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‘Radicle' biochemistry: the biology of root-specific metabolism
trends in plant science reviews
ÔRadicleÕ biochemistry: the biology of root-specific metabolism Hector E. Flores, Jorge M. Vivanco and Victor M. Loyola-Vargas The roots of higher plants are a fascinating and largely unexplored biological frontier. One of their features is the ability to synthesize a remarkable diversity of secondary metabolites, and to adjust their metabolic activities in response to biotic and abiotic stress. This includes the ability to exude a complex array of micro- and macromolecules into the rhizosphere, with the potential to affect the inter-relationships between plants and beneficial or deleterious soil-borne organisms. In the past, research on root biology has been hampered by the underground growth habit of roots and by the lack of a suitable experimental system. However, recent progess in growing roots in isolation has greatly facilitated the study of root-specific metabolism and contributed to our understanding of this remarkable plant organ.
R
esearch in root biology has experienced a renaissance in recent years due to a combination of factors. The availability of Arabidopsis mutants, impaired in root development and function, has led to valuable insights and is allowing the genetic dissection of root processes to an extent not possible just a few years ago. Novel non-destructive methods and instruments for analysis of root growth and architecture (magnetic resonance imaging, portable minirhizotrons, new modeling software) are likewise available. In addition to providing valuable new information, these recent developments have catalyzed a dramatic reconceptualization of the role of roots in the life of plants and in the biosphere. Below-ground plant processes are now recognized as essential components of ecosystem productivity and stability. It is estimated that the surface area represented by plant roots exceeds that of the aerial plant organs in every ecosystem that has been examined1. This review addresses a most neglected aspect of root biology, namely, that roots have evolved a unique array of biochemical abilities, some of which have little if any parallel in above-aground plant organs. Chemistry and biology of root-specific metabolites
There are several ways in which we can visualize roots as chemical factories2, from their uses as staples (cassava, sweet potato) and vegetables (carrots, radishes) to their ability to exude a vast array of compounds into the rhizosphere3. But perhaps the most dramatic examples of root biochemical diversity come from our knowledge of roots as medicines. For example, the root of Mandragora (mandrake) was a favorite means of inducing halucinations and poisoning enemies in the Middle Ages. Relatives of mandrake have been used for medicinal purposes in many traditional cultures worldwide, from the sacred Datura of Ayurveda in India, to the medicinal and cosmetic Atropa belladona of the Mediterranean and the tree Datura (Brugmansia) of Amazonia. The medicinal uses of these Solanaceae species have a defined chemical basis, namely the tropane alkaloids found in roots and leaves. These secondary metabolites, exemplified by hyoscyamine (Fig. 1) and scopolamine, are derived from phenylalanine and ornithine or arginine and are synthesized almost exclusively in the root. Their mode of action is based on their binding to acetylcholine receptors, and accounts for their use as smooth muscle relaxants, nerve gas antidotes and in the treatment of seasickness. The tropane alkaloids and their chemical cousins nicotine and nicotine derivatives, are just one of many compounds that are found in plant roots (Fig. 1). For example, the fatty acid-derived 220
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polyacetylenes, such as thiarubrine and terthienyl, are classic examples of nematicides that are found mostly in the roots of marigold (Calendula officinalis) and related Asteraceae. Over 800 compounds have been reported in this family and include phototoxins, antifungals and antibacterials. The shikonins are a group of naphthoquinones found in the roots of Boraginaceae, and also show a wide spectrum of antimicrobial activities. Emetine is an alkaloid from the roots of a South American vine, Cephaelis ipecacuanha, which is now incorporated into Western pharmacopoeia to induce vomiting in cases of poisoning. The isoflavonoid rotenone is found in the roots of several woody legumes (Derris, Lonchocarpus) and used widely as a fish poison and pesticide3. The diterpene ginkgolides of the maidenhair tree (Ginkgo biloba) are the major active principles of root or leaf extracts used to treat heart disease, dementia and senility. The labdane diterpenoid forskolin, from the roots of the Indian herb Coleus forskolii, is a potential activator of adenylate cyclase, which is used to treat bronchial asthma. Finally, the alkaloid camptothecin, from the roots of the Chinese medicinal shrub Camptotheca acuminata, is one of the most recent anticancer drugs of plant origin3. In spite of their diversity in chemistry and biological activities, we know very little about root-specific metabolism and its significance for the plant. A summary of what is known about the biology of the beststudied compounds produced in Solanaceae roots is shown in Fig. 2. Hairy root culture as an experimental system
The underground growth habit of roots poses major technical difficulties for their study and has hindered biochemical research in particular. A recent reincarnation of a classic plant organ culture system has proven extremely useful in reinvigorating research on root metabolism. Experiments revealed that hairy roots of Egyptian henbane (Hyoscyamus muticus) could be induced by transformation of shoot cultures with the soil-borne pathogen Agrobacterium rhizogenes, and established as long-term aseptic root clones4. Remarkably, these hairy roots were able to synthesize hyoscyamine at levels equal to or greater than the roots in planta, while showing growth rates comparable to those of the fastest growing cell suspension cultures. The biosynthetic capacity of hairy root cultures was strictly correlated with a differentiated state and, in contrast with undifferentiated culture systems, remained stable for an indefinite number of passages (subcultures) The original hyoscyamine-producing hairy root clones have been maintained for .15 years through monthly culture passages, with no indication of a change in biosynthetic capacity. Similar results have
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trends in plant science reviews been obtained with Datura stramonium5 and Catharanthus roseus6 and are reflected in part by stable chromosome numbers. The ability to convert root cultures into disorganized cell suspension and to readily regenerate the organized phenotype has allowed for selection of root clones with desirable characteristics. Roots regenerated from Hyoscyamus muticus suspension cultures selected for resistance to p-fluorophenylalanine, showed significantly higher and stable levels of hyoscyamine than the parental hairy roots7. Many other laboratories have confirmed and extended these findings8. Understanding and manipulating root-specific metabolism
Hairy root cultures from ~200 species of higher plants, mostly dicots, representing at least 30 plant families, have been reported, and represent a truly remarkable range of biosynthetic capabilities (see Table 1). Hairy root culture is thus well established as an experimental system (Fig. 3) and, most importantly, it has provided many insights into root-specific metabolism and its regulation. For example, even though roots usually grow as heterotrophic organs, and are dependent on leaves for photosynthate supply, they can express their photosynthetic potential, as in the specialized aerial roots of orchids3. In vitro, hairy roots can also express their photosynthetic abilities (Fig. 3), and in some cases they can grow under full carbon photoautotrophy9. The change in carbon assimilation mode has dramatic effects on the patterns of phytochemicals produced by root cultures, thus providing a system to study the coordination between primary and secondary metabolism. Hairy roots are able to biotransform xenobiotics, as in the case of Asteraceae roots, which can efficiently uptake and dimerize butylated hydroxytoluene into a stilbene quinone10 (Fig. 3). The biotransformation product appears to have antifungal activity (H. Flores and Y.R. Dai, unpublished), in contrast with the relatively inert xenobiotic precursor.
a-Terthienyl
Thiarubrine A H3C
C C
C C
C
C
CH
CH2
SÐS
S
S
L-Hyoscyamine
Shikonin
CH3 N O
O
H
C
C
S
CH2OH
OH
O
OH
O
Emetine
OH
Rotenone
CH3O N
CH3O
H O
O
O
H
H
H
H
O H
OCH3
HN
OCH OCH
OCH3
Ginkgolide A CH3 OH
O
H
Forskolin CH3 OH O CH3
O O
O
CH
CH3 O
O
O O
O H H H
H
Nicotine
OH C(CH3)3
CH2
OH OCCH3 H3C
H OH CH3
Camptothecin OH
H
N CH3
N
O O
N
N O
Fig. 1. Chemical structures of selected secondary metabolites synthesized in plant roots.
RootÐorganism co-culture
In addition to expressing root-specific pathways, hairy roots can also be established as co-cultures with other organisms. Some of these co-culture systems might have applied potential, as in the case of vesicular-arbuscular mycorrhizal (VAM) fungi. Over 90% of higher plants in nature develop VAM symbioses, which are useful in scavenging nutrients in limited supply, such as phosphate, but the axenic cultivation of VAM has been a major challenge until recently. Using hairy root cultures of carrot (Daucus carota), it has been possible to infect these roots with several species of Glomus, a VAM fungus11 (Fig. 3). In some cases, sporulation of VAM fungi in large numbers has been possible, signaling the potential of this system for large-scale production of axenic mycorrhizal inoculum through bioreactor culture2. The establishment of VAM symbioses
in vitro appears to affect root secondary metabolism. It has been reported that Glomus intraradices induced accumulation of a terpenoid glycoside in Hordeum vulgare roots12. Also, the co-culture of Acaulospora scrobiculata with Catharanthus roseus hairy roots reverts the inhibitory effect of phosphorus on indole alkaloid production (V.M. Loyola-Vargas and B. Canto, unpublished). In addition to fungal co-cultures, it is possible to co-culture nematodes with hairy roots. We have established co-cultures of D. carota roots with the wound nematode Pratylenchus spp., which is able to complete its life cycle and induce the same root tip hypertrophy symptoms that are characteristic of the infection in the soil (Fig. 3). Using a similar system, a detailed analysis has been made of Pratylenchus penetrans reproduction in root cultures of Ladino clover June 1999, Vol. 4, No. 6
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trends in plant science reviews Manipulation of alkaloid biosynthesis
Several strategies have been used to modify the alkaloid content of hairy root cultures. The exposure of C. roseus hairy roots to elicitors, such as pectinase or jasmonic acid15, led to increases in tabersonine, ajmalicine, serpentine, lochnericine and hörhammericine. The use of amberlite resins, such as XAD-7, greatly enhanced the release of catharanthine and ajmalicine from hairy root cultures of C. roseus16. A similar effect was detected in Datura stramonium hairy root cultures when the culture medium pH was lowered17. Hyoscyamine Herbivore attack Alkaloid accumulation (increased alkaloid) and proline accumulate in water-stressed and modification Hyoscyamus muticus hairy roots18. In conHerbivore signal trast, exposure of H. muticus hairy roots to transport Alkaloid transport an elicitor preparation from Rhizoctonia (xylem) solani did not have any effect on tropane alkaloid production, but induced the secretion of massive amounts of sesquiterpene phytoalexins2. A hydroxymethylglutarylAlkaloid synthesis Pathogen infection CoA reductase (HMGR) gene from (nicotine or tropane) Secretion of bioactive proteins C. roseus was introduced into H. muticus Nutrient deficiency Phytoalexin induction hairy roots via A. rhizogenes, under the Organic acid secretion control of the 35S CaMV promoter Phosphatase release (H.E. Flores and F. Medina-Bolivar, Fig. 2. Drawing of a generic tropane alkaloid-producing Solanaceae species showing the unpublished). One transformed root line central role played by roots in determining the spatial and temporal patterns of bioactive showed a higher constitutive level of secondary metabolites and macromolecule synthesis and induction, either constitutively, or in HMGR activity than the control roots, and response to biotic stress. Also included is the generalized response of roots to nutrient higher levels of sesquiterpene elicitation. deficiency, exemplified by phosphate depletion in the soil. As shown above, Agrobacterium rhizogenes might also be used to introduce foreign genes into hairy roots. The expression (Trifolium repens f. lodigense) roots13. Finally, it is possible to of a bacterial lysine decarboxylase gene under the regulation of grow insects and roots in co-culture as well. Recently several the 35S CaMV promoter in hairy roots of Nicotiana tabacum19 aseptic long-term aphid-root co-cultures have been established in resulted in a threefold increase in the anabasine alkaloids. The vitro14 (Fig. 3). These phloem-feeding insects are able to reproduce gene for hyoscyamine 6b-hydroxylase (H6H), which catalyzes a parthenogenetically on roots of safflower (Carthamus tinctorius), two-step hydroxylation and epoxidation of hyoscyamine to and stable populations have been maintained for .two years. The scopolamine (a more commercially valuable alkaloid), was aphids also appear to induce changes in root metabolism, from the cloned from Hyoscyamus niger hairy roots. Remarkably, expresbrowning of roots at times of heavy herbivory, to induction of spe- sion of H6H is restricted only to the pericycle cell layer, and this cific polyacetylenes, a potential defense response. is presumably related to the transformation of hysocyamine into scopolamine before transfer to the xylem and transport to the shoots20. The H6H cDNA was placed under the control of the 35S CaMV promoter and introduced to Atropa belladonna roots, which normally produce hyoscyamine but little, if any, scopolTable 1. Recent reports of secondary amine. The resulting hairy roots and plant regenerants were able metabolites synthesized in hairy root cultures to express H6H and, most remarkably, the pattern of tropane alkaloids was changed, so that scopolamine now was the predominant Genus Metabolite Ref. product21. To date, this is the most dramatic example of the potential for metabolic engineering of root-specific metabolism22. Ajuga Hydroxyecydsone 37 Armoracia Artemisia Echinacea Lithospermum Lobelia Rauwolfia Rubia Salvia Serratula Trichosanthes Valeriana
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Fusicoccin Artemisinin Alkamides Naphthoquinones (shikonin) Polyacetylenes Indole alkaloids Anthraquinone Diterpenoids Ecdysteroids Bryonolic acid Iridoid diesters
38 39 40 23 41 42 43 44 45 46 47
Bioactive root exudates
The ability to secrete a vast array of compounds into the rhizosphere is the most remarkable metabolic feature of plant roots. Perhaps with the exception of germinating pollen grains, no other plant organ cell type rivals the root in this respect. Extracellular phytoalexins can be elicited in root cultures of Solanaceae upon exposure to root-rot fungi3. Root exudates, such as organic acids, which might be induced in response to phosphate limitation, might affect soil microbial populations indirectly through their effects on soil pH. In addition to low molecular weight metabolites, we also know that roots are capable of secreting a discrete,
trends in plant science reviews
Fig. 3. The many faces of root cultures and their use in the study of root biosynthetic capabilities. (a) Hairy root culture of ko-shikon (Lithospermum erythrorhizon) (photograph courtesy of Koichiro Shimomura). (b) Secretion of antimicrobial naphthoquinones by root hairs of L. erythrorhizon root cultures (photograph courtesy of Lindy Brigham; reprinted, with permission, from Ref. 23). (c) Detail of secretion of antimicrobial naphthoquinones in the epidermal cell walls of L. erythrorhizon root cultures (reprinted, with permission, from Ref. 23). (d) Hairy root cultures of safflower (Carthamus tinctorius). (e) Hairy root cultures of Bidens sulphureus. (f) Hairy root cultures of B. sulphureus grown in normal culture medium (control) or treated with butylated hydroxytoluene (BHT) for 24 h. (g) Co-culture of carrot (Daucus carota) hairy roots and the wound nematode Pratylenchus spp. (h) The plant aphid Rhopalosiphum padi feeding on a hairy root culture of C. tinctorius. (i) Co-culture of D. carota hairy roots and the vesicular-arbuscular mycorrhizal fungus Glomus spp. (photograph courtesy of Roger Koide). (j) Plant of mauka (Mirabilis expansa) showing its storage root and hairy root culture derived by infection with Agrobacterium rhizogenes. (k) Photoheterotrophic hairy root cultures of Trichosanthes kirilowii (photograph courtesy of Brett Savary). (l) Detail of 15 l glass column trickle-bed bioreactor with hairy roots of Egyptian henbane (Hyoscyamus muticus), at the end of a one-month batch culture period (photograph courtesy of Wayne Curtis).
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trends in plant science reviews species-specific pattern of extracellular proteins. We illustrate this root exudation capacity with a graphic example of ethnobotanical origin. The roots of ko-shikon (Lithospermum erythrorhizon) produce a small cohort of naphthoquinones collectively known as shikonins. These phenylalanine and geranyl pyrophosphatederived compounds have been used as dyes and as antibacterials in the treatment of skin infections, and can accumulate to high levels (3–5% dry wt) in the tap roots of three-to-five-year-old plants. It is also possible to express this pathway in hairy root culture (Fig. 3). Recent work indicates that the synthesis of shikonins is tightly controlled in space and time, as well as in response to biotic stress23. There are three sites of pigment production in primary roots: • So-called root border cells, released from the root cap as the root grows. • Trichoblasts, the epidermal root cells destined to become root hairs (as the root hairs expand, the pigments are left forming a ring at the base of the root hair) (Fig. 3). • The cell wall spaces of mature epidermal cells (Fig. 3). The above pattern is seen in root cultures grown on production medium that contains nitrate as the sole nitrogen source and high concentrations of copper23,24. Production of shikonins is inhibited by the presence of ammonium and by light, consistent with the view that it is under tight physiological and developmental control. The secretion of shikonin in root culture is in striking contrast with the situation in unorganized cell cultures, which are only able to accumulate the pigment24. Root exudation might thus be biologically relevant in the context of root–rhizosphere interactions. We know something about the medicinal properties of shikonins, but little about their biological significance. Based on the secretion of shikonins into the root culture medium and the rhizosphere (L. Brigham and H.E. Flores, unpublished), and their antimicrobial activity against human pathogens, their possible effects on soil microorganisms were investigated23. It is clear that shikonins show selective inhibitory activity against a range of soil-borne bacteria and fungi. For example, several bacterial species involved in bacterial root-rot disease, such as Pseudomonas solanacearum, are inhibited by shikonin, but other species of Pseudomonas spp. used as biocontrol agents are not affected. Similarly, the hyphal growth of root pathogenic fungi, such as Pythium aphanidermatum, P. ultimum and Rhizoctonia solani, is dramatically inhibited, but the growth of mycorrhizal fungi, such as Glomus intraradices, is unaffected. A localized and systemic elicitation of shikonin production has also been observed by coculturing root pathogenic fungi with L. erythrorhizon roots, consistent with the hypothesis that these compounds might, in fact, be part of the defense mechanism against soil-borne pathogens. Root-specific proteins
In addition to low-molecular weight compounds, roots can accumulate polymers, such as starch, fructans and proteins25. The most abundant polypeptides found in storage roots appear to fulfill the role of vegetative storage proteins, which break down to provide nitrogen and sulfur as the shoots sprout after a period of quiescence or stress. It is intriguing that some such proteins have also been shown to have biological activity. The expression of sporamin, the main storage protein of sweet potato roots can respond to challenge by root pests26. We can thus speculate that some major proteins found in underground storage organs might have evolved more than one function. In addition to storage proteins, a diverse array of defense proteins are produced in roots, including glucanases, chitinases and ribosome inactivating proteins (RIPs)3,25,27. Ribosome inactivating proteins are widely distributed among higher plants28 and inhibit protein synthesis by virtue of their 224
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N-glycosidase activity, selectively cleaving an adenine residue at a conserved site of the 28S rRNA (Ref. 29). This cleavage prevents the binding of elongation factor 2 (Ref. 30), with the consequent arrest of protein synthesis. Some type I (single polypeptide) RIPs have been shown to inhibit fungal growth31. Other RIPs have been found to have insecticidal activity against Coleopteran and Lepidopteran species32. The roots of the native Peruvian ornamental plant Mirabilis jalapa were found to contain an antiviral protein active against mechanically transmitted plant viruses and a viroid33. The active protein, named MAP, is a single-chain RIP. More recently, two novel type I RIPs have been isolated from the storage root of the Andean root crop species Mirabilis expansa. These proteins, named ME1 and ME2, are active against root-rot fungi and bacteria34. Related Mirabilis species have been shown to produce similar root-specific RIPs (J.M. Vivanco and H.E. Flores, unpublished). In addition to studying secondary metabolism, the hairy root system has been used to follow the production of bioactive proteins. We recently established root cultures of Trichosanthes spp.27,35, the storage roots of which have been used in traditional medicine to induce abortions and treat diabetes. The abortifacient principle has been identified as a type I RIP, named trichosanthin. The Trichosanthes spp. hairy root cultures express a protein that crossreacts with an antibody against trichosanthin, but is of higher molecular weight, presumably a precursor form27. Although storage roots of Trichosanthes spp. are able to accumulate large quantities of trichosanthin (.25% total soluble protein), the quantities of the cross-reactive protein obtained in root cultures are rather modest. The ability to express large quantities of trichosanthin thus appears to be highly correlated with the development of secondary growth, a feature that is generally lacking in root cultures. In the course of these studies, it was also observed that Trichosanthes roots are able to secrete species-specific patterns of proteins into the culture medium. Among these are several classes of chitinases, one of which showed antifungal activity and was inducible by salicylic acid35, and at least one class of permatin27. It is thus possible that roots might be secreting bioactive proteins into the rhizosphere in response to microbial or herbivory stress. We have recently developed hairy root cultures of several Mirabilis spp. which, in contrast with Trichosanthes hairy roots, produce RIPs both intra- and extracellularly (J.M. Vivanco and H.E. Flores, unpublished). Interestingly, the hairy roots of Mirabilis longiflora were able to produce RIPs only after elicitation with fungal cell wall preparations, salicylic acid, abscisic acid or methyl jasmonate. Future perspectives
The recognition that the functions of plant roots involve much more than water and nutrient uptake or the establishment of symbiotic associations opens fascinating prospects for research. Roots must now be reckoned with as the site of unique metabolic activities and, in many cases, as major contributors to the makeup of secondary metabolites in the whole plant. The production of biologically active metabolites and proteins by roots has important implications for the study of root–organism interactions in the rhizosphere. The ability to grow root cultures from many plant species in isolation and to manipulate root metabolism, allows the isolation and characterization of enzymes involved in rootspecific pathways, cloning of the corresponding genes and understanding of pathway regulation. This basic information provides the necessary background to predictably manipulate root biosynthetic potential in the whole plant, as well as in scaled-up root cultures. The potential for metabolic engineering of root-specific pathways has already been demonstrated. The long-term expression of a fully functional murine IgG1 monoclonal antibody
trends in plant science reviews (which binds to the surface protein of Streptococcus mutans, the causative agent of dental caries) in Nicotiana tabacum hairy roots36 is a good indication of things to come. In the future, the close integration of molecular, biochemical and ecological approaches, together with mathematical modeling, will help to reveal the chemistry and biology of the plant’s hidden half. Acknowledgements
We would like to thank the numerous undergraduate, graduate and postdoctoral collaborators that have been part of our laboratories over the past 15 years and contributed toward a better understanding of radical biochemistry. We are grateful to Lindy Brigham, Roger Koide, Brett Savary and Wayne Curtis, for kindly providing pictures of their work on root culture. The work in H.E.F’s laboratory has been supported by research and training grants from the National Science Foundation and the McKnight Foundation (Minneapolis, MN). Work in V.M.L-V.’s laboratory has been supported by the Consejo Nacional de Ciencia y Tecnologia (CONACYT, Mexico, D.F.). References 1 Jackson, R.B. et al. (1997) A global budget for fine root biomass, surface area and nutrient contents, Proc. Natl. Acad. Sci. U. S. A. 94, 7362–7366 2 Flores, H.E. and Curtis, W.R. (1992) Approaches to understanding and manipulating the biosynthetic potential of plant roots, Ann. NY Acad. Sci. 665, 188–209 3 Flores, H.E., Weber, C. and Puffett, J. (1996) Underground metabolism: the biosynthetic potential of roots, in Roots: The Hidden Half (2nd edn) (Waisel, Y. et al., eds), pp. 931–956, Marcel Dekker 4 Flores, H.E. and Filner, P. (1985) Metabolic relationships of putrescine, GABA, and alkaloids in cell and root cultures of Solanaceae, in Primary and Secondary Metabolism of Plant Cell Cultures (Neumann, K-H., Barz, W. and Reinhard, E., eds), pp. 174–186, Springer-Verlag 5 Maldonado-Mendoza, I.E., Ayora-Talavera, T. and Loyola-Vargas, V.M. (1993) Establishment of hairy root cultures of Datura stramonium. Characterization and stability of tropane alkaloid production during long periods of subculturing, Plant Cell, Tissue Organ Cult. 33, 321–329 6 Ciau-Uitz, R. et al. (1994) Indole alkaloid production by transformed and non-transformed root cultures of Catharanthus roseus, In vitro Cell. Dev. Biol. 30P, 84–88 7 Medina-Bolivar, F.B. and Flores, H.E. (1995) Selection for hyoscyamine and cinnamoyl putrescine overproduction in cell and root cultures of Hyoscyamus muticus, Plant Physiol. 108, 1553–1560 8 Porter, J.R. (1991) Host range and implications of plant infection by Agrobacterium rhizogenes, CRC Crit. Rev. Plant Sci. 10, 387–421 9 Flores, H.E. et al. (1993) Green roots: photosynthesis and photoautotrophy in an underground plant organ, Plant Physiol. 101, 363–371 10 Flores, H.E. et al. (1994) Biotransformation of butylated hydroxytoluene in ‘hairy root’ cultures, Plant Physiol. Biochem. 32, 511–519 11 Bécard, G., Douds, D.D. and Pfeffer, P.E. (1992) Extensive in vitro hyphal growth of vesicular-arbuscular mycorrhizal fungi in the presence of CO2 and flavonoids, Appl. Environ. Microbiol. 58, 821–825 12 Peipp, H. (1997) Arbuscular mycorrhizal fungus-induced changes in the accumulation of secondary compounds in barley roots, Phytochemistry 44, 581–587 13 Mizukubo, T. (1997) Effect of temperature on Pratylenchus penetrans development, J. Nematol. 29, 306–314 14 Wu, T., Wittkamper, J. and Flores, H.E. Root herbivory in vitro: interactions between roots and aphids grown in aseptic co-culture, In vitro Cell. Dev. Biol. (in press) 15 Rijhwani, S.K. and Shanks, J.V. (1998) Effect of elicitor dosage and exposure time on biosynthesis of indole alkaloids by Catharanthus roseus hairy root cultures, Biotechnol. Prog. 14, 442–449
16 Sim, S.J. et al. (1994) Production and secretion of indole alkaloids in hairy root cultures of Catharanthus roseus: effects of in situ adsorption, cell permeabilization, J. Ferment. Bioeng. 78, 229–234 17 Sáenz-Carbonell, L. et al. (1993) Effect of the medium pH on the release of secondary metabolites from roots of Datura stramonium, Catharanthus roseus and Tagetes patula cultured in vitro, Appl. Biochem. Biotechnol. 38, 257–267 18 Halperin, S.J. and Flores, H.E. (1997) Hyoscyamine and proline accumulation in water-stressed Hyoscyamus muticus hairy root cultures, In vitro Cell. Dev. Biol. 33P, 240–244 19 Fecker, L.F., Rügenhagen, C. and Berlin, J. (1993) Increased production of cadaverine and anabasine in hairy root cultures of Nicotiana tabacum expressing a bacterial lysine decarboxylase gene, Plant Mol. Biol. 23, 11–21 20 Hashimoto, T. et al. (1991) Hyoscyamine 6b-hydroxylase, an enzyme involved in tropane alkaloid biosynthesis, is localized at the pericycle of the root, J. Biol. Chem. 266, 4648–4653 21 Yun, D-J., Hashimoto, T. and Yamada, Y. (1992) Metabolic engineering of medicinal plants: transgenic Atropa belladona with an improved alkaloid composition, Proc. Natl. Acad. Sci. U. S. A. 89, 11799–11803 22 Verpoorte, R. et al. (1998) Metabolic engineering for the improvement of plant secondary metabolite production, Plant Tissue Cult. Biotechnol. 4, 3–20 23 Brigham, L.A., Michaels, P.J. and Flores, H.E. (1999) Cell-specific production and antimicrobial activity of naphthoquinones in the roots of Lithospermum erythrorhizon, Plant Physiol. 119, 417–428 24 Flores, H.E., Brigham, L.A. and Vivanco, J.M. (1998) The future of radical biology? Connecting roots, people and scientists, in Radical Biology: Advances and Perspectives on the Function of Plant Roots (Flores, H.E., Lynch, J. and Eissenstat, D., eds), pp. 320–329, American Society of Plant Physiologists 25 Flores, H.E. and Flores, T. (1997) Biochemistry of underground plant storage organs, in Functionality of Food Phytochemicals (Johns, T. and Romeo, J.T., eds.) pp. 113–132, Plenum Press, NY, USA 26 Yeh, K. et al. (1997) Functional activity of sporamin from sweet potato (Ipomoea batatas Lam.): a tuber storage protein with trypsin inhibitor activity, Plant Mol. Biol. 33, 565–570 27 Savary, B.J. and Flores, H.E. (1994) Biosynthesis of defense-related proteins in transformed root cultures of Trichosanthes kirilowii Maxim. var. japonicum (Kitam.), Plant Physiol. 106, 1195–1204 28 Mehta, A.D. and Boston, R.S. (1998) Ribosome-inactivating proteins, in A Look Beyond Transcription: Mechanisms Determining mRNA Stability and Translation in Plants (Bailey-Serres, J. and Gallie, D.R., eds), pp. 145–152, American Society of Plant Physiologists 29 Endo, Y. and Tsurigi, K. (1988) RNA N-glycosidase activity of ricin A-chain, mechanism of action of the toxic lectin ricin on eukaryotic ribosomes, J. Biol. Chem. 262, 8128–8130 30 Stirpe, F. et al. (1992) Ribosome inactivating proteins from plants: present status and future prospects, Biotechnology 10, 405–412 31 Roberts, W.K. and Selitrennikoff, C.P. (1986) Isolation and characterization of two antifungal proteins from barley, Biochim. Biophys. Acta 880, 161–170 32 Gatehouse, A.M.R. et al. (1990) Effects of ribosome inactivating proteins on insect development – differences between Lepidoptera and Coleoptera, Entomol. Exp. Appl. 54, 43–51 33 Kubo, S. et al. (1990) A potent plant virus inhibitor found in Mirabilis jalapa L., Ann. Phytopathol. Soc. Japan 56, 481–487 34 Vivanco, J.M., Savary, B.J. and Flores, H.E. (1999) Characterization of two novel type I ribosome inactivating proteins from the storage roots of the Andean crop species Mirabilis expansa (Ruiz & Pavon), Plant Physiol. 119, 1447–1456 35 Savary, B.J., Flores, H.E. and Hill, J.J. (1997) Isolation of a class III chitinase produced in root cultures of Tricosanthes kirilowii and related species, Plant Physiol. Biochem. 35, 543–551 36 Wongsamuth, R. and Doran, P.M. (1997) Hairy root as an expression system for production of antibodies, in Hairy Roots Culture and Applications (Doran, P.M., ed.), pp. 89–97, Harwood Academic Press 37 Tanaka, N. and Matsumoto, T. (1993) Regenerants from Ajuga hairy roots with high productivity of 20-hydroxyecdysone, Plant Cell Rep. 13, 87–90
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trends in plant science reviews 38 Babakov, A.V. et al. (1995) Culture of transformed horseradish roots as a source of fusicoccin-like ligands, J. Plant Growth Regul. 14, 163–167 39 Qin, M.B. et al. (1994) Induction of hairy root from Artemisia annua with Agrobacterium rhizogenes and its culture in vitro, Acta Bot. Sinica 36 (Suppl.), 165–170 40 Trypsteen, M. et al. (1991) Agrobacterium rhizogenes-mediated transformation of Echinaceae purpurea, Plant Cell Rep. 10, 85–89 41 Yamanaka, M. et al. (1996) Polyacetylene glucosides in hairy root cultures of Lobelia cardinalis, Phytochemistry 41, 183–185 42 Benjamin, B.D., Roja, G. and Heble, M.R. (1994) Alkaloid synthesis by root cultures of Rauwolfia serpentina transformend with Agrobacterium rhizogenes, Phytochemistry 35, 381–383 43 Sato, K. et al. (1991) Anthraquinone production by transformed root cultures of Rubia tinctorum: influence of phytohormones and sucrose concentration, Phytochemistry 30, 1507–1510 44 Hu, Z-B. and Alfermann, A.W. (1993) Diterpenoid production in hairy root cultures of Salvia miltiorrhiza, Phytochemistry 32, 699–703 45 Delbecque, J.P. et al. (1995) In vitro incorporation of radiolabelled cholesterol and mevalonic acid into ecdysteroid by hairy root cultures of a plant, Serratula tictoria, Eur. J. Entomol. 92, 301–307
46 Takeda, T. et al. (1994) Bryonolic acid production in hairy roots of Trichosanthes kirilowii Max. var. japonica Kitam. transformed with Agrobacterium rhizogenes and its cytotoxic activity, Chem. Pharm. Bull. (Tokyo) 42, 730–732 47 Gränicher, F. et al. (1995) An iridoid diester from Valeriana officinalis var. sambucifolia hairy roots, Phytochemistry 38, 103–105
Hector E. Flores* and Jorge M. Vivanco are at the Dept of Plant Pathology and Life Sciences Consortium, 315 Wartik Building, The Pennsylvania State University, University Park, PA 16802, USA; Victor M. Loyola-Vargas is at the Centro de Investigacion Cientifica de Yucatan, Merida, Mexico (tel 152 99 81 3961; fax 152 99 81 3900; e-mail [email protected]).
*Author for correspondence (tel 11 814 865 2955; fax 11 814 863 7217; e-mail [email protected]).
Recent advances in the transformation of plants Genevi•ve Hansen and Martha S. Wright Plant transformation technology has become a versatile platform for cultivar improvement as well as for studying gene function in plants. This success represents the culmination of many years of effort in tissue culture improvement, in transformation techniques and in genetic engineering. The next challenge is to develop technology that minimizes or eliminates the tissue culture steps, and provides predictable transgene expression.
G
enetic engineering has opened new avenues to modify crops, and provided new solutions to solve specific needs1. In the future, the proportion of acreage planted with transgenic crops, and the range of transgenic crops, is sure to increase. For example, in the USA, the proportion of acreage planted with commercial transgenic cotton, soybean and corn was ~25, 18 and 10, respectively, in 1997 (Ref. 2). Forty-eight transgenic crop products had been approved for commercialization in various countries by the end of 1997. The powerful combination of genetic engineering and conventional breeding programs permits useful traits encoded by transgenes to be introduced into commercial crops within an economically viable time frame. There is great potential for genetic manipulation of crops to enhance productivity through increasing resistance to diseases, pests and environmental stress and by qualitatively changing the seed composition. Plant ‘factories’ are also being designed for high volume production of pharmaceuticals, nutraceuticals and other beneficial chemicals. Transgenic plants might become drug-delivery devices, with both HIV and rabies vaccines being synthesized in plants3, and bananas have been engineered to produce edible vaccines4. Moreover, with the establishment and expansion of genomics programs, a much broader range of genes with potential for crop improvement are being identified and, in some cases, tailored and/or re-designed for further enhancement of their properties within specific crops1. This has further intensified the interest in developing efficient plant transformation technologies to be able to concurrently test and capture the value of these genes. 226
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Beyond crop improvement, the ability to engineer transgenic plants is also a powerful and informative means for studying gene function and the regulation of physiological and developmental processes. Transgenic plants are being used as an assay system for the modification of endogenous metabolism or gene inactivation. Advances in tissue culture, combined with improvements in transformation technology, have resulted in increased transformation efficiencies. In recent years, many crops, previously classified as recalcitrant because they were stubbornly resistant to the overtures of genetic engineering, have now been transformed. In many instances, the technology has been pushed even further to introduce the gene of interest directly into elite cultivars5. Tissue culture prerequisite
A tissue culture stage is required in most current transformation protocols to ultimately recover plants. Indeed, it is the totipotency of plant cells that underlies most plant transformation systems. Plants are regenerated from cell culture via two methods, somatic embryogenesis and organogenesis. Both are controlled by plant hormones and other factors added to the culture medium. Some species, such as soybeans6, banana4,7 and sugar beet8,9 can be regenerated via either method so the choice is dependent upon which gives the best yield or the easiest outcome. Somatic embryogenesis is the generation of embryos from somatic tissues, such as embryos, microspores or leaves. Proliferating somatic embryos in liquid culture or on solid medium are suitable targets for transformation because the origin of proliferating
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ÔRadicleÕ biochemistry: the biology of root-specific metabolism Hector E. Flores, Jorge M. Vivanco and Victor M. Loyola-Vargas The roots of higher plants are a fascinating and largely unexplored biological frontier. One of their features is the ability to synthesize a remarkable diversity of secondary metabolites, and to adjust their metabolic activities in response to biotic and abiotic stress. This includes the ability to exude a complex array of micro- and macromolecules into the rhizosphere, with the potential to affect the inter-relationships between plants and beneficial or deleterious soil-borne organisms. In the past, research on root biology has been hampered by the underground growth habit of roots and by the lack of a suitable experimental system. However, recent progess in growing roots in isolation has greatly facilitated the study of root-specific metabolism and contributed to our understanding of this remarkable plant organ.
R
esearch in root biology has experienced a renaissance in recent years due to a combination of factors. The availability of Arabidopsis mutants, impaired in root development and function, has led to valuable insights and is allowing the genetic dissection of root processes to an extent not possible just a few years ago. Novel non-destructive methods and instruments for analysis of root growth and architecture (magnetic resonance imaging, portable minirhizotrons, new modeling software) are likewise available. In addition to providing valuable new information, these recent developments have catalyzed a dramatic reconceptualization of the role of roots in the life of plants and in the biosphere. Below-ground plant processes are now recognized as essential components of ecosystem productivity and stability. It is estimated that the surface area represented by plant roots exceeds that of the aerial plant organs in every ecosystem that has been examined1. This review addresses a most neglected aspect of root biology, namely, that roots have evolved a unique array of biochemical abilities, some of which have little if any parallel in above-aground plant organs. Chemistry and biology of root-specific metabolites
There are several ways in which we can visualize roots as chemical factories2, from their uses as staples (cassava, sweet potato) and vegetables (carrots, radishes) to their ability to exude a vast array of compounds into the rhizosphere3. But perhaps the most dramatic examples of root biochemical diversity come from our knowledge of roots as medicines. For example, the root of Mandragora (mandrake) was a favorite means of inducing halucinations and poisoning enemies in the Middle Ages. Relatives of mandrake have been used for medicinal purposes in many traditional cultures worldwide, from the sacred Datura of Ayurveda in India, to the medicinal and cosmetic Atropa belladona of the Mediterranean and the tree Datura (Brugmansia) of Amazonia. The medicinal uses of these Solanaceae species have a defined chemical basis, namely the tropane alkaloids found in roots and leaves. These secondary metabolites, exemplified by hyoscyamine (Fig. 1) and scopolamine, are derived from phenylalanine and ornithine or arginine and are synthesized almost exclusively in the root. Their mode of action is based on their binding to acetylcholine receptors, and accounts for their use as smooth muscle relaxants, nerve gas antidotes and in the treatment of seasickness. The tropane alkaloids and their chemical cousins nicotine and nicotine derivatives, are just one of many compounds that are found in plant roots (Fig. 1). For example, the fatty acid-derived 220
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polyacetylenes, such as thiarubrine and terthienyl, are classic examples of nematicides that are found mostly in the roots of marigold (Calendula officinalis) and related Asteraceae. Over 800 compounds have been reported in this family and include phototoxins, antifungals and antibacterials. The shikonins are a group of naphthoquinones found in the roots of Boraginaceae, and also show a wide spectrum of antimicrobial activities. Emetine is an alkaloid from the roots of a South American vine, Cephaelis ipecacuanha, which is now incorporated into Western pharmacopoeia to induce vomiting in cases of poisoning. The isoflavonoid rotenone is found in the roots of several woody legumes (Derris, Lonchocarpus) and used widely as a fish poison and pesticide3. The diterpene ginkgolides of the maidenhair tree (Ginkgo biloba) are the major active principles of root or leaf extracts used to treat heart disease, dementia and senility. The labdane diterpenoid forskolin, from the roots of the Indian herb Coleus forskolii, is a potential activator of adenylate cyclase, which is used to treat bronchial asthma. Finally, the alkaloid camptothecin, from the roots of the Chinese medicinal shrub Camptotheca acuminata, is one of the most recent anticancer drugs of plant origin3. In spite of their diversity in chemistry and biological activities, we know very little about root-specific metabolism and its significance for the plant. A summary of what is known about the biology of the beststudied compounds produced in Solanaceae roots is shown in Fig. 2. Hairy root culture as an experimental system
The underground growth habit of roots poses major technical difficulties for their study and has hindered biochemical research in particular. A recent reincarnation of a classic plant organ culture system has proven extremely useful in reinvigorating research on root metabolism. Experiments revealed that hairy roots of Egyptian henbane (Hyoscyamus muticus) could be induced by transformation of shoot cultures with the soil-borne pathogen Agrobacterium rhizogenes, and established as long-term aseptic root clones4. Remarkably, these hairy roots were able to synthesize hyoscyamine at levels equal to or greater than the roots in planta, while showing growth rates comparable to those of the fastest growing cell suspension cultures. The biosynthetic capacity of hairy root cultures was strictly correlated with a differentiated state and, in contrast with undifferentiated culture systems, remained stable for an indefinite number of passages (subcultures) The original hyoscyamine-producing hairy root clones have been maintained for .15 years through monthly culture passages, with no indication of a change in biosynthetic capacity. Similar results have
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trends in plant science reviews been obtained with Datura stramonium5 and Catharanthus roseus6 and are reflected in part by stable chromosome numbers. The ability to convert root cultures into disorganized cell suspension and to readily regenerate the organized phenotype has allowed for selection of root clones with desirable characteristics. Roots regenerated from Hyoscyamus muticus suspension cultures selected for resistance to p-fluorophenylalanine, showed significantly higher and stable levels of hyoscyamine than the parental hairy roots7. Many other laboratories have confirmed and extended these findings8. Understanding and manipulating root-specific metabolism
Hairy root cultures from ~200 species of higher plants, mostly dicots, representing at least 30 plant families, have been reported, and represent a truly remarkable range of biosynthetic capabilities (see Table 1). Hairy root culture is thus well established as an experimental system (Fig. 3) and, most importantly, it has provided many insights into root-specific metabolism and its regulation. For example, even though roots usually grow as heterotrophic organs, and are dependent on leaves for photosynthate supply, they can express their photosynthetic potential, as in the specialized aerial roots of orchids3. In vitro, hairy roots can also express their photosynthetic abilities (Fig. 3), and in some cases they can grow under full carbon photoautotrophy9. The change in carbon assimilation mode has dramatic effects on the patterns of phytochemicals produced by root cultures, thus providing a system to study the coordination between primary and secondary metabolism. Hairy roots are able to biotransform xenobiotics, as in the case of Asteraceae roots, which can efficiently uptake and dimerize butylated hydroxytoluene into a stilbene quinone10 (Fig. 3). The biotransformation product appears to have antifungal activity (H. Flores and Y.R. Dai, unpublished), in contrast with the relatively inert xenobiotic precursor.
a-Terthienyl
Thiarubrine A H3C
C C
C C
C
C
CH
CH2
SÐS
S
S
L-Hyoscyamine
Shikonin
CH3 N O
O
H
C
C
S
CH2OH
OH
O
OH
O
Emetine
OH
Rotenone
CH3O N
CH3O
H O
O
O
H
H
H
H
O H
OCH3
HN
OCH OCH
OCH3
Ginkgolide A CH3 OH
O
H
Forskolin CH3 OH O CH3
O O
O
CH
CH3 O
O
O O
O H H H
H
Nicotine
OH C(CH3)3
CH2
OH OCCH3 H3C
H OH CH3
Camptothecin OH
H
N CH3
N
O O
N
N O
Fig. 1. Chemical structures of selected secondary metabolites synthesized in plant roots.
RootÐorganism co-culture
In addition to expressing root-specific pathways, hairy roots can also be established as co-cultures with other organisms. Some of these co-culture systems might have applied potential, as in the case of vesicular-arbuscular mycorrhizal (VAM) fungi. Over 90% of higher plants in nature develop VAM symbioses, which are useful in scavenging nutrients in limited supply, such as phosphate, but the axenic cultivation of VAM has been a major challenge until recently. Using hairy root cultures of carrot (Daucus carota), it has been possible to infect these roots with several species of Glomus, a VAM fungus11 (Fig. 3). In some cases, sporulation of VAM fungi in large numbers has been possible, signaling the potential of this system for large-scale production of axenic mycorrhizal inoculum through bioreactor culture2. The establishment of VAM symbioses
in vitro appears to affect root secondary metabolism. It has been reported that Glomus intraradices induced accumulation of a terpenoid glycoside in Hordeum vulgare roots12. Also, the co-culture of Acaulospora scrobiculata with Catharanthus roseus hairy roots reverts the inhibitory effect of phosphorus on indole alkaloid production (V.M. Loyola-Vargas and B. Canto, unpublished). In addition to fungal co-cultures, it is possible to co-culture nematodes with hairy roots. We have established co-cultures of D. carota roots with the wound nematode Pratylenchus spp., which is able to complete its life cycle and induce the same root tip hypertrophy symptoms that are characteristic of the infection in the soil (Fig. 3). Using a similar system, a detailed analysis has been made of Pratylenchus penetrans reproduction in root cultures of Ladino clover June 1999, Vol. 4, No. 6
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trends in plant science reviews Manipulation of alkaloid biosynthesis
Several strategies have been used to modify the alkaloid content of hairy root cultures. The exposure of C. roseus hairy roots to elicitors, such as pectinase or jasmonic acid15, led to increases in tabersonine, ajmalicine, serpentine, lochnericine and hörhammericine. The use of amberlite resins, such as XAD-7, greatly enhanced the release of catharanthine and ajmalicine from hairy root cultures of C. roseus16. A similar effect was detected in Datura stramonium hairy root cultures when the culture medium pH was lowered17. Hyoscyamine Herbivore attack Alkaloid accumulation (increased alkaloid) and proline accumulate in water-stressed and modification Hyoscyamus muticus hairy roots18. In conHerbivore signal trast, exposure of H. muticus hairy roots to transport Alkaloid transport an elicitor preparation from Rhizoctonia (xylem) solani did not have any effect on tropane alkaloid production, but induced the secretion of massive amounts of sesquiterpene phytoalexins2. A hydroxymethylglutarylAlkaloid synthesis Pathogen infection CoA reductase (HMGR) gene from (nicotine or tropane) Secretion of bioactive proteins C. roseus was introduced into H. muticus Nutrient deficiency Phytoalexin induction hairy roots via A. rhizogenes, under the Organic acid secretion control of the 35S CaMV promoter Phosphatase release (H.E. Flores and F. Medina-Bolivar, Fig. 2. Drawing of a generic tropane alkaloid-producing Solanaceae species showing the unpublished). One transformed root line central role played by roots in determining the spatial and temporal patterns of bioactive showed a higher constitutive level of secondary metabolites and macromolecule synthesis and induction, either constitutively, or in HMGR activity than the control roots, and response to biotic stress. Also included is the generalized response of roots to nutrient higher levels of sesquiterpene elicitation. deficiency, exemplified by phosphate depletion in the soil. As shown above, Agrobacterium rhizogenes might also be used to introduce foreign genes into hairy roots. The expression (Trifolium repens f. lodigense) roots13. Finally, it is possible to of a bacterial lysine decarboxylase gene under the regulation of grow insects and roots in co-culture as well. Recently several the 35S CaMV promoter in hairy roots of Nicotiana tabacum19 aseptic long-term aphid-root co-cultures have been established in resulted in a threefold increase in the anabasine alkaloids. The vitro14 (Fig. 3). These phloem-feeding insects are able to reproduce gene for hyoscyamine 6b-hydroxylase (H6H), which catalyzes a parthenogenetically on roots of safflower (Carthamus tinctorius), two-step hydroxylation and epoxidation of hyoscyamine to and stable populations have been maintained for .two years. The scopolamine (a more commercially valuable alkaloid), was aphids also appear to induce changes in root metabolism, from the cloned from Hyoscyamus niger hairy roots. Remarkably, expresbrowning of roots at times of heavy herbivory, to induction of spe- sion of H6H is restricted only to the pericycle cell layer, and this cific polyacetylenes, a potential defense response. is presumably related to the transformation of hysocyamine into scopolamine before transfer to the xylem and transport to the shoots20. The H6H cDNA was placed under the control of the 35S CaMV promoter and introduced to Atropa belladonna roots, which normally produce hyoscyamine but little, if any, scopolTable 1. Recent reports of secondary amine. The resulting hairy roots and plant regenerants were able metabolites synthesized in hairy root cultures to express H6H and, most remarkably, the pattern of tropane alkaloids was changed, so that scopolamine now was the predominant Genus Metabolite Ref. product21. To date, this is the most dramatic example of the potential for metabolic engineering of root-specific metabolism22. Ajuga Hydroxyecydsone 37 Armoracia Artemisia Echinacea Lithospermum Lobelia Rauwolfia Rubia Salvia Serratula Trichosanthes Valeriana
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Fusicoccin Artemisinin Alkamides Naphthoquinones (shikonin) Polyacetylenes Indole alkaloids Anthraquinone Diterpenoids Ecdysteroids Bryonolic acid Iridoid diesters
38 39 40 23 41 42 43 44 45 46 47
Bioactive root exudates
The ability to secrete a vast array of compounds into the rhizosphere is the most remarkable metabolic feature of plant roots. Perhaps with the exception of germinating pollen grains, no other plant organ cell type rivals the root in this respect. Extracellular phytoalexins can be elicited in root cultures of Solanaceae upon exposure to root-rot fungi3. Root exudates, such as organic acids, which might be induced in response to phosphate limitation, might affect soil microbial populations indirectly through their effects on soil pH. In addition to low molecular weight metabolites, we also know that roots are capable of secreting a discrete,
trends in plant science reviews
Fig. 3. The many faces of root cultures and their use in the study of root biosynthetic capabilities. (a) Hairy root culture of ko-shikon (Lithospermum erythrorhizon) (photograph courtesy of Koichiro Shimomura). (b) Secretion of antimicrobial naphthoquinones by root hairs of L. erythrorhizon root cultures (photograph courtesy of Lindy Brigham; reprinted, with permission, from Ref. 23). (c) Detail of secretion of antimicrobial naphthoquinones in the epidermal cell walls of L. erythrorhizon root cultures (reprinted, with permission, from Ref. 23). (d) Hairy root cultures of safflower (Carthamus tinctorius). (e) Hairy root cultures of Bidens sulphureus. (f) Hairy root cultures of B. sulphureus grown in normal culture medium (control) or treated with butylated hydroxytoluene (BHT) for 24 h. (g) Co-culture of carrot (Daucus carota) hairy roots and the wound nematode Pratylenchus spp. (h) The plant aphid Rhopalosiphum padi feeding on a hairy root culture of C. tinctorius. (i) Co-culture of D. carota hairy roots and the vesicular-arbuscular mycorrhizal fungus Glomus spp. (photograph courtesy of Roger Koide). (j) Plant of mauka (Mirabilis expansa) showing its storage root and hairy root culture derived by infection with Agrobacterium rhizogenes. (k) Photoheterotrophic hairy root cultures of Trichosanthes kirilowii (photograph courtesy of Brett Savary). (l) Detail of 15 l glass column trickle-bed bioreactor with hairy roots of Egyptian henbane (Hyoscyamus muticus), at the end of a one-month batch culture period (photograph courtesy of Wayne Curtis).
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trends in plant science reviews species-specific pattern of extracellular proteins. We illustrate this root exudation capacity with a graphic example of ethnobotanical origin. The roots of ko-shikon (Lithospermum erythrorhizon) produce a small cohort of naphthoquinones collectively known as shikonins. These phenylalanine and geranyl pyrophosphatederived compounds have been used as dyes and as antibacterials in the treatment of skin infections, and can accumulate to high levels (3–5% dry wt) in the tap roots of three-to-five-year-old plants. It is also possible to express this pathway in hairy root culture (Fig. 3). Recent work indicates that the synthesis of shikonins is tightly controlled in space and time, as well as in response to biotic stress23. There are three sites of pigment production in primary roots: • So-called root border cells, released from the root cap as the root grows. • Trichoblasts, the epidermal root cells destined to become root hairs (as the root hairs expand, the pigments are left forming a ring at the base of the root hair) (Fig. 3). • The cell wall spaces of mature epidermal cells (Fig. 3). The above pattern is seen in root cultures grown on production medium that contains nitrate as the sole nitrogen source and high concentrations of copper23,24. Production of shikonins is inhibited by the presence of ammonium and by light, consistent with the view that it is under tight physiological and developmental control. The secretion of shikonin in root culture is in striking contrast with the situation in unorganized cell cultures, which are only able to accumulate the pigment24. Root exudation might thus be biologically relevant in the context of root–rhizosphere interactions. We know something about the medicinal properties of shikonins, but little about their biological significance. Based on the secretion of shikonins into the root culture medium and the rhizosphere (L. Brigham and H.E. Flores, unpublished), and their antimicrobial activity against human pathogens, their possible effects on soil microorganisms were investigated23. It is clear that shikonins show selective inhibitory activity against a range of soil-borne bacteria and fungi. For example, several bacterial species involved in bacterial root-rot disease, such as Pseudomonas solanacearum, are inhibited by shikonin, but other species of Pseudomonas spp. used as biocontrol agents are not affected. Similarly, the hyphal growth of root pathogenic fungi, such as Pythium aphanidermatum, P. ultimum and Rhizoctonia solani, is dramatically inhibited, but the growth of mycorrhizal fungi, such as Glomus intraradices, is unaffected. A localized and systemic elicitation of shikonin production has also been observed by coculturing root pathogenic fungi with L. erythrorhizon roots, consistent with the hypothesis that these compounds might, in fact, be part of the defense mechanism against soil-borne pathogens. Root-specific proteins
In addition to low-molecular weight compounds, roots can accumulate polymers, such as starch, fructans and proteins25. The most abundant polypeptides found in storage roots appear to fulfill the role of vegetative storage proteins, which break down to provide nitrogen and sulfur as the shoots sprout after a period of quiescence or stress. It is intriguing that some such proteins have also been shown to have biological activity. The expression of sporamin, the main storage protein of sweet potato roots can respond to challenge by root pests26. We can thus speculate that some major proteins found in underground storage organs might have evolved more than one function. In addition to storage proteins, a diverse array of defense proteins are produced in roots, including glucanases, chitinases and ribosome inactivating proteins (RIPs)3,25,27. Ribosome inactivating proteins are widely distributed among higher plants28 and inhibit protein synthesis by virtue of their 224
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N-glycosidase activity, selectively cleaving an adenine residue at a conserved site of the 28S rRNA (Ref. 29). This cleavage prevents the binding of elongation factor 2 (Ref. 30), with the consequent arrest of protein synthesis. Some type I (single polypeptide) RIPs have been shown to inhibit fungal growth31. Other RIPs have been found to have insecticidal activity against Coleopteran and Lepidopteran species32. The roots of the native Peruvian ornamental plant Mirabilis jalapa were found to contain an antiviral protein active against mechanically transmitted plant viruses and a viroid33. The active protein, named MAP, is a single-chain RIP. More recently, two novel type I RIPs have been isolated from the storage root of the Andean root crop species Mirabilis expansa. These proteins, named ME1 and ME2, are active against root-rot fungi and bacteria34. Related Mirabilis species have been shown to produce similar root-specific RIPs (J.M. Vivanco and H.E. Flores, unpublished). In addition to studying secondary metabolism, the hairy root system has been used to follow the production of bioactive proteins. We recently established root cultures of Trichosanthes spp.27,35, the storage roots of which have been used in traditional medicine to induce abortions and treat diabetes. The abortifacient principle has been identified as a type I RIP, named trichosanthin. The Trichosanthes spp. hairy root cultures express a protein that crossreacts with an antibody against trichosanthin, but is of higher molecular weight, presumably a precursor form27. Although storage roots of Trichosanthes spp. are able to accumulate large quantities of trichosanthin (.25% total soluble protein), the quantities of the cross-reactive protein obtained in root cultures are rather modest. The ability to express large quantities of trichosanthin thus appears to be highly correlated with the development of secondary growth, a feature that is generally lacking in root cultures. In the course of these studies, it was also observed that Trichosanthes roots are able to secrete species-specific patterns of proteins into the culture medium. Among these are several classes of chitinases, one of which showed antifungal activity and was inducible by salicylic acid35, and at least one class of permatin27. It is thus possible that roots might be secreting bioactive proteins into the rhizosphere in response to microbial or herbivory stress. We have recently developed hairy root cultures of several Mirabilis spp. which, in contrast with Trichosanthes hairy roots, produce RIPs both intra- and extracellularly (J.M. Vivanco and H.E. Flores, unpublished). Interestingly, the hairy roots of Mirabilis longiflora were able to produce RIPs only after elicitation with fungal cell wall preparations, salicylic acid, abscisic acid or methyl jasmonate. Future perspectives
The recognition that the functions of plant roots involve much more than water and nutrient uptake or the establishment of symbiotic associations opens fascinating prospects for research. Roots must now be reckoned with as the site of unique metabolic activities and, in many cases, as major contributors to the makeup of secondary metabolites in the whole plant. The production of biologically active metabolites and proteins by roots has important implications for the study of root–organism interactions in the rhizosphere. The ability to grow root cultures from many plant species in isolation and to manipulate root metabolism, allows the isolation and characterization of enzymes involved in rootspecific pathways, cloning of the corresponding genes and understanding of pathway regulation. This basic information provides the necessary background to predictably manipulate root biosynthetic potential in the whole plant, as well as in scaled-up root cultures. The potential for metabolic engineering of root-specific pathways has already been demonstrated. The long-term expression of a fully functional murine IgG1 monoclonal antibody
trends in plant science reviews (which binds to the surface protein of Streptococcus mutans, the causative agent of dental caries) in Nicotiana tabacum hairy roots36 is a good indication of things to come. In the future, the close integration of molecular, biochemical and ecological approaches, together with mathematical modeling, will help to reveal the chemistry and biology of the plant’s hidden half. Acknowledgements
We would like to thank the numerous undergraduate, graduate and postdoctoral collaborators that have been part of our laboratories over the past 15 years and contributed toward a better understanding of radical biochemistry. We are grateful to Lindy Brigham, Roger Koide, Brett Savary and Wayne Curtis, for kindly providing pictures of their work on root culture. The work in H.E.F’s laboratory has been supported by research and training grants from the National Science Foundation and the McKnight Foundation (Minneapolis, MN). Work in V.M.L-V.’s laboratory has been supported by the Consejo Nacional de Ciencia y Tecnologia (CONACYT, Mexico, D.F.). References 1 Jackson, R.B. et al. (1997) A global budget for fine root biomass, surface area and nutrient contents, Proc. Natl. Acad. Sci. U. S. A. 94, 7362–7366 2 Flores, H.E. and Curtis, W.R. (1992) Approaches to understanding and manipulating the biosynthetic potential of plant roots, Ann. NY Acad. Sci. 665, 188–209 3 Flores, H.E., Weber, C. and Puffett, J. (1996) Underground metabolism: the biosynthetic potential of roots, in Roots: The Hidden Half (2nd edn) (Waisel, Y. et al., eds), pp. 931–956, Marcel Dekker 4 Flores, H.E. and Filner, P. (1985) Metabolic relationships of putrescine, GABA, and alkaloids in cell and root cultures of Solanaceae, in Primary and Secondary Metabolism of Plant Cell Cultures (Neumann, K-H., Barz, W. and Reinhard, E., eds), pp. 174–186, Springer-Verlag 5 Maldonado-Mendoza, I.E., Ayora-Talavera, T. and Loyola-Vargas, V.M. (1993) Establishment of hairy root cultures of Datura stramonium. Characterization and stability of tropane alkaloid production during long periods of subculturing, Plant Cell, Tissue Organ Cult. 33, 321–329 6 Ciau-Uitz, R. et al. (1994) Indole alkaloid production by transformed and non-transformed root cultures of Catharanthus roseus, In vitro Cell. Dev. Biol. 30P, 84–88 7 Medina-Bolivar, F.B. and Flores, H.E. (1995) Selection for hyoscyamine and cinnamoyl putrescine overproduction in cell and root cultures of Hyoscyamus muticus, Plant Physiol. 108, 1553–1560 8 Porter, J.R. (1991) Host range and implications of plant infection by Agrobacterium rhizogenes, CRC Crit. Rev. Plant Sci. 10, 387–421 9 Flores, H.E. et al. (1993) Green roots: photosynthesis and photoautotrophy in an underground plant organ, Plant Physiol. 101, 363–371 10 Flores, H.E. et al. (1994) Biotransformation of butylated hydroxytoluene in ‘hairy root’ cultures, Plant Physiol. Biochem. 32, 511–519 11 Bécard, G., Douds, D.D. and Pfeffer, P.E. (1992) Extensive in vitro hyphal growth of vesicular-arbuscular mycorrhizal fungi in the presence of CO2 and flavonoids, Appl. Environ. Microbiol. 58, 821–825 12 Peipp, H. (1997) Arbuscular mycorrhizal fungus-induced changes in the accumulation of secondary compounds in barley roots, Phytochemistry 44, 581–587 13 Mizukubo, T. (1997) Effect of temperature on Pratylenchus penetrans development, J. Nematol. 29, 306–314 14 Wu, T., Wittkamper, J. and Flores, H.E. Root herbivory in vitro: interactions between roots and aphids grown in aseptic co-culture, In vitro Cell. Dev. Biol. (in press) 15 Rijhwani, S.K. and Shanks, J.V. (1998) Effect of elicitor dosage and exposure time on biosynthesis of indole alkaloids by Catharanthus roseus hairy root cultures, Biotechnol. Prog. 14, 442–449
16 Sim, S.J. et al. (1994) Production and secretion of indole alkaloids in hairy root cultures of Catharanthus roseus: effects of in situ adsorption, cell permeabilization, J. Ferment. Bioeng. 78, 229–234 17 Sáenz-Carbonell, L. et al. (1993) Effect of the medium pH on the release of secondary metabolites from roots of Datura stramonium, Catharanthus roseus and Tagetes patula cultured in vitro, Appl. Biochem. Biotechnol. 38, 257–267 18 Halperin, S.J. and Flores, H.E. (1997) Hyoscyamine and proline accumulation in water-stressed Hyoscyamus muticus hairy root cultures, In vitro Cell. Dev. Biol. 33P, 240–244 19 Fecker, L.F., Rügenhagen, C. and Berlin, J. (1993) Increased production of cadaverine and anabasine in hairy root cultures of Nicotiana tabacum expressing a bacterial lysine decarboxylase gene, Plant Mol. Biol. 23, 11–21 20 Hashimoto, T. et al. (1991) Hyoscyamine 6b-hydroxylase, an enzyme involved in tropane alkaloid biosynthesis, is localized at the pericycle of the root, J. Biol. Chem. 266, 4648–4653 21 Yun, D-J., Hashimoto, T. and Yamada, Y. (1992) Metabolic engineering of medicinal plants: transgenic Atropa belladona with an improved alkaloid composition, Proc. Natl. Acad. Sci. U. S. A. 89, 11799–11803 22 Verpoorte, R. et al. (1998) Metabolic engineering for the improvement of plant secondary metabolite production, Plant Tissue Cult. Biotechnol. 4, 3–20 23 Brigham, L.A., Michaels, P.J. and Flores, H.E. (1999) Cell-specific production and antimicrobial activity of naphthoquinones in the roots of Lithospermum erythrorhizon, Plant Physiol. 119, 417–428 24 Flores, H.E., Brigham, L.A. and Vivanco, J.M. (1998) The future of radical biology? Connecting roots, people and scientists, in Radical Biology: Advances and Perspectives on the Function of Plant Roots (Flores, H.E., Lynch, J. and Eissenstat, D., eds), pp. 320–329, American Society of Plant Physiologists 25 Flores, H.E. and Flores, T. (1997) Biochemistry of underground plant storage organs, in Functionality of Food Phytochemicals (Johns, T. and Romeo, J.T., eds.) pp. 113–132, Plenum Press, NY, USA 26 Yeh, K. et al. (1997) Functional activity of sporamin from sweet potato (Ipomoea batatas Lam.): a tuber storage protein with trypsin inhibitor activity, Plant Mol. Biol. 33, 565–570 27 Savary, B.J. and Flores, H.E. (1994) Biosynthesis of defense-related proteins in transformed root cultures of Trichosanthes kirilowii Maxim. var. japonicum (Kitam.), Plant Physiol. 106, 1195–1204 28 Mehta, A.D. and Boston, R.S. (1998) Ribosome-inactivating proteins, in A Look Beyond Transcription: Mechanisms Determining mRNA Stability and Translation in Plants (Bailey-Serres, J. and Gallie, D.R., eds), pp. 145–152, American Society of Plant Physiologists 29 Endo, Y. and Tsurigi, K. (1988) RNA N-glycosidase activity of ricin A-chain, mechanism of action of the toxic lectin ricin on eukaryotic ribosomes, J. Biol. Chem. 262, 8128–8130 30 Stirpe, F. et al. (1992) Ribosome inactivating proteins from plants: present status and future prospects, Biotechnology 10, 405–412 31 Roberts, W.K. and Selitrennikoff, C.P. (1986) Isolation and characterization of two antifungal proteins from barley, Biochim. Biophys. Acta 880, 161–170 32 Gatehouse, A.M.R. et al. (1990) Effects of ribosome inactivating proteins on insect development – differences between Lepidoptera and Coleoptera, Entomol. Exp. Appl. 54, 43–51 33 Kubo, S. et al. (1990) A potent plant virus inhibitor found in Mirabilis jalapa L., Ann. Phytopathol. Soc. Japan 56, 481–487 34 Vivanco, J.M., Savary, B.J. and Flores, H.E. (1999) Characterization of two novel type I ribosome inactivating proteins from the storage roots of the Andean crop species Mirabilis expansa (Ruiz & Pavon), Plant Physiol. 119, 1447–1456 35 Savary, B.J., Flores, H.E. and Hill, J.J. (1997) Isolation of a class III chitinase produced in root cultures of Tricosanthes kirilowii and related species, Plant Physiol. Biochem. 35, 543–551 36 Wongsamuth, R. and Doran, P.M. (1997) Hairy root as an expression system for production of antibodies, in Hairy Roots Culture and Applications (Doran, P.M., ed.), pp. 89–97, Harwood Academic Press 37 Tanaka, N. and Matsumoto, T. (1993) Regenerants from Ajuga hairy roots with high productivity of 20-hydroxyecdysone, Plant Cell Rep. 13, 87–90
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trends in plant science reviews 38 Babakov, A.V. et al. (1995) Culture of transformed horseradish roots as a source of fusicoccin-like ligands, J. Plant Growth Regul. 14, 163–167 39 Qin, M.B. et al. (1994) Induction of hairy root from Artemisia annua with Agrobacterium rhizogenes and its culture in vitro, Acta Bot. Sinica 36 (Suppl.), 165–170 40 Trypsteen, M. et al. (1991) Agrobacterium rhizogenes-mediated transformation of Echinaceae purpurea, Plant Cell Rep. 10, 85–89 41 Yamanaka, M. et al. (1996) Polyacetylene glucosides in hairy root cultures of Lobelia cardinalis, Phytochemistry 41, 183–185 42 Benjamin, B.D., Roja, G. and Heble, M.R. (1994) Alkaloid synthesis by root cultures of Rauwolfia serpentina transformend with Agrobacterium rhizogenes, Phytochemistry 35, 381–383 43 Sato, K. et al. (1991) Anthraquinone production by transformed root cultures of Rubia tinctorum: influence of phytohormones and sucrose concentration, Phytochemistry 30, 1507–1510 44 Hu, Z-B. and Alfermann, A.W. (1993) Diterpenoid production in hairy root cultures of Salvia miltiorrhiza, Phytochemistry 32, 699–703 45 Delbecque, J.P. et al. (1995) In vitro incorporation of radiolabelled cholesterol and mevalonic acid into ecdysteroid by hairy root cultures of a plant, Serratula tictoria, Eur. J. Entomol. 92, 301–307
46 Takeda, T. et al. (1994) Bryonolic acid production in hairy roots of Trichosanthes kirilowii Max. var. japonica Kitam. transformed with Agrobacterium rhizogenes and its cytotoxic activity, Chem. Pharm. Bull. (Tokyo) 42, 730–732 47 Gränicher, F. et al. (1995) An iridoid diester from Valeriana officinalis var. sambucifolia hairy roots, Phytochemistry 38, 103–105
Hector E. Flores* and Jorge M. Vivanco are at the Dept of Plant Pathology and Life Sciences Consortium, 315 Wartik Building, The Pennsylvania State University, University Park, PA 16802, USA; Victor M. Loyola-Vargas is at the Centro de Investigacion Cientifica de Yucatan, Merida, Mexico (tel 152 99 81 3961; fax 152 99 81 3900; e-mail [email protected]).
*Author for correspondence (tel 11 814 865 2955; fax 11 814 863 7217; e-mail [email protected]).
Recent advances in the transformation of plants Genevi•ve Hansen and Martha S. Wright Plant transformation technology has become a versatile platform for cultivar improvement as well as for studying gene function in plants. This success represents the culmination of many years of effort in tissue culture improvement, in transformation techniques and in genetic engineering. The next challenge is to develop technology that minimizes or eliminates the tissue culture steps, and provides predictable transgene expression.
G
enetic engineering has opened new avenues to modify crops, and provided new solutions to solve specific needs1. In the future, the proportion of acreage planted with transgenic crops, and the range of transgenic crops, is sure to increase. For example, in the USA, the proportion of acreage planted with commercial transgenic cotton, soybean and corn was ~25, 18 and 10, respectively, in 1997 (Ref. 2). Forty-eight transgenic crop products had been approved for commercialization in various countries by the end of 1997. The powerful combination of genetic engineering and conventional breeding programs permits useful traits encoded by transgenes to be introduced into commercial crops within an economically viable time frame. There is great potential for genetic manipulation of crops to enhance productivity through increasing resistance to diseases, pests and environmental stress and by qualitatively changing the seed composition. Plant ‘factories’ are also being designed for high volume production of pharmaceuticals, nutraceuticals and other beneficial chemicals. Transgenic plants might become drug-delivery devices, with both HIV and rabies vaccines being synthesized in plants3, and bananas have been engineered to produce edible vaccines4. Moreover, with the establishment and expansion of genomics programs, a much broader range of genes with potential for crop improvement are being identified and, in some cases, tailored and/or re-designed for further enhancement of their properties within specific crops1. This has further intensified the interest in developing efficient plant transformation technologies to be able to concurrently test and capture the value of these genes. 226
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Beyond crop improvement, the ability to engineer transgenic plants is also a powerful and informative means for studying gene function and the regulation of physiological and developmental processes. Transgenic plants are being used as an assay system for the modification of endogenous metabolism or gene inactivation. Advances in tissue culture, combined with improvements in transformation technology, have resulted in increased transformation efficiencies. In recent years, many crops, previously classified as recalcitrant because they were stubbornly resistant to the overtures of genetic engineering, have now been transformed. In many instances, the technology has been pushed even further to introduce the gene of interest directly into elite cultivars5. Tissue culture prerequisite
A tissue culture stage is required in most current transformation protocols to ultimately recover plants. Indeed, it is the totipotency of plant cells that underlies most plant transformation systems. Plants are regenerated from cell culture via two methods, somatic embryogenesis and organogenesis. Both are controlled by plant hormones and other factors added to the culture medium. Some species, such as soybeans6, banana4,7 and sugar beet8,9 can be regenerated via either method so the choice is dependent upon which gives the best yield or the easiest outcome. Somatic embryogenesis is the generation of embryos from somatic tissues, such as embryos, microspores or leaves. Proliferating somatic embryos in liquid culture or on solid medium are suitable targets for transformation because the origin of proliferating
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