(S )-norcoclaurine

ODC Putrescine

(S )-coclaurine

PMT

N -methylputrescine

(S )-N -methylcoclaurine

N -methyl-∆1 pyrrolinium cation

(S )-3′-hydroxy-N -methylcoclaurine

CYP80B1

Tropinone TR-1

Nicotine N H Me

(S )-reticuline BBE

Tropine

Codeinone (S )-scoulerine COR Codeine Sanguinarine Morphine O O HO CH3

N (–)-Hyoscyamine H6H Scopolamine Me N

N+

CH2OH

H

O

O

O

(c)

O

NMe H

O

H

HO

Shikimate pathway

O

Pyruvate + glyceraldehyde 3-phosphate DXS

Chorismate

Geranyl-diphosphate

AS Anthranilate

Geraniol G10H

CPR

10-Hydroxy-geraniol

Tryptophan TDC Tryptamine

Loganin SLS

STR

Secologanin

Tabersonine

Cathenamine

H

N Ajmalicine N H H

T16H 16-Hydroxytabersonine Catharantine

O H CO2Me

D4H N

Deacetylvindoline DAT Vindoline

Vinblastine

N H MeO2C MeO

HO

N H

OAc OH

N H Me CO2Me TRENDS in Plant Science

Fig. 2. Alkaloid biosynthesis pathways. Unbroken arrows indicate single enzymatic conversions and broken arrows indicate multiple enzymatic conversions. Enzymes for which the corresponding genes have been cloned are indicated. Dark-blue font indicates that the corresponding genes have been tested for jasmonate responsiveness; enzymes encoded by jasmonate-responsive genes are shown in darkblue font in a light-blue box. (a) Biosynthesis pathway for nicotine and tropane alkaloids (which are found in different plant species): H6H, hyoscyamine 6β-hydroxylase; ODC, ornithine decarboxylase; PMT, putrescine-N-methyltransferase; TR-1, tropinone reductase 1. (b) Biosynthesis pathway for benzylisoquinoline alkaloids in opium poppy: BBE, berberine bridge enzyme; CYP80B1, (S)-Nmethylcoclaurine 3′-hydroxylase; TYDC, tyrosine decarboxylase. (c) Biosynthesis pathway for terpenoid indole alkaloids in periwinkle (enzymes regulated by ORCA3 are in a light-blue box outlined in dark-blue): AS, anthranilate synthase; CPR, cytochrome P450 reductase; D4H, desacetoxyvindoline 4-hydroxylase; DAT, acetyl-CoA:4-O-deacetylvindoline 4-O-acetyltransferase; DXS, D-1-deoxyxylulose 5-phosphate synthase; G10H, geraniol 10-hydroxylase; SGD, strictosidine β-D-glucosidase; SLS, secologanin synthase; STR, strictosidine synthase; TDC, tryptophan decarboxylase; T16H, tabersonine 16-hydroxylase.

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California poppy. The Tydc (Ref. 20), Bbe (Refs 21,22) and CYP80B1 (Ref. 22) genes were induced in opium poppy by MeJA treatment. However, expression of the Cor gene (encoding the enzyme codeinone reductase, which is involved in morphine biosynthesis) did not respond to MeJA (Ref. 22), which is consistent with the observation that MeJA induces sanguinarine but not morphine production in cell suspensions of opium poppy. Also, in California poppy, Bbe (Refs 7,23) and CYP80B1 (Ref. 23) expression was induced by treatment with MeJA. In cell suspension cultures of Thalictrum tuberosum (meadow rue; Ranunculaceae family), berberine biosynthesis is induced by MeJA as a result of increases in the activities of four different O-methyltransferases that act in the biosynthesis pathway to berberine. This regulation of methyltransferase activities in berberine-producing species is in contrast with the regulation of benzophenanthridine alkaloid biosynthesis in California poppy, in which MeJA does not affect methyltransferase activities24. Four cDNAs for MeJA-inducible genes were isolated from meadow rue that encoded O-methyltransferases that can use different benzylisoquinoline alkaloid compounds as substrates, including (S)-norcoclaurine24. Thus, the biosynthesis of both benzophenanthridine and berberine alkaloids is induced by MeJA as a result of the coordinated activation of biosynthesis genes. However, it seems that MeJA induces distinct sets of genes for rate-limiting enzymes in different plant species depending on the benzylisoquinoline alkaloid subclass. Terpenoid indole alkaloids

Strictosidine SGD

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TIAs are found in a limited number of plant species that belong to the plant families Apocynaceae, Loganiaceae, Rubiaceae and Nyssaceae. Most progress on molecular characterization of the pathway has been made with Madagascar periwinkle, a member of the Apocynaceae family. Periwinkle cells have the genetic potential to synthesize over 100 TIAs. Several have pharmaceutical applications, including the monomeric alkaloids serpentine and ajmalicine, which are used as a tranquillizer and to reduce hypertension, respectively, and the dimeric alkaloids vincristine and vinblastine, which are potent antitumour drugs. Physiologically active TIAs from other plant species include the poison strychnine from Strychnos nux-vomica and the antineoplastic agent camptothecin from the Chinese ‘happy’ tree Camptotheca acuminata. The initial step in TIA biosynthesis is the condensation of the tryptophan derivative tryptamine with the terpenoid derivative secologanin (Fig. 2c). This condensation is performed by the enzyme strictosidine synthase (STR) and results in the synthesis of 3α(S)strictosidine. Strictosidine is the common precursor of a wide range of different TIAs in various plant species.

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Additional species-specific enzymes and spontaneous conversions determine the types of TIAs that are eventually formed. In periwinkle, dimeric alkaloids are formed by a peroxidase-catalysed condensation of vindoline and catharanthine (Fig. 2c). Biosynthesis of TIAs is highly regulated and depends on tissue-specific factors3 as well as environmental signals. TIA biosynthesis is induced by MeJA in developing seedlings25 and in cell cultures26. Nine genes encoding enzymes involved in TIA biosynthesis have now been cloned from periwinkle (Fig. 2c). In addition, genes acting in primary precursor pathways leading to the formation of tryptophan and geraniol have also been cloned (Fig. 2c). This places periwinkle in a unique position for studying coordinately controlled expression of genes in primary and alkaloid metabolism during development or upon stress. All TIA biosynthesis genes tested are induced by MeJA (Refs 27,28). In addition, MeJA induces genes in primary metabolism leading to the formation of TIA precursors28. This presents a good example of the profound effect of jasmonates on plant metabolism at the level of gene expression. The promoter of the Str gene has been studied in great detail to identify elicitor- and jasmonateresponsive sequences29,30. Two regions were identified that dictated elicitor- and jasmonate-responsive reporter gene activation: the so-called BA region and a region close to the TATA box called jasmonate- and elicitor-responsive element (JERE) (Fig. 1). An Str promoter derivative with a deletion of the BA region is still active and responds to stress signals, but at a lower level. Mutation or removal of the JERE results in an inactive and unresponsive Str promoter derivative29. A tetramer of the JERE fused to a minimal promoter confers jasmonic acid-responsive gene expression on a reporter gene29, showing that the JERE is an autonomous jasmonic acid-responsive sequence. The JERE interacts with two jasmonic acidresponsive transcription factors called octadecanoidresponsive Catharanthus AP2-domain proteins (ORCAs). ORCA2 was isolated by yeast one-hybrid screening with the JERE as bait29 and ORCA3 was isolated by a genetic T-DNA activation tagging approach28. Both belong to the AP2/ERF family of transcription factors, which are unique to plants and characterized by the AP2/ERF DNA-binding domain31. Significantly, Orca gene expression was rapidly induced by MeJA (Refs 29,32). Furthermore, ORCA proteins transactivate Str gene expression via specific binding to the JERE (Refs 29,32). Use of the BA region in a yeast one-hybrid screen resulted in the isolation of a periwinkle homologue of the MYB-like factor BPF-1 from parsley (Petroselinum crispum)30. Like its parsley counterpart33, transcription of periwinkle CrBPF-1 is induced by a fungal elicitor. However, jasmonates had no effect on its expression. CrBPF1 elicitation depends on protein kinase activity and an increase in cytosolic calcium concentration, but is independent of http://plants.trends.com

jasmonate biosynthesis (Fig. 1), indicating that the yeast elicitor induces gene expression in periwinkle cells via jasmonic acid-dependent and jasmonic acidindependent pathways. Figure 1 shows a model that summarizes the activation of Str gene expression by a yeast elicitor. Upon perception of the elicitor, protein phosphorylation is required to induce a calcium influx. This transient increase in cytosolic Ca2+ concentration is necessary for activation of the ODA pathway. Jasmonate induces Orca gene expression and activates pre-existing ORCA proteins32. Then, activated ORCAs switch on Str expression via their interaction with the JERE. In addition, Ca2+ influx is required for jasmonic acid-independent induction of CrBPF-1 mRNA accumulation30. CrBPF-1 is putatively involved in regulation of the Str gene via interaction with the jasmonate- and elicitorresponsive BA region. In conclusion, the Str promoter contains the JERE, which forms an ORCA-dependent switch for elicitor- and jasmonate-responsive activity, and other cis-acting elements with their cognate binding factors (such as BA/CrBPF-1), which might be important for enhancing the elicitor and/or jasmonate response. ORCA3 is a master regulator of metabolism

Overexpression of ORCA3 increased expression of the TIA biosynthesis genes Tdc, Str, Cpr and D4h (Ref. 28; Fig. 2c). The TIA biosynthesis genes G10h, Sgd and Dat were not induced, suggesting that these genes are not controlled by ORCA3. Although previous studies report different regulation of genes involved in early (i.e. Tdc and Str) and late (i.e. D4h) steps in vindoline biosynthesis in periwinkle plants3, genes of both classes are controlled by Orca3 in suspension-cultured cells. Plant cells must regulate their primary metabolic pathways to accommodate the biosynthesis of secondary metabolites. MeJA induces genes in terpenoid indole alkaloid metabolism as well as genes in primary metabolism leading to TIA precursor formation28, indicating that these processes are coordinately regulated. Orca3 overexpression induced genes encoding the α subunit of anthranilate synthase (ASα) and D-1-deoxyxylulose 5-phosphate synthase, enzymes involved in primary metabolism leading to TIA precursor formation28 (Fig. 2c). Apparently, ORCA3 acts as a central regulator to direct metabolic fluxes into the TIA biosynthesis pathway by regulation of genes involved in primary as well as secondary metabolism. Cell cultures overexpressing Orca3 accumulated significantly more tryptophan and tryptamine, providing the indole moiety of TIAs. Because no TIAs were detected in these cultures, it was concluded that the terpenoid branch of the pathway was limiting for TIA production, and that this limitation could not be overcome by Orca3 overexpression. A possible bottleneck was G10h, because G10h expression was

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AvrPto

Ethylene

Cold

Drought

Jasmonate

Receptor (-kinase)

Pto

Ethylene receptor

?

?

?

AP2/ERFdomain transcription factor

Pti

EREBP/ERF

DREB1/CBF

DREB2

ORCA

PR genes

PR genes

Coldresponsive genes

Droughtresponsive genes

TIA genes

Signal

Response genes

TRENDS in Plant Science

Fig. 3. Models showing the involvement of AP2/ERF-domain transcription factors in regulation of stress and defence gene expression in plants. The stress signal is perceived by interaction with a receptor (kinase). Induction of de novo synthesis and/or modulation of pre-existing AP2/ERF-domain transcription factor(s) activates gene expression. Unidentified signal transduction components are indicated with a question mark. Broken arrows indicate the possible involvement of multiple signalling steps. DREB1/CBF and DREB2 proteins have distinct but overlapping target gene sets35. Abbreviations: AvrPto, bacterial avirulence gene product recognized by Pto; CBF, C-repeat-binding factor; DRE, dehydration-responsive element; DREB, DRE-binding protein; EREBP, ethyleneresponsive-element binding protein; ERF, ethylene-responsive element binding factor; ORCA, octadecanoid-responsive Catharanthus AP2-domain protein; PR, pathogenesis-related; Pti, Ptointeracting protein; Pto, tomato protein kinase conferring resistance to Pseudomonas syringae pv tomato expressing AvrPto; TIA, terpenoid indole alkaloid.

not observed in the Orca3-overexpressing cell cultures. When the terpenoid precursor loganin was added to the cell cultures, Orca3 overexpression caused an increase in TIA production28. This demonstrates the potential of using central transcriptional regulators for metabolic engineering of alkaloid biosynthesis. Specific AP2/ERF family members respond to distinct stress signals

The AP2/ERF-domain (also called AP2/EREBP or ERF) proteins form a subfamily of AP2-domain proteins that is characterized by the presence of a single copy of the AP2 DNA-binding domain. The other members of the AP2 family contain two AP2 DNA-binding domains that, although similar, are clearly distinct from the AP2/ERF domain31. AP2/ERF-domain transcription factors occupy central positions in the regulation of plant stress responses. In gene expression induced by the stress hormone ethylene (ethene), AP2/ERF-domain transcription factors called ethylene-responsiveelement binding proteins (EREBPs) or factors (ERFs) play crucial roles34 (Fig. 3). Overexpression of ERF1 resulted in the induction of several ethyleneresponsive defence genes in Arabidopsis34. Resistance of tomato to the bacterial pathogen Pseudomonas syringae pv tomato depends on the recognition of the bacterial avirulence gene product AvrPto by the tomato receptor kinase Pto (Ref. 9). Yeast two-hybrid screening with Pto as a bait identified three Ptointeracting tomato proteins (Pti4, Pti5 and Pti6), each of which contained an AP2/ERF-domain9. Pti4–6 are thought to be involved in elicitor-induced activation of defence genes in tomato (Fig. 3). http://plants.trends.com

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Another set of AP2/ERF-domain proteins, represented by DREB2A and DREB2B, is involved in drought-responsive gene expression, whereas the CBF/DREB1 group are central regulators of coldregulated gene expression in Arabidopsis35 (Fig. 3). Upon overexpression of CBF1/DREB1B, the expression of a set of cold-induced genes was induced, resulting in increased freezing tolerance35. In periwinkle, the stress hormone jasmonate coordinately induces the expression of TIA biosynthesis genes11,27,28. Many of these genes are controlled by the jasmonate-responsive transcription factor ORCA3 (Ref. 28). Therefore, the spectrum of defence genes regulated by AP2/ERF-domain transcription factors includes genes involved in jasmonate-responsive secondary metabolism, in addition to elicitor- and ethylene-responsive pathogenesis-related genes and cold- and droughtresponsive genes (Fig. 3). Perspectives

In terpenoid indole alkaloid metabolism, jasmonic acid-responsive gene expression is conferred by members of the AP2/ERF-domain family of transcription factors. Overexpression of ORCA3 in suspension-cultured cells results in increased levels of tryptophan and, upon feeding of a terpenoid precursor, TIAs. Although ORCA3 regulates multiple genes in primary and secondary metabolism, several genes in TIA metabolism do not seem to be targets of ORCA3. A good candidate transcription factor involved in controlling these genes is ORCA2. Preliminary results indicate that ORCA2 and ORCA3 regulate overlapping but distinct sets of target genes. This indicates that these ORCA proteins have different functions and that both are required for the full spectrum of jasmonate-induced metabolic changes. It will be interesting to see whether the fitness of periwinkle plants can be improved by modulating alkaloid metabolism via ectopic expression of ORCAs. It is also possible that ectopic expression of these regulators will affect plants negatively. For example, overexpression of the cold-responsive AP2/ERFdomain protein DREB1A/CBF3 under control of the constitutive cauliflower mosaic virus 35S RNA promoter resulted in transgenic Arabidopsis plants with a dwarf phenotype35. However, use of the stressinducible promoter of the rd29A gene to express DREB1A/CBF3 resulted in phenotypically normal plants that were more tolerant of drought, salt and freezing35. Possible negative effects of constitutive ORCA overexpression could be circumvented with a similar approach, using an inducible promoter to drive ORCA expression. It is still unknown whether jasmonic acidresponsive master regulators such as ORCA3 control other metabolic pathways or defence gene sets. For several plant secondary metabolic pathways, effects of jasmonate have been found on the activity of a

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pathway enzyme, the abundance of the corresponding transcript or on metabolite accumulation. These effects are not restricted to alkaloids but are also found for flavonoids, terpenoids, anthraquinones and other metabolites (Table 1). Jasmonate can also induce multiple secondary metabolite classes in a single plant species, for example alkaloids and phenolics in tobacco, anthocyanins and gum in peach and tulip, and naphthoquinones and rosmarinic acid in Lithospermum erythrorhizon (Table 1). It is tempting to speculate that these pathways in other plant species are controlled by ORCA-like AP2/ERF-domain transcription factors. If so, isolation and overexpression of Orca homologues might provide a general approach for genetic engineering of jasmonic acid-responsive metabolism to improve plant fitness for agricultural purposes or to modify metabolite levels for industrial purposes. Overexpression of maize MYB and basic helix–loop–helix regulatory proteins in heterologous plant species resulted in increased production of flavonoids36, showing the possibilities for interspecies exchange of regulatory proteins. Expression of the periwinkle ORCA proteins in other plant species might result in the induction of jasmonate-responsive metabolic pathways and consequently in increased metabolite production. Jasmonate-induced expression of the Orca genes might occur via autoregulation of gene expression, as References 1 Kutchan, T.M. (1995) Alkaloid biosynthesis – the basis for metabolic engineering of medicinal plants. Plant Cell 7, 1059–1070 2 Baldwin, I.T. (1999) Inducible nicotine production in native Nicotiana as an example of adaptive phenotypic plasticity. J. Chem. Ecol. 25, 3–30 3 De Luca, V. and St-Pierre, B. (2000) The cell and developmental biology of alkaloid biosynthesis. Trends Plant Sci. 5, 168–173 4 Reymond, P. and Farmer, E.E. (1998) Jasmonate and salicylate as global signals for defense gene expression. Curr. Opin. Plant Biol. 1, 404–411 5 Gundlach, H. et al. (1992) Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proc. Natl. Acad. Sci. U. S. A. 89, 2389–2393 6 Müller, M.J. (1997) Enzymes involved in jasmonic acid biosynthesis. Physiol. Plant. 100, 653–663 7 Blechert, S. et al. (1995) The octadecanoic pathway: signal molecules for the regulation of secondary pathways. Proc. Natl. Acad. Sci. U. S. A. 92, 4099–4105 8 Boller, T. (1995) Chemoperception of microbial signals in plant cells. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 189–214 9 Martin, G.B. (1999) Functional analysis of plant disease resistance genes and their downstream effectors. Curr. Opin. Plant Biol. 2, 273–279 10 Gómez-Gómez, L. and Boller, T. (2000) FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol. Cell 5, 1003–1011 11 Menke, F.L.H. et al. (1999) Involvement of the octadecanoid pathway and protein phosphorylation in fungal elicitor-induced http://plants.trends.com

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shown for the elicitor-responsive WRKY proteins in parsley33,37, or via a transcriptional cascade involving an upstream transcription factor, as shown for ethylene-responsive AP2/ERF-domain transcription factors34. Jasmonic acid-responsive expression of TIA biosynthesis genes does not depend on de novo ORCA protein synthesis32, indicating that pre-existing ORCA proteins are activated via protein modification. MeJA-induced Tdc and Str expression is sensitive to protein kinase inhibitors11, and therefore activation of ORCA proteins could occur via phosphorylation, thereby increasing their nuclear localization, DNAbinding affinity or activation potential. Questions that need to be answered include how the jasmonic acid signal is transduced from the receptor to activate the ORCAs and how the ORCAs themselves are activated. The tomato transcription factor Pti4 is phosphorylated by the receptor kinase Pto (Ref. 9), indicating that phosphorylation might be a common modification to activate AP2/ERF-domain transcription factors. With the complete Arabidopsis genome sequence available, it is now clear that AP2domain proteins form a large family of ~150 members in this tiny weed. A significant proportion of these are involved in responses to diverse stress signals (Fig. 3). It will be challenging to discover how different stress signals activate specific subsets of genes and to what extent AP2-domain proteins provide a platform for crosstalk between signal transduction pathways.

expression of terpenoid indole alkaloid biosynthetic genes in Catharanthus roseus. Plant Physiol. 119, 1289–1296 Scheel, D. (1998) Resistance response physiology and signal transduction. Curr. Opin. Plant Biol. 1, 305–310 Meskiene, I. and Hirt, H. (2000) MAP kinase pathways: molecular plug-and-play chips for the cell. Plant Mol. Biol. 42, 791–806 Xie, D-X. et al. (1998) COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility. Science 280, 1091–1094 Imanishi, S. et al. (1998) Differential induction by methyl jasmonate of genes encoding ornithine decarboxylase and other enzymes involved in nicotine biosynthesis in tobacco cell cultures. Plant Mol. Biol. 38, 1101–1111 Shoji, T. et al. (2000) Jasmonate induction of putrescine N-methyltransferase genes in the root of Nicotiana sylvestris. Plant Cell Physiol. 41, 831–839 Zabetakis, I. et al. (1999) Elicitation of tropane alkaloid biosynthesis in transformed root cultures of Datura stramonium. Phytochemistry 50, 53–56 Biondi, S. et al. (2000) Jasmonates induce overaccumulation of methylputrescine and conjugated polyamines in Hyoscyamus muticus L. root cultures. Plant Cell Rep. 19, 691–697 Suzuki, K. et al. (1999) Expression of Atropa belladonna putrescine N-methyltransferase gene in root pericycle. Plant Cell Physiol. 40, 289–297 Facchini, P.J. et al. (2000) Plant aromatic L-amino acid decarboxylases: evolution, biochemistry, regulation, and metabolic engineering applications. Phytochemistry 54, 121–138 Facchini, P.J. et al. (1996) Molecular

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characterization of berberine bridge enzyme genes from opium poppy. Plant Physiol. 112, 1669–1677 Huang, F. and Kutchan, T.M. (2000) Distribution of morphinan and benzo[c]phenanthridine alkaloid gene transcript accumulation in Papaver somniferum. Phytochemistry 53, 555–564 Pauli, H.H. and Kutchan, T.M. (1998) Molecular cloning and functional heterologous expression of two alleles encoding (S)-N-methylcoclaurine 3′hydroxylase (CYP80B1), a new methyl jasmonate-inducible cytochrome P-450dependent mono-oxygenase of benzylisoquinoline alkaloid biosynthesis. Plant J. 13, 793–801 Frick, S. and Kutchan, T.M. (1999) Molecular cloning and functional expression of Omethyltransferases common to isoquinoline alkaloid and phenylpropanoid biosynthesis. Plant J. 17, 329–339 Aerts, R.J. et al. (1994) Methyl jasmonate vapor increases the developmentally controlled synthesis of alkaloids in Catharanthus and Cinchona seedlings. Plant J. 5, 635–643 Gantet, P. et al. (1998) Necessity of a functional octadecanoic pathway for indole alkaloid synthesis by Catharanthus roseus cell suspensions cultured in an auxin-starved medium. Plant Cell Physiol. 39, 220–225 Geerlings, A. et al. (2000) Molecular cloning and analysis of strictosidine β-D-glucosidase, an enzyme in terpenoid indole alkaloid biosynthesis in Catharanthus roseus. J. Biol. Chem. 275, 3051–3056 van der Fits, L. and Memelink, J. (2000) ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science 289, 295–297

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29 Menke, F.L.H. et al. (1999) A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2. EMBO J. 18, 4455–4463 30 van der Fits, L. et al. (2000) A Catharanthus roseus BPF-1 homologue interacts with an elicitor-responsive region of the secondary metabolite biosynthetic gene Str and is induced by elicitor via a jasmonate-independent signal transduction pathway. Plant Mol. Biol. 44, 675–685

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31 Riechmann, J.L. and Meyerowitz, E.M. (1998) The AP2/EREBP family of plant transcription factors. Biol. Chem. 379, 633–646 32 van der Fits, L. and Memelink, J. (2001) The jasmonate-inducible AP2/ERF-domain transcription factor ORCA3 activates gene expression via interaction with a jasmonateresponsive promoter element. Plant J. 25, 43–53 33 Rushton, P.J. and Somssich, I.E. (1998) Transcriptional control of plant genes responsive to pathogens. Curr. Opin. Plant Biol. 1, 311–315 34 Stepanova, A.N. and Ecker, J.R. (2000) Ethylene signaling: from mutants to molecules. Curr. Opin.

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Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants Henry Daniell, Stephen J. Streatfield and Keith Wycoff The use of plants for medicinal purposes dates back thousands of years but genetic engineering of plants to produce desired biopharmaceuticals is much more recent. As the demand for biopharmaceuticals is expected to increase, it would be wise to ensure that they will be available in significantly larger amounts, on a cost-effective basis. Currently, the cost of biopharmaceuticals limits their availability. Plant-derived biopharmaceuticals are cheap to produce and store, easy to scale up for mass production, and safer than those derived from animals. Here, we discuss recent developments in this field and possible environmental concerns.

Henry Daniell* Dept Molecular Biology and Microbiology and Center for Discovery of Drugs and Diagnostics, University of Central Florida, 12 722 Research Parkway, Orlando, FL 32826, USA. *e-mail: [email protected] Stephen J. Streatfield ProdiGene, 101 Gateway Boulevard, College Station, TX 77845, USA. Keith Wycoff Planet Biotechnology, 2462 Wyandotte Street, Mountain View, CA 94043, USA.

Research in the past few decades has revolutionized the use of therapeutically valuable proteins in a variety of clinical treatments. Because most genes can be expressed in many different systems, it is essential to determine which system offers the most advantages for the production of the recombinant protein. The ideal expression system would be the one that produces the most safe, biologically active material at the lowest cost. The use of modified mammalian cells with recombinant DNA techniques has the advantage of resulting in products that are identical to those of natural origin; however, culturing these cells is expensive and can only be carried out on a limited scale. The use of microorganisms such as bacteria permits manufacture on a larger scale, but introduces the disadvantage of producing products that differ appreciably from the products of natural origin. For example, proteins that are usually glycosylated in humans are not glycosylated by bacteria. Furthermore, human proteins that are

expressed at high levels in E. coli frequently acquire an unnatural conformation accompanied by intracellular precipitation, owing to lack of proper folding and disulfide bridges. The production of recombinant proteins in plants has many potential advantages for generating biopharmaceuticals relevant to clinical medicine. First, plant systems are more economical than industrial facilities using fermentation or bioreactor systems. Second, the technology is already available for harvesting and processing plants and plant products on a large scale. Third, the purification requirement can be eliminated when the plant tissue containing the recombinant protein is used as a food (edible vaccines). Fourth, plants can be directed to target proteins into intracellular compartments in which they are more stable, or even to express them directly in certain compartments (chloroplasts). Fifth, the amount of recombinant product that can be produced approaches industrial-scale levels. Last, health risks arising from contamination with potential human pathogens or toxins are minimized. Antibody production in plants

In the decade since the expression and assembly of immunoglobulin (Ig) heavy and light chains into functional antibodies was first shown in transgenic tobacco, plants have proven to be versatile production systems for many forms of antibodies. These include full-sized IgG and IgA, chimeric IgG and IgA,

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