Skip to main content
Log in

Association with arbuscular mycorrhizal fungi influences alkaloid synthesis and accumulation in Catharanthus roseus and Nicotiana tabacum plants

  • Original Paper
  • Published:
Acta Physiologiae Plantarum Aims and scope Submit manuscript

Abstract

Frequently disregarded, plant associations with arbuscular mycorrhizal fungi (AMF) can influence plant specialized metabolism with important ecological and/or economic implications. In this study, we report on both the influence of mycorrhization on the content of a wide range of alkaloids and differential gene expression of some enzymes involved in alkaloid biosynthetic pathways in the leaves and roots of Catharanthus roseus and Nicotiana tabacum plants. These plants were divided into several treatments: mycorrhizal, inoculated with AMF; non-AMF inoculated plants; and non-AMF inoculated plants with an extra supply of phosphorus. The contents of vindoline, vinblastine, vincristine, catharanthine, ajmalicine and serpentine in C. roseus and of nicotine, anabasine and nornicotine in N. tabacum tobacco plants were determined. Mycorrhizal inoculation increased ajmalicine and serpentine contents in C. roseus roots suggesting that mycorrhization had a greater influence on the accumulation of alkaloids in roots than it did in shoots. The youngest leaves of mycorrhizal C. roseus plants showed lower transcript levels of the genes analysed; however, in older leaves, the expression levels were higher when compared with the leaves of non-mycorrhizal plants. In the case of tobacco, higher leaf to root ratios for nicotine and anabasine were found in plants with a mycorrhizal association. Our results showed that mycorrhization changed the alkaloid content and expression pattern of the genes analysed in both species; however, differences were found between the roots and shoots. In nature, such changes may have a direct influence on the interactions between plants and insects (herbivory) and pathogens. These interactions must be studied further to reveal the ecological influence mycorrhizae may have on chemical defences in a broader sense.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Explore related subjects

Discover the latest articles and news from researchers in related subjects, suggested using machine learning.

References

  • Abu-Zeyad R, Khan AG, Khoo C (1999) Occurrence of arbuscular mycorrhiza in Castanospermum australe A. Cunn. & C. Fraser and effects on growth and production of castanospermine. Mycorrhiza 9:111–117

    CAS  Google Scholar 

  • Asensio D, Rapparini F, Peñuelas J (2012) AM fungi root colonization increases the production of essential isoprenoids vs. nonessential isoprenoids especially under drought stress conditions or after jasmonic acid application. Phytochemistry 77:149–161

    Article  PubMed  CAS  Google Scholar 

  • Baldwin IT, Schmelz EA (1996) Immunological” memory” in the induced accumulation of nicotine in wild tobacco. Ecology 77:236–246

    Article  Google Scholar 

  • Baldwin IT, Schmelz EA, Ohnmeiss TE (1994) Wound-induced changes in root and shoot jasmonic acid pools correlate with induced nicotine synthesis in Nicotiana sylvestris spegazzini and comes. J Chem Ecol 20:2139–2157

    Article  CAS  Google Scholar 

  • Balsevich J., Bishop G (1989) Distribution of catharanthine, vindoline and 3′, 4′,-anhydrovinblastine in the aerial parts of some Catharanthus roseus plants and the significance thereof in relation to alkaloid production in cultured cells. In: Kurz WGW (ed) Primary and secondary metabolism of plant cell cultures. New York, Springer, pp 149–153

  • Biastoff S, Brandt W, Drager B (2009) Putrescine N-methyltransferase-the start for alkaloids. Phytochemistry 70:1708–1718

    Article  PubMed  CAS  Google Scholar 

  • Blom T, Sierra M, Vliet T, Franke-van Dijk M, Koning P, Iren F, Verpoorte R, Libbenga K (1991) Uptake and accumulation of ajmalicine into isolated vacuoles of cultured cells of Catharanthus roseus (L.) G. Don. and its conversion into serpentine. Planta 183:170–177

    Article  CAS  Google Scholar 

  • Brundrett MC (2002) Coevolution of roots and mycorrhizas of land plants. New Phytol 154:275–304

    Article  Google Scholar 

  • Burlat V, Oudin A, Courtois M, Rideau M, St-Pierre B (2004) Co-expression of three MEP pathway genes and geraniol 10-hydroxylase in internal phloem parenchyma of Catharanthus roseus implicates multicellular translocation of intermediates during the biosynthesis of monoterpene indole alkaloids and isoprenoid-derived primary metabolites. Plant J 38:131–141

    Article  PubMed  CAS  Google Scholar 

  • Cartmill AD, Valdez-Aguilar LA, Bryan DL, Alarcón A (2008) Arbuscular mycorrhizal fungi enhance tolerance of vinca to high alkalinity in irrigation water. Scientia Horticulturae 115:275–284

    Google Scholar 

  • Chakrabarti M, Bowen SW, Coleman NP, Meekins KM, Dewey RE, Siminszky B (2008) CYP82E4-mediated nicotine to nornicotine conversion in tobacco is regulated by a senescence-specific signaling pathway. Plant Mol Biol 66:415–427

    Article  PubMed  CAS  Google Scholar 

  • Copetta A, Lingua G, Berta G (2006) Effects of three AM fungi on growth, distribution of glandular hairs, and essential oil production in Ocimum basilicum L. var Genovese. Mycorrhiza 16:485–494

    Article  PubMed  CAS  Google Scholar 

  • Costa M, Hilliou F, Duarte P, Pereira L, Almeida I, Leech M, Memelink J, Barcelo A, Sottomayor M (2008) Molecular cloning and characterization of a vacuolar class III peroxidase involved in the metabolism of anticancer alkaloids in Catharanthus roseus. Plant Physiol 146:403

    Article  PubMed  CAS  Google Scholar 

  • Dawson RF (1942) Accumulation of nicotine in reciprocal grafts of tomato and tobacco. Am J Bot 29:66–71

    Article  CAS  Google Scholar 

  • De la Rosa-Mera CJ, Ferrera-Cerrato R, Alarcón A, de Jesús Sánchez-Colín M, Muñoz-Muñiz OD (2011) Arbuscular mycorrhizal fungi and potassium bicarbonate enhance the foliar content of the vinblastine alkaloid in Catharanthus roseus. Plant Soil 1–10

  • DeBoer KD, Lye JC, Aitken CD, Su AKK, Hamill JD (2009) The A622 gene in Nicotiana glauca (tree tobacco): evidence for a functional role in pyridine alkaloid synthesis. Plant Mol Biol 69:299–312

    Article  PubMed  CAS  Google Scholar 

  • DiCosmo F, Misawa M (1995) Plant cell and tissue culture: alternatives for metabolite production. Biotechnol Adv 13:425–453

    Article  PubMed  CAS  Google Scholar 

  • El-Sayed M, Verpoorte R (2007) Catharanthus terpenoid indole alkaloids: biosynthesis and regulation. Phytochem Rev 6:277–305

    Article  CAS  Google Scholar 

  • Erb M, Ton J, Degenhardt J, Turlings TCJ (2008) Interactions between arthropod-induced aboveground and belowground defenses in plants. Plant Physiol 146:867–874

    Article  PubMed  CAS  Google Scholar 

  • Fester T, Maier W, Strack D (1999) Accumulation of secondary compounds in barley and wheat roots in response to inoculation with an arbuscular mycorrhizal fungus and co-inoculation with rhizosphere bacteria. Mycorrhiza 8:241–246

    Article  CAS  Google Scholar 

  • Friesen J, Leete E (1990) Nicotine synthase: an enzyme from Nicotiana species which catalyzes the formation of (S)-nicotine from nicotinic acid and 1-methyl-D’-pyrrolinium chloride. Tetrahedron Lett 31:6295–6298

    Article  CAS  Google Scholar 

  • Gange AC, Ayres RL (1999) On the relation between arbuscular mycorrhizal colonization and plant ‘benefit’. Oikos 615–621

  • Gavilano LB, Siminszky B (2007) Isolation and characterization of the cytochrome P450 gene CYP82E5v2 that mediates nicotine to nornicotine conversion in the green leaves of tobacco. Plant Cell Physiol 48:1567–1574

    Article  PubMed  CAS  Google Scholar 

  • Gavilano LB, Coleman NP, Burnley LE, Bowman ML, Kalengamaliro NE, Hayes A, Bush L, Siminszky B (2006) Genetic engineering of Nicotiana tabacum for reduced nornicotine content. J Agric Food Chem 54:9071–9078

    Article  PubMed  CAS  Google Scholar 

  • Giovannetti M, Mosse B (1980) Evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytol 84:489–500

    Article  Google Scholar 

  • Hanounik S, Osborne W (1977) The relationships between population density of Meloidogyne incognita and nicotine content of tobacco. Nematologica 23:147–152

    Article  CAS  Google Scholar 

  • Hartley SE, Gange AC (2009) Impacts of plant symbiotic fungi on insect herbivores: mutualism in a multitrophic context. Ann Rev Entomol 54:323–342

    Article  CAS  Google Scholar 

  • Hause B, Maier W, Miersch O, Kramell R, Strack D (2002) Induction of jasmonate biosynthesis in arbuscular mycorrhizal barley roots. Plant Physiol 130:1213–1220

    Article  PubMed  CAS  Google Scholar 

  • Hause B, Mrosk C, Isayenkov S, Strack D (2007) Jasmonates in arbuscular mycorrhizal interactions. Phytochemistry 68:101–110

    Article  PubMed  CAS  Google Scholar 

  • Hol WHG (2011) The effect of nutrients on pyrrolizidine alkaloids in Senecio plants and their interactions with herbivores and pathogens. Phytochem Rev 10:119–126

    Article  PubMed  CAS  Google Scholar 

  • Hughes E, Hong S, Gibson S, Shanks J, San K. (2004) Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels. Biotechnol Bioeng 86

  • Janouskova M, Vosatka M, Rossi L, Lugon-Moulin N (2007) Effects of arbuscular mycorrhizal inoculation on cadmium accumulation by different tobacco (Nicotiana tabacum L.) types. Appl Soil Ecol 35:502–510

    Article  Google Scholar 

  • Kapoor R, Chaudhary V, Bhatnagar A (2007) Effects of arbuscular mycorrhiza and phosphorus application on artemisinin concentration in Artemisia annua L. Mycorrhiza 17:581–587

    Article  PubMed  CAS  Google Scholar 

  • Karthikeyan B, Jaleel C, Changxing Z, Joe M, Srimannarayan J, Deiveekasundaram M (2008) The effect of AM fungi and phosphorous level on the biomass yield and ajmalicine production in Catharanthus roseus. Eur Asian J Biosci 2:26–33

    Google Scholar 

  • Kempel A, Schmidt A, Brandl R, Schädler M (2010) Support from the underground: induced plant resistance depends on arbuscular mycorrhizal fungi. Funct Ecol 24:293–300

    Article  Google Scholar 

  • Khaosaad T, Krenn L, Medjakovic S, Ranner A, Lössl A, Nell M, Jungbauer A, Vierheilig H (2008) Effect of mycorrhization on the isoflavone content and the phytoestrogen activity of red clover. J Plant Physiol 165:1161–1167

    Article  PubMed  CAS  Google Scholar 

  • Koricheva J (1999) Interpreting phenotypic variation in plant allelochemistry: problems with the use of concentrations. Oecologia 119:467–473

    Article  Google Scholar 

  • Lata B (2007) Cultivation, mineral nutrition and seed production of Catharanthus roseus (L.) G. Don in the temperate climate zone. Phytochem Rev 6:403–411

    Article  CAS  Google Scholar 

  • Leete E (1983) Biosynthesis and metabolism of the tobacco alkaloids. Alkaloids Chem Biol Perspect 1:85–151

    Google Scholar 

  • Lewis RS, Bowen SW, Keogh MR, Dewey RE (2010) Three nicotine demethylase genes mediate nornicotine biosynthesis in Nicotiana tabacum L.: functional characterization of the CYP82E10 gene. Phytochemistry 71:1988–1998

    Article  PubMed  CAS  Google Scholar 

  • Liscombe DK, Usera AR, O′Connor SE (2010) Homolog of tocopherol C methyltransferases catalyzes N methylation in anticancer alkaloid biosynthesis. Proc Natl Acad Sci USA 107:18793–18798

    Article  PubMed  CAS  Google Scholar 

  • Liu J, Maldonado-Mendoza I, Lopez-Meyer M, Cheung F, Town CD, Harrison MJ (2007) Arbuscular mycorrhizal symbiosis is accompanied by local and systemic alterations in gene expression and an increase in disease resistance in the shoots. Plant J 50:529–544

    Article  PubMed  CAS  Google Scholar 

  • López-Ráez J, Verhage A, Fernandez I, Garcia J, Azcón-Aguilar C, Flors V, Pozo M (2010) Hormonal and transcriptional profiles highlight common and differential host responses to arbuscular mycorrhizal fungi and the regulation of the oxylipin pathway. J Exp Bot 61:2589–2601

    Article  PubMed  Google Scholar 

  • Luijendijk TJC, van der Meijden E, Verpoorte R (1996) Involvement of strictosidine as a defensive chemical in Catharanthus roseus. J Chem Ecol 22:1355–1366

    Article  CAS  Google Scholar 

  • Mahroug S, Burlat V, St-Pierre B (2007) Cellular and sub-cellular organisation of the monoterpenoid indole alkaloid pathway in Catharanthus roseus. Phytochem Rev 6:363–381

    Article  CAS  Google Scholar 

  • Marschner H, Dell B (1994) Nutrient uptake in mycorrhizal symbiosis. Plant Soil 159:89–102

    CAS  Google Scholar 

  • Mazzafera P (1999) Mineral nutrition and caffeine content in coffee leaves. Bragantia 58:387–391

    Article  CAS  Google Scholar 

  • Moreno P, Heijden R, Verpoorte R (1993) Effect of terpenoid precursor feeding and elicitation on formation of indole alkaloids in cell suspension cultures of Catharanthus roseus. Plant Cell Rep 12:702–705

    Article  CAS  Google Scholar 

  • Morone-Fortunato I, Avato P (2008) Plant development and synthesis of essential oils in micropropagated and mycorrhiza inoculated plants of Origanum vulgare L. ssp. hirtum (Link) Ietswaart. Plant Cell, Tissue Organ Cult 93:139–149

    Article  CAS  Google Scholar 

  • Pan Q, Chen Y, Wang Q, Yuan F, Xing S, Tian Y, Zhao J, Sun X, Tang K (2010) Effect of plant growth regulators on the biosynthesis of vinblastine, vindoline and catharanthine in Catharanthus roseus. Plant Growth Regul 60:133–141

    Article  CAS  Google Scholar 

  • Pasquali G, Goddijn OJM, Waal A, Verpoorte R, Schilperoort RA, Hoge JHC, Memelink J (1992) Coordinated regulation of two indole alkaloid biosynthetic genes from Catharanthus roseus by auxin and elicitors. Plant Mol Biol 18:1121–1131

    Article  PubMed  CAS  Google Scholar 

  • Peebles CAM, Hughes EH, Shanks JV, San KY (2009) Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time. Metab Eng 11:76–86

    Article  PubMed  CAS  Google Scholar 

  • Phillips J, Hayman D (1970) Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans Br Mycol Soc 55:158–160

    Article  Google Scholar 

  • Pozo M, Azcón-Aguilar C (2007) Unraveling mycorrhiza-induced resistance. Curr Opin Plant Biol 10:393–398

    Article  PubMed  CAS  Google Scholar 

  • Ratti N, Verma H, Gautam S (2010) Effect of Glomus species on physiology and biochemistry of Catharanthus roseus. Ind J Microbiol 50:355–360

    Article  Google Scholar 

  • Rischer H, Oresic M, Seppänen-Laakso T, Katajamaa M, Lammertyn F, Ardiles-Diaz W, Van Montagu MCE, Inzé D, Oksman-Caldentey KM, Goossens A (2006) Gene-to-metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells. Proc Natl Acad Sci 103:5614–5619

    Article  PubMed  CAS  Google Scholar 

  • Roepke J, Salim V, Wu M, Thamm AMK, Murata J, Ploss K, Boland W, De Luca V (2010) Vinca drug components accumulate exclusively in leaf exudates of Madagascar periwinkle. Proc Natl Acad Sci USA 107:15287–15292

    Article  PubMed  CAS  Google Scholar 

  • Schmidt GW, Delaney SK (2010) Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress. Mol Genet Genomics 283:233–241

    Article  PubMed  CAS  Google Scholar 

  • Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative Ct method. Nat Protocols 3:1101–1108

    Article  CAS  Google Scholar 

  • Shi Q, Li C, Zhang F (2006) Nicotine synthesis in Nicotiana tabacum L. induced by mechanical wounding is regulated by auxin. J Exp Bot 57:2899–2907

    Article  PubMed  CAS  Google Scholar 

  • Shoji T, Yamada Y, Hashimoto T (2000) Jasmonate induction of putrescine N-methyltransferase genes in the root of Nicotiana sylvestris. Plant Cell Physiol 41:831–839

    Article  PubMed  CAS  Google Scholar 

  • Siminszky B, Gavilano L, Bowen SW, Dewey RE (2005) Conversion of nicotine to nornicotine in Nicotiana tabacum is mediated by CYP82E4, a cytochrome P450 monooxygenase. Proc Natl Acad Sci USA 102:14919

    Article  PubMed  CAS  Google Scholar 

  • Smith S, Read D (2008) Mycorrhizal symbiosis. Academic Press, Amsterdam, p 787

    Google Scholar 

  • Smith SE, Jakobsen I, Grønlund M, Smith FA (2011) Roles of arbuscular mycorrhizas in plant phosphorus nutrition: interactions between pathways of phosphorus uptake in arbuscular mycorrhizal roots have important implications for understanding and manipulating plant phosphorus acquisition. Plant Physiol 156:1050

    Article  PubMed  CAS  Google Scholar 

  • Sottomayor M, Lopes Cardoso I, Pereira L, Ros Barceló A (2004) Peroxidase and the biosynthesis of terpenoid indole alkaloids in the medicinal plant Catharanthus roseus (L.) G. Don. Phytochem Rev 3:159–171

    Article  CAS  Google Scholar 

  • Steppuhn A, Gase K, Krock B, Halitschke R, Baldwin I (2004) Nicotine-defensive function in nature. PLoS Biol 2:e217

    Article  PubMed  Google Scholar 

  • St-Pierre B, Vazquez-Flota FA, De Luca V (1999) Multicellular compartmentation of Catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate. Plant Cell Online 11:887–900

    CAS  Google Scholar 

  • Subhashini D, Krishnamurty V (1995) Influence of vesicular-arbuscular mycorrhiza on phosphorus economy, yield and quality of flue-cured Virginia tobacco in rain-fed Alfisols. In Adholeya A, Singh S (eds) Mycorrhizae: biofertilizers for the future. Proceedings of the third national conference on mycorrhiza (13–15 March 1995). Tata Energy Research Institute, New Delhi, pp 328–330

  • Thurston R, Smith WT, Cooper BP (1966) Alkaloid secretion by trichomes of Nicotiana species and resistance to aphids. Entomol Exp Appl 9:428–432

    Article  CAS  Google Scholar 

  • Tikhomiroff C, Jolicoeur M (2002) Screening of Catharanthus roseus secondary metabolites by high-performance liquid chromatography. J Chromatogr A 955:87–93

    Article  PubMed  CAS  Google Scholar 

  • Toussaint J (2007) Investigating physiological changes in the aerial parts of AM plants: what do we know and where should we be heading? Mycorrhiza 17:349–353

    Article  PubMed  Google Scholar 

  • Toussaint J, Smith F, Smith S (2007) Arbuscular mycorrhizal fungi can induce the production of phytochemicals in sweet basil irrespective of phosphorus nutrition. Mycorrhiza 17:291–297

    Article  PubMed  CAS  Google Scholar 

  • Tyler VE (1988) Medicinal plant research: 1953–1987. Plant Med 54:95–100

    Article  CAS  Google Scholar 

  • Vannette R, Hunter M (2009) Mycorrhizal fungi as mediators of defence against insect pests in agricultural systems. Agric For Entomol 11:351–358

    Article  Google Scholar 

  • Verma P, Mathur AK, Srivastava A, Mathur A (2012) Emerging trends in research on spatial and temporal organization of terpenoid indole alkaloid pathway in Catharanthus roseus: a literature update. Protoplasma 24:255–268

    Article  Google Scholar 

  • Wang CT, Liu H, Gao XS, Zhang HX (2010) Overexpression of G10H and ORCA3 in the hairy roots of Catharanthus roseus improves catharanthine production. Plant Cell Rep 29:887–894

    Article  PubMed  CAS  Google Scholar 

  • Wei S (2008) Methyl jasmonic acid induced expression pattern of terpenoid indole alkaloid pathway genes in Catharanthus roseus seedlings. Plant Growth Regul 61:243–251

    Article  Google Scholar 

  • Wooley SC, Paine TD (2011) Infection by mycorrhizal fungi increases natural enemy abundance on tobacco (Nicotiana rustica). Environ Entomol 40:36–41

    Article  PubMed  Google Scholar 

  • Xu D, Shen Y, Chappell J, Cui M, Nielsen M (2007) Biochemical and molecular characterizations of nicotine demethylase in tobacco. Physiol Plant 129:307–319

    Article  CAS  Google Scholar 

  • Yuan Z, Dai C, Chen L (2007) Review—regulation and accumulation of secondary metabolites in plant–fungus symbiotic system. Afr J Biotechnol 6:1266–1271

    CAS  Google Scholar 

  • Ziegler J, Facchini P (2008) Alkaloid biosynthesis: metabolism and trafficking. Ann Rev Plant Biol 59:735

    Article  CAS  Google Scholar 

  • Zubek S, Blaszkowski J (2009) Medicinal plants as hosts of arbuscular mycorrhizal fungi and dark septate endophytes. Phytochem Rev 8:571–580

    Article  CAS  Google Scholar 

  • Zubek S, Stojakowska A, Anielska T, Turnau K (2010) Arbuscular mycorrhizal fungi alter thymol derivative contents of Inula ensifolia L. Mycorrhiza 20:497–504

    Article  PubMed  CAS  Google Scholar 

  • Zubek S, Mielcarek S, Turnau K (2011) Hypericin and pseudohypericin concentrations of a valuable medicinal plant Hypericum perforatum L. are enhanced by arbuscular mycorrhizal fungi. Mycorrhiza 1–8

Download references

Acknowledgments

The authors thank FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for financial support through a postdoctoral and research fellowship to Andrade SAL and Mazzafera P, respectively.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to S. A. L. Andrade.

Additional information

Communicated by M. H. Walter.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 47 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Andrade, S.A.L., Malik, S., Sawaya, A.C.H.F. et al. Association with arbuscular mycorrhizal fungi influences alkaloid synthesis and accumulation in Catharanthus roseus and Nicotiana tabacum plants. Acta Physiol Plant 35, 867–880 (2013). https://doi.org/10.1007/s11738-012-1130-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11738-012-1130-8

Keywords

Profiles

  1. S. A. L. Andrade