Modulation of artemisinin biosynthesis by elicitors, inhibitor, and precursor in hairy root cultures of Artemisia annua L.

References

• Aftab T, Khan MMA, Idrees M, Naeem M, Moinuddin, Hashmi N, Varshney L. 2011. Enhancing the growth, photosynthetic capacity and artemisinin content in Artemisia annua L. by irradiated sodium alginate. Radiat Phys Chem. 80:833–836.
   Web of Science ®Google Scholar
• Aftab T, Khan MMA, Idrees M, Naeem M, Singh M, MaujiRam. 2010. Stimulation of crop productivity, photosynthesis and artemisinin production in Artemisia annua L. by triacontanol and gibberellic acid application, J Plant Interact. 5:273–281. 10.1080/17429141003647137
   Web of Science ®Google Scholar
• Ahlawat S, Saxena P, MaujiRam, Alam P, Nafis T, Mohd A, Abdin MZ. 2012. Influence of Agrobacterium rhizogenes on induction of hairy roots for enhanced production of artemisinin in Artemisia annua L. plants. Afr J Biotechnol. 11:8684–8691.
   Google Scholar
• Alam P, Abdin MZ. 2011. Over-expression of HMG-CoA reductase and amorpha-4,11-diene synthase genes in Artemisia annua L. and its influence on artemisinin content. Plant Cell Rep. 30:1919–1928. 10.1007/s00299-011-1099-6
   PubMed Web of Science ®Google Scholar
• Alam P, Kamaluddin, Khan MA, Mohammad A, Khan R, Abdin MZ. 2014. Enhanced artemisinin accumulation and metabolic profiling of transgenic Artemisia annua L. plants over-expressing by rate-limiting enzymes from isoprenoid pathway. J Plant Interact. 9:655–665. 10.1080/17429145.2014.893030
   Web of Science ®Google Scholar
• Bais HP, Govindaswamy S, Ravishankar GA. 2000. Enhancement of growth and coumarin production in hairy root cultures of Witloof chicory (Cichorium intybus L. cv Lucknow local) under the influence of fungal elicitors. J Biosci Bioeng. 90:648–653.
   PubMed Web of Science ®Google Scholar
• Bais HP, Sudha G, George J, Ravishankar GA. 2001. Influence of exogenous hormones on growth and secondary metabolite production in hairy root cultures of Cichorium intybus L. cv. Lucknow Local. In Vitro Cell Dev Biol Plant. 37:293–299. 10.1007/s11627-001-0052-8
   Web of Science ®Google Scholar
• Baldi A, Dixit VK. 2008. Yield enhancement strategies for artemisinin production by suspension cultures of Artemisia annua. Bioresource Technol. 99:4609–4614. 10.1016/j.biortech.2007.06.061
   PubMed Web of Science ®Google Scholar
• Baldi A, Jain A, Gupta N, Srivastava AK, Bisaria VS. 2008. Co-culture of mycorrhiza-like fungi (Piriformospora indica and Sebacina vermifera) with plant cells of Linum album for enhanced production of podophyllotoxins: a first report. Biotechnol Lett. 30:1671–1677. 10.1007/s10529-008-9736-z
   PubMed Web of Science ®Google Scholar
• Bertea CM, Freije JR, Van der WH, Verstappen FW, Perk L, Marwquez V, Kraker JW, Posthumus MA, Jansen B. 2005. Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med. 71:40–47.
   PubMed Web of Science ®Google Scholar
• Bianchini GM, Stermer BA, Paiva NL. 1996. Induction of early mevalonate pathway enzymes and biosynthesis of end products in potato (Solanum tuberosum) tubers by wounding and elicitation. Phytochemistry. 42:1563–1571. 10.1016/0031-9422(96)00139-2
   Web of Science ®Google Scholar
• Bohm H, Mack G. 2004. Betaxanthin formation and free amino acids in hairy roots of Beta vulgaris var. lutea depending on nutrient medium and blutamate or glutamine feeding. Phytochemistry. 65:1361–1368. 10.1016/j.phytochem.2004.03.008
   PubMed Web of Science ®Google Scholar
• Borrmann S, Szlezák N, Faucher JF, Matsiegui PB, Neubauer R, Binder R, Lell B, Kremsner PG. 2001. Artesunate and praziquantel for the treatment of Schistosoma haematobium infections: a double-blind, randomized, placebo-controlled study. J Infect Dis. 184:1363–1366. 10.1086/324004
   PubMed Web of Science ®Google Scholar
• Bouwmeester HJ, Wallaart TE, Janssen MH, van Loo B, Jansen BJ, Posthumus MA, Schmidt CO, de Kraker JW, Knig WA, Franssen MC, et al. 1999. Amorpha-4,11-diene synthase catalyses the first probable step in artemisinin biosynthesis. Phytochemistry. 52:843–854. 10.1016/S0031-9422(99)00206-X
   PubMed Web of Science ®Google Scholar
• Camilleri C, Jouanin L. 1991. The Tr-DNA region carrying the auxin synthesis genes of the Agrobacterium rhizogenes agropine-type plasmid pRiA4: nucleotide sequence analysis and introduction in to tobacco plants. Mol Plant Microbe In. 4:155–162.
   PubMed Web of Science ®Google Scholar
• Caretto S, Quarta A, Durante M, Nisi R, de Paolis A, Blando F, Mita G. 2011. Methyl jasmonate and miconazole differently affect arteminisin production and gene expression in Artemisia annua suspension cultures. Plant Biol. 13:51–58. 10.1111/j.1438-8677.2009.00306.x
   PubMed Web of Science ®Google Scholar
• Creelman RA, Mullet J. 1997. Biosynthesis and action of jasmonates in plants. Annu Rev Plant Physiol Plant Mol Biol. 48:355–381.
   PubMed Web of Science ®Google Scholar
• Efferth T, Dunstan H, Sauerbrey A, Miyachi H, Chitambar CR. 2001. The anti-malarial artesunate is also active against cancer. Int J Oncol. 18:767–773.
   PubMed Web of Science ®Google Scholar
• Gamborg OL, Miller RA, Ojima K. 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res. 50:151–158. 10.1016/0014-4827(68)90403-5
   PubMed Web of Science ®Google Scholar
• Giri A, Ravindra ST, Dhingra V, Narasu ML. 2001. Influence of different strains of Agrobacterium rhizogenes on induction of hairy roots and artemisinin production in Artemisia annua. Curr Sci. 81:378–382.
   Web of Science ®Google Scholar
• Guillon S, Tremouillaux GJ, Pati PK, Rideau M, Gantet P. 2006. Hairy root research: recent scenario and exciting prospects - Commentary. Curr Opin Plant Biol. 9:341–346. 10.1016/j.pbi.2006.03.008
   PubMed Web of Science ®Google Scholar
• Gundlach H, Muller MJ, Kutchan TM, Zenk MH. 1992. Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proc Natl Acad Sci. 89:2389–2393. 10.1073/pnas.89.6.2389
   PubMed Web of Science ®Google Scholar
• Hamill JD, Lidgett JA. 1997. Hairy root cultures – Opportunities and key protocols for studies in metabolic engineering. In: Doran PM, editor. Hairy roots-culture and applications. Amsterdam (The Netherlands): Harwood Academic Publishers; p. 1–31.
   Google Scholar
• Haynes RK. 2006. From artemisinin to new artemisinin antimalarials: biosynthesis, extraction, old and new derivatives, stereochemistry and medicinal chemistry requirements. Curr Top Med Chem. 6:509–537.
   PubMed Web of Science ®Google Scholar
• Jaleel CA, Manivannan P, Wahid A, Farooq M, Somasundaram R, Panneerselvam R. 2009. Drought stress in plants: A review on morphological characteristics and pigments composition. Int J Agr Biol. 11:100–105.
   Web of Science ®Google Scholar
• Kaefer E. 1977. Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations. Adv Genet. 19:33–131.
   PubMedGoogle Scholar
• Kang SM, Min JY, Kim YD, Park DJ, Jung HN, Karigar CS, Ha YL, Kim SW, Choi MS. 2006. Effect of supplementing terpenoid biosynthetic precursors on the accumulation of bilobalide and ginkgolides in Ginkgo biloba cell cultures. J Biotechnol. 123:85–92. 10.1016/j.jbiotec.2005.10.021
   PubMed Web of Science ®Google Scholar
• Ketchum REB, Gibson DM, Croteau RB, Shuler ML. 1999. The kinetics of taxoid accumulation in cell suspension culture of taxus following elicitation with methyl jasmonate. Biotechnol Bioeng. 62:97–105. 10.1002/(SICI)1097-0290(19990105)62:1%3C97::AID-BIT11%3E3.0.CO;2-C
   PubMed Web of Science ®Google Scholar
• Kim Y, Wyslouzil BE, Weathers PJ. 2002. Secondary metabolism of hairy root cultures in bioreactors. In Vitro Cell Dev Biol Plant. 38:1–10. 10.1079/IVP2001243
   Web of Science ®Google Scholar
• Kiong ALP, Mahmood M, Fadzillah NM, Daud SK. 2005. Effects of precursor supplementation on the production of triterpenes by Centella asiatica Callus Cultures. Pakistan J Biol Sci. 8:1160–1169. 10.3923/pjbs.2005.1160.1169
   Google Scholar
• Kudakasseril GJ, Lam L, Staba EJ. 1987. Effect of sterol inhibitors on the incorporation of 14C-isopentenyl pyrophosphate into artemisinin by a cell-free system from Artemisia annua tissue cultures and plants. Planta Med. 53:280–284. 10.1055/s-2006-962706
   PubMed Web of Science ®Google Scholar
• Kumar V, Rajauria G, Sahai V, Bisaria VS. 2012. Culture filtrate of root endophytic fungus Piriformospora indica promotes the growth and lignan production of Linum album hairy root cultures. Process Biochem. 47:901–907. 10.1016/j.procbio.2011.06.012
   Web of Science ®Google Scholar
• Lacroix B, Tzfira T, Vainstein A, Citovsky V. 2006. A case of promiscuity: Agrobacterium's endless hunt for new partners. Trends Genet. 22:29–37. 10.1016/j.tig.2005.10.004
   PubMed Web of Science ®Google Scholar
• Liu C, Wang Y, Xu X, Ouyang F, Ye H, Li G. 1999. Improvement of artemisinin accumulation in hairy root cultures of Artemisia annua L by fungal elicitor. Bioproc Biosyst Eng. 20:161–164.
   Web of Science ®Google Scholar
• Liu C, Zhao Y, Wang Y. 2006. Artemisinin: current state and perspectives for biotechnological production of an antimalarial drug. Appl Microbiol Biotechnol. 72:11–20. 10.1007/s00253-006-0452-0
   PubMed Web of Science ®Google Scholar
• Liu CZ, Guo C, Wang YC, Ouyang F. 2002. Effect of light irradiation on hairy root growth and artemisinin biosynthesis of Artemisia annua L. Process Biochem. 38:581–585. 10.1016/S0032-9592(02)00165-6
   Web of Science ®Google Scholar
• Liu CZ, Wang YC, Ouyang F, Ye HC, Li GF. 1997. Production of artemisinin by hairy root cultures of Artemisia annua L. Biotechnol Lett. 19:927–930. 10.1023/A:1018362309677
   Web of Science ®Google Scholar
• Liu S, Tian N, Li J, Huang J, Liu Z. 2009. Isolation and identification of novel genes involved in artemisinin production from flowers of Artemisia annua using suppression subtractive hybridization and metabolite analysis. Planta Med. 14:1542–1547. 10.1055/s-0029-1185809
   Web of Science ®Google Scholar
• Luo XD, Shen CC. 1987. The chemistry, pharmacology, and clinical applications of qinghaosu (Artemisinin) and its derivatives. Med Res Rev. 7:29–52. 10.1002/med.2610070103
   PubMed Web of Science ®Google Scholar
• MaujiRam, Khan MA, Jha P, Khan S, Kiran U, Ahmad MM, Javed S, Abdin MZ. 2010. HMG-CoA reductase limits artemisinin biosynthesis and accumulation in Artemisia annua L. plants. Acta Physiol Plant. 32:859–866.
   Web of Science ®Google Scholar
• Miziorko HM. 2011. Enzymes of the mevalonate pathway of isoprenoid biosynthesis. Arch Biochem Biophys. 505:131–143. 10.1016/j.abb.2010.09.028
   PubMed Web of Science ®Google Scholar
• Murashige T, Skoog F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Plant Physiol. 15:473–497. 10.1111/j.1399-3054.1962.tb08052.x
   Web of Science ®Google Scholar
• Murphy JG, Rafferty SM, Cassells AC. 2000. Stimulation of wild strawberry (Fragaria vesca) arbuscular mycorrhizas by addition of shellfish waste to the growth substrate: interaction between mycorrhization, substrate amendment and susceptibility to red core (Phytophthora fragariae). Appl Soil Ecol. 15:153–158. 10.1016/S0929-1393(00)00091-3
   Web of Science ®Google Scholar
• Namdeo A, Patil S, Fulzele DP. 2002. Influence of fungal elicitors on production of ajmalicine by cell cultures of Catharanthus roseus. Biotechnol Prog. 18:159–162. 10.1021/bp0101280
   PubMed Web of Science ®Google Scholar
• Oelmuller R, Sherameti I, Tripathi S, Varma A. 2009. Piriformospora indica, a cultivable root endophyte with multiple biotechnological applications. Symbiosis. 49:1–17. 10.1007/s13199-009-0009-y
   Web of Science ®Google Scholar
• Patra N, Srivastava AK, Sharma S. 2013. Study of various factors for enhancement of artemisinin in Artemisia annua hairy roots. Int J Chem Eng App. 4:157–160.
   Google Scholar
• Pauwels L, Morreel K, De Witte E, Lammertyn F, Van Montagu M, Boerjan W, Inze D, Goossens A. 2008. Mapping methyl jasmonate-mediated transcriptional reprogramming of metabolism and cell cycle progression in cultured Arabidopsis cells. Proc Natl Acad Sci. 105:1380–1385. 10.1073/pnas.0711203105
   PubMed Web of Science ®Google Scholar
• Peebles CA, Gibson SI, Shanks JV, San KY. 2007. Long-term maintenance of a transgenic Catharanthus roseus hairy root line. Biotechnol Prog. 23:1517–1518. 10.1021/bp0702166
   PubMed Web of Science ®Google Scholar
• Pozo M, Cordier C, Dumas-Gautod E, Gianinazzi SJ, Barea JM, Azcón-Aguilar C. 2002. Localized versus systemic effect of arbuscular mycorrhizal fungi on defence responses to Phytophthora infection in tomato plants. J Exp Bot. 53:525–534. 10.1093/jexbot/53.368.525
   PubMed Web of Science ®Google Scholar
• Putalun W, Luealon W, De-Eknamkul W, Tanaka H, Shoyama Y. 2007. Improvement of artemisinin production by chitosan in hairy root cultures of Artemisia annua L. Biotech Lett. 29:1143–1146. 10.1007/s10529-007-9368-8
   PubMed Web of Science ®Google Scholar
• Rao SR, Ravishankar GA. 2002. Plant cell cultures: chemical factories of secondary metabolites. Biotechnol Adv. 20:101–153. 10.1016/S0734-9750(02)00007-1
   PubMed Web of Science ®Google Scholar
• Rijhwani S, Shanks JV. 1998. Effect of elicitor dosage and exposure time on biosynthesis of indole alkaloids by Catharanthus roseus hairy root cultures. Biotechnol Prog. 14:442–449. 10.1021/bp980029v
   PubMed Web of Science ®Google Scholar
• Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL., Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, et al. 2006. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature. 440:940–943. 10.1038/nature04640
   PubMed Web of Science ®Google Scholar
• Robins RJ. 1998. The application of root cultures to problems of biological chemistry. Nat Prod Rep. 15:549–570. 10.1039/a815549y
   Web of Science ®Google Scholar
• Roewer IA, Cloutier N, Nessler CL, Luca V. 1992. Transient induction of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) genes in cell suspension cultures of Catharanthus roseus. Plant Cell Rep. 11:86–89. 10.1007/BF00235259
   PubMed Web of Science ®Google Scholar
• Romero MR, Efferth T, Serrano MA, Castaño B, Macias RL, Briz O, Marin JJ. 2005. Effect of artemisinin/artesunate as inhibitors of hepatitis B virus production in an “in vitro” system. Antiviral Res. 68:75–83. 10.1016/j.antiviral.2005.07.005
   PubMed Web of Science ®Google Scholar
• Sanz MK, Hernandez XE, Tonn CE, Guerreio E. 2000. Enhancement of tessaric acid production in Tessaria absinthioides cell suspension cultures. Plant Cell Rep. 19:821–824. 10.1007/s002990000192
   PubMed Web of Science ®Google Scholar
• Savitha BC, Thimmaraju R, Bhagyalakshmi N, Ravishankar GA. 2006. Different biotic and abiotic elicitors influence betalain production in hairy root cultures of Beta vulgaris in shake-flask and bioreactor. Process Biochem. 41:50–60. 10.1016/j.procbio.2005.03.071
   Web of Science ®Google Scholar
• Schaller H, Grausem B, Benveniste P, Chye ML, Tan CT, Song YH, Chua NH. 1995. Expression of the Hevea brasiliensis (H.B.K.) müll. Arg. 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 in tobacco results in sterol overproduction. Plant Physiol. 109:761–770.
   PubMed Web of Science ®Google Scholar
• Schramek N, Wang H, Romisch-Margl W, Keil B, Radykewicz T, Winzenhörlein B, Beerhues L, Bacher A, Rohdich F, Gershenzon J, et al. 2010. Artemisinin biosynthesis in growing plants of Artemisia annua. A 13CO2 study. Phytochemistry. 71:179–187. 10.1016/j.phytochem.2009.10.015
   PubMed Web of Science ®Google Scholar
• Sen R, Bandyopadhyay S, Dutta A, Mandal G, Ganguly S, Saha P, Chatterjee M. 2007. Artemisinin triggers induction of cell-cycle arrest and apoptosis in Leishmania dovani promastigotes. J Med Microbiol. 56:1213–1218. 10.1099/jmm.0.47364-0
   PubMed Web of Science ®Google Scholar
• Sharan M, Taguchi G, Gonda K, Jouke T, Shimosaka M, Hayashida N. 1998. Effects of methyl jasmonate and elicitor on the activation of phenylalanine ammonia-lyase and the accumulation of scopoletin and scopolin in tobacco cell cultures. Plant Sci. 132:13–19. 10.1016/S0168-9452(97)00260-4
   Web of Science ®Google Scholar
• Sharma G, Agrawal V. 2013. Marked enhancement in the artemisinin content and biomass productivity in Artemisia annua L. shoots co-cultivated with Piriformospora indica. World J Microbiol Biotechnol. 29:1133–1138. 10.1007/s11274-013-1263-y
   PubMed Web of Science ®Google Scholar
• Singh A, Rajpal K, Singh M, Kharkwal AC, Arora M, Varma A. 2013. Mass cultivation of Piriformospora indica and Sebacina species. In: Varma A, Kost G, Oelmüller R, editors, Piriformospora indica, Sebacinales and their biotechnological applications. Germany: Springer-Verlag; p. 377–392.
   Google Scholar
• Srivastava N, Akhila A. 2011. Biosynthesis of artemisinin––revisited. J Plant Interact. 6:265–273. 10.1080/17429145.2011.570869
   Web of Science ®Google Scholar
• Suresh B, Thimmaraju R, Bhagyalakshmi N, Ravishankar GA. 2004. Polyamines and methyl jasmonate-influenced enhancement of betalain production in hairy root cultures of Beta vulgaris grown in a bubble column reactor and studies on efflux of pigments. Process Biochem. 39:2091–2096. 10.1016/j.procbio.2003.10.009
   Web of Science ®Google Scholar
• Tang K, Shen Q, Yan T, Fu X. 2014. Transgenic approach to increase artemisinin content in Artemisia annua L. Plant Cell Rep. 33:605–615. 10.1007/s00299-014-1566-y
   PubMed Web of Science ®Google Scholar
• Tenea GN, Calin A, Gavrila L, Cucu N. 2008. Manipulation of root biomass and biosynthetic potential of Glycyrrhiza glabra L. plants by Agrobacterium rhizogenes mediated transformation. Rom Biotechnol Lett. 13:3922–3932.
   Web of Science ®Google Scholar
• Teoh KH, Polichuk DR, Reed DW, Nowak G, Covello PS. 2006. Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin. FEBS Lett. 580:1411–1416. 10.1016/j.febslet.2006.01.065
   PubMed Web of Science ®Google Scholar
• Towler MJ, Weathers PJ. 2007. Evidence of artemisinin production from IPP stemming from both the mevalonate and the nonmevalonate pathways. Plant Cell Rep. 26:2129–2136. 10.1007/s00299-007-0420-x
   PubMed Web of Science ®Google Scholar
• Varma A, Singh A, Sudha, Sahay NS, Sharma J, Roy A, Kumari M, Rana D, Thakran S, Deka D, et al. 2001. Piriformospora indica: an axenically culturable mycorrhiza-like endosymbiotic fungus. In: Hock B, editor. Fungal associations. Berlin, Heidelberg, New York: Springer; p. 123–150.
   Google Scholar
• Varma A, Verma S, Sudha Sahay N, Bütehorn B, Franken P. 1999. Piriformospora indica, a cultivable plant-growth-promoting root endophyte. Appl Environ Microbiol. 65:2741–2744.
   PubMed Web of Science ®Google Scholar
• Waller F, Achatz B, Baltruschat H, Fodor J, Becker K, Fischer M, Heier T, Hückelhoven R, Neumann C, von Wettstein D, et al. 2005. The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance and higher yield. Proc Natl Acad Sci USA 102:13386–13391. 10.1073/pnas.0504423102
   PubMed Web of Science ®Google Scholar
• Wang H, Ma C, Li Z, Ma L, Wang H, Ye H, Xu G, Liu B. 2009. Effects of exogenous methyl jasmonate on artemisinin biosynthesis and secondary metabolites in Artemisia annua L. Ind Crop Prod. 31:214–218. 10.1016/j.indcrop.2009.10.008
   Web of Science ®Google Scholar
• Wang JW, Xia ZH, Tan RX. 2002. Elicitation on artemisinin biosynthesis in Artemisia annua hairy roots by the oligosaccharide extract from the endophytic Colletotrichum sp. B501. Acta Bot Sinica. 44:1233–1238.
   Google Scholar
• Wang JW, Zheng LP, Tan RX. 2006. The preparation of an elicitor from a fungal endophyte to enhance artemisinin production in hairy root cultures of Artemisia annua L. Sheng Wu Gong Cheng Xue Bao. 22:829–834.
   PubMedGoogle Scholar
• White NJ. 2008. Qinghaosu (artemisinin): the price of success. Science. 320:330–334. 10.1126/science.1155165
   PubMed Web of Science ®Google Scholar
• Xie D, Ye H, Li G, Guo Z, Zou Z, Guo Z. 2001. Selection of hairy root clones of Artemisia annua L. for artemisinin production. Isr J Plant Sci. 49:129–134. 10.1560/N11N-6BLG-ER7C-XKCT
   Web of Science ®Google Scholar
• Zarn JA, Bru schweiler BJ, Schlatter JR. 2003. Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14-ademethylase and aromatase. Environ Health Persp. 111:255–261. 10.1289/ehp.5785
   PubMed Web of Science ®Google Scholar
• Zhao SS, Zeng MY. 1986. Determination of Qinghaosu in Artemisia annua L. by high performance liquid chromatography. Chin J Pharm Ana. 6:3–5.
   Google Scholar
• Zheng LP, Guo YT, Wang JW, Tan RX. 2008. Nitric oxide potentiates oligosaccharide-induced artemisinin production in Artemisia annua hairy roots. J Intg Plant Biol. 50:49–55.
   PubMed Web of Science ®Google Scholar