Responses of Transformed Roots of Atropa Belladonna to Treatment

Note Plant Biotechuology, 20 (1), 97- 100 (2003)

Responses of Transformed Roots ofAtropa Belladonna to Treatment with Salicylic Acid and other Phenolic Compounds

Hiroshi HIRANOl, Koichiro SHIMOMURA2~ and Takashi YAMAKAWAI *

ILaboratory ofPlant Biotechnology, Department of GlobalAgricultural Sciences, The University of Tokyo, Yayoi, Bunkyoku, Tokyo 1138657, Japan 2Tsukuba Medicinal Plant Research Station, National Jnstitute of Ilealth S'ciences, Jlachimandai, /'sukuba 305 0843, Japan ' present address: Faculty ofLife Sciences, Toyo University, ltakura. Gunma 374-0193, Japan *Corresponding author E-mail address: [email protected]

Received 27 septembe~ 2002; accepted 15 october 2002

Abstract

Treatment of transformed Atropa belladonna root cultures with high concentrations of salicylic acid (SA) resulted in the production of methylated salicylic acid derivatives but not of glucosylated derivatives. This response was induced by O.1 mM SA whereas treatment with SA at concentraticns higher than I.2 mM caused growth retardation. Nontransformed roots of A. belladonna responded to the addition of SA in almost the same manner as transformed roots, which suggests that this is a natural response inA. belladonna. We could not detect methylated SA in cultures of Nicotiana plambagimfolia and Ilyoscyamus niger. Besides SA, several aromatic compounds were methylated by the transformed roots of A. belladonna, and other responses, such as the conversion of benzoi.c acid to benzyl alcohol, were also observed.

Key words: Atropa belladonna, bioconversion, methylation, methylOmethoxybenzoate, methyl salicylate, methylation, O methyltransferase, sa]icylic acid.

Salicylic acid (SA) is a phenolic compound pro- subsequently MSA was converted to MMB. In this duced and metabolized in the phenylpropanoid paper we report the changes in the concentration of pathway in higher plants, and has several physio- methylated SAS in the medium over time, and the logical functions including acting as the signal for specific substrates which A. belladonna can methy- systemic acquired resistance (Malamy et al, 1990; late. , Metraux et al. 1990). However, excessive is Transformed root cultures incubated in , SA were 200 harmful to plant growth and development (Manthe ml of hormonefree halfstrength Murashige and et al., 1992). In plant cells, SA is detoxified by Skoog (1/2 MS) Iiquid medium containing 3(~/o (w/v) several processes, including glucosylation (Tanaka sucrose in 300 ml conical flasks capped with alu- et al. 1990; Edwards, 1994), methylation (Schulaev foil. (fresh weight) of , minum One gram roots was et al. 1997), and binding with amino aeids (Bourne inoculated flask and subcultured weeks. , per every 2 et al., 1991). Glucosylation of SA is reported in All cultures were maintained on a rotary shaker many plants, and is the most dominant response to (100 strokes per minute) at 25 'C in the dark. SA feeding (Pierpoint, 1994). The concentration of SA in medium was varied to We reported previously that the SA was monome- determine the amount of SA that is required to thylated and dimethylated without any glucosy- induce this response. Sterilized SA solution was lation when transformed roots of Atropa added to 18-dayold root cultures to give a final belladonna M8 were cultured in the presence of concentration ranging from 0.01 to 2.0 mM. SA high concentrations (O.2mM) of SA (Lee et al., derivatives in the culture medium were quantified 2001). While the concentrations of SA decreased by HPLC analysis (Simadzu SCL10Avp, SPD with time, methyl salicylate (MSA) and methyl O MIOAvp). Ten microliters of culturing medium was methoxybenzoate (MMB) were detected 12 and 14 injected into a Tosoh ODS 1_20A column (4.6 mm h after the addition of SA, respectively. Therefore, i.d. x 250 mm) and carried by the mobile solution we presumed that SA was converted to MSA, and [acetonitrile : 10 mM SDS (adjusted to pH 3.4 with 98 la/o H3P04) = 2:3] at 1.2ml min~. The concen- subcultured every month. Sterilized SA solution trations of SA and its derivatives were recorded at was added to 30dayold cultures to give a final 215 nm absorbance. concentration of 0.8 mM. Two compounds were Fig, I shows the production of MSA and MMB found in the medium 5 days after the addition of over time following the addition of SA. SA at a SA, and these were identified as MSA and MMB concentration of at least 0.1 mM SA was required to (Fig. 2) using the HPLC system. These results induce a positive response, and at concentrations of 1.2 1.6 mM SA caused negative effccts to the root, A such as tissue browning and growth retardation. A similar experiment was performed for nontrans- 1; e:l formed roots of A. belladonna, which was estab- ~ g: ~ 1; lished and maintained at Tsukuba Medicinal Plant ~ ~e g S: Research Station (Nakanishi et al. 1998). The eq* ~ , ~ cultures were incubated in 100 ml of 1/2 MS Iiquid ~ ~~ medium containing 3~~/o (w/v) sucrose in 200 ml ~ o (:) conical flasks, and maintained rotatry shaker ~ on a e (75 strokes per minute) at 25 ~C in the dark. Roots (3 ~ g fresh weight) were inoculated in each flask and 6.0 8.0 ro.o I~.O 14.0 A Retention time (miu) lO B ~: ~:~ ~ 1~~ 5 v_

I~*.~

~) ~:~ $~ ~~; ~~{ ~ o ~~~ c~O ~i O IOO 200 300 400 500 600 Colnpound A Time (h)

B Methyl-O-methoxybenzoate 20 200 250 aoo 3~;o ~_ Wavelength (nln) ~:~ ~ Ns:!

~;~ lo X ,~e

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e~e o f~ ;~ eEl J~ ~~ o e ~ ~~e O 100 2CO 300 400 500 600 700 800 900 Compound B Time (h) Fig. I Time courscs of methyl saiicylate (A) and methylOmethoxybenzoate production (B) in Methyl Salicylate A. belladonna transformed root culture medium 200 2so 300 3~;o treated with O.O1, O.1, O.4, 0.8, and 1.2 mM SA. Wavelength (nm) The transformed roct culture treated with 1.2 mM Fig. 2 HPLC profile of the culture medium of damaged. SA was adventitious roots with photodiode array Symbols: x, O~ .OI SA; O.1 SA; mM I , mM e , detection (A), and abso~rption spectra of O.4 SA; O.8 SA; 1.2 SA. ar]d (B). mM I , mM O , mM compounds A B 99 suggest that T DNA integration is not the cause of column (0.25 mm i,d, x 30 m, 0.25 f!m; J&W the methylation response, and therefore it appears Scientific DB 1) using helium gas as the carrier at a that this is a natural response byA. belladonna. flow rate of 1.7ml min{. The oven temperature We investigated whether a similar response to the was increased from 30 'C to 250 'C at a rate of 20 "C addition of SA to culture media could be observed min ~, and the ionization voltage was 70 eV. in other plant species. We tested Nicotiana plumba- As shown in Table l, two compounds (MSA and gimfolia and Ilyoscyamus niger (Solanaceae), salicyl alcohol) were methylated by A. belladonna which taxonomically is close relative of A. bella- transformed roots. The ability of methylation of donna. We employed transformed roots of N. M.SA supports the hypothesis of the conversion plumbagintfolia, which were established by co- sequence described above (i.e., SA - MSA - culture of leaf discs with Agrobacterium rhizo- MMB). However, SA was detected in the culture genes 1610, and nontransformed roots of H. niger, medium to which MMB had been added (Table 1). which were established and maintained at Tsukuba This result suggests that MMB is converted to SA in Medicinal Plant Research Station (Jung et al., A, belladonna. Interestingly, the result also suggests 2001.). that A. belladonna was able to remove the acetyl Sterilized SA solution was added to 14-day-old group from the aromatic compounds, because the transformed root cultures of N, plumbagimfolia and addition of acetyl SA induced the production of 30day old nontransformed root cultures of II. MSA and MMB (Table l). Another manner of niger. They were incubated under the same condi- modification was also detected, when some other tions as the transformed root cultures of A. bella- compounds (such as benzoic acid) were added donna. In both cases the concentration of SA in the (Table l). medium was decreased, but methylated SAS were We examined the ability of the crude cell extract not detected (data not shown). of transformed roots of A. belladonna to methylate We als.o investigated the responses of A. bella- the aromatic compounds in vitro. The extraction donna to the addition of compounds which are procedure basically followed the protocol of Akashi structurally similar to SA. For this purpose we et al. (2000). Sterilized SA was added to the trans- examined the responses of transformed roots of A. formed root cultures to give a final concentration of belladonna to treatment with 13 aromatic com- 0.4 mM, and roots (about 50 g) were homcgenized pounds (Table l). Seventytwo h after the addition in a mortar and pestle with twofold volume (w/v) of of each compound, any compounds in the medium extraction buffer and O.5--fold weight of sea sand were extracted by hexane and identified by a GC and polyclar VT. The extraction buffer consisted of (Shimadzun 0.2 sodium phosphate buffer 7.5) containing - MS system GCMSQP5050A). A 5 M (pH into fl I extract was injected a chromatographic 10% (w/v) sucrose, 14mM 2mercaptoethanol, 2

Table l The compounds detected in the culture media of transformed roots of A. belladonna after the addition of aromatic compounds, and the products of conversion with the crude cellfree extracts. (n.d.; not detected, n.t.; not tested)

Compound Cultl'fe mediurn Crude cell extract MSA SA, MMB MMB MMB SA n.d. acetyl salicylic acid MSA, MMB MSA, MMB benzoic acid benzyl alcohoi n.d.

catechol n.d. n.t.

cyclohexane carboxylic acid hydroxymethyl cyclohexane n.t.

ferulic acid n,d. n.t.

gallic acid n.d. n.t. m - hydroxy benzoic acid n.d. n.d. p hydroxy benzoic acid n.d. n.d. l. - hydroxy 2 naphthoic acid 2 hydroxy I naphthalenecarboxaldehyde n.t. phloroglucinol n.d. n.t. pyrogallol 4 carboxylic acid n.d. n,t. salicyl alcohol 2 methoxy benzylaleohol 2 methoxy benzylalcoho lOO

mM EDTA, 0.5%, (w/v) sodium aseorbate, 10 llg Subsidy of the Ministry of Health and Welfare that ml-~ ~ pepstatin A, and O.5 mM (p Amidinophenyl) was granted to the Japan Health Science Foun- methan esulfonyl fluoride hydrochloride (p dation. The work was carried out in part using the APMSF). Pepstatin A, p-- APMSF, and polyclar VT facilities of the Biotechno]ogy Research Center, were obtained from Wako Pure Chemical Indus- The University of Tokyo. tries. The homogenate filtered through eight was References layers of cheesecloth, and the filtrate was centri- fugcd at 15,000g for 15 min. The supernatant was Akashi. T., Sawada, Y., Aoki, T., Ayabe, S., 2000. New stirred of the biosynthesis of formononetin involving for 30 min with half the amount of Dowcx I- scheme x2 (equilibrated with 0.2 M K-Pi buffer, pH 7.5). 2,7,4'-trihydroxyisoflavanone but ~ot daidzein as the Biosci. Biotechnol. 64: The solution obtained by filtration through a glass- methyi acceptor. Biochem., 2276 2279. sintered filter was fractionated with (NH~)2S.04' The Bourne. D. J., Rarrow, K. D., Milbortow, B. V., 1991. precipitate between 5_5 to 750/~ saturation was dis- Salicyloyiaspartate as an endogenous conrponent in the solved in extraction buffer, passed through Sepha- a leaves of Phaseolus vulgaris. Phytoche~nistry, 30: 4041 dex 25 column, and the resultant solution G - was 4044. for the the reaction, used assay. For a mixture Edwaids. R., 1994. Conjugation and metabolism of salicylic containing of cellfree extract, /ll of 2- Iml 50 acid in tobacco. Plant Physiol., 143: 609 61. 4. methoxyethanol, 65.2 f~:M substrate (SA, MSA, Fuka~ni. H., Asakura, T., Iiir~~o, H.. Abe, K.. Shimo~nura, salicyl alcohol, acetyl SA, and benzoic acid were K., Yarnakawa, T., 2002. Saiicylic acid carboxyl tested), and 932.4 ~:M SadenosylLmethionine methyltra~lsferase indueed in hairy root cultures of in a total volume of 1110 f!1 was incubated for 3h Atropa belladonna after treatment with exogenously added salicylic acid. Plant Ceil Physiol., 43: 1054-- at 28 'C The products were extracted (and the reac- l058. tion was stopped) by nhexane, and identified by Jung, D.W., Sung, C. K., Touno, K., Yoshirnatsu, K., the GC-MS system. As shown in Table l, SA, Shimomura, K., 2001. Cryepreservaiion of Hyosryamus MSA, and salicyl alcohol methylated, and were niger adventitious roots hy vitrification. J. Plant acethyl SA were converted to and MMB. On MSA Physiol., 158: 801 - 805. the other hand, benzoic acid, hydroxy when m -- Lee, K.T., Hirano, H., Yamakawa, T.. Kodarna, T., lga- acid, acid bcnzoic or p" hydroxy benzoic were used rashi, Y., Shimcmura, K., 2001. Responses of trans- s.ubstrate, as a we could detect neither methylated formed ~oot cuhure of. Atropa belladonna to salicylic. benzoic acids nor any other modified bcnzoic acids. acid strcss. J. Biosci. Biocngineer., 91: 586- 589. Further investigation will be needed to elucidate Malamy, J., Carr. J. P.. Klessig, D. F., Raskin, l., 1990. the function of these methylation responses in A. Salicylic acid: A Iikely endogenous signal in the re- belladonna. SA methyl transferase of A, bella- sistance response of tobacco to viral infection. Science, 250~ 1004. donna (AbSAMT1), a homolog of SA methyl trans- 1002 Manthe, B., Schulz, M., Schnabl, }1., 1992. Effects of ferase of Clarkia breweri, was isolated recently salicylic ~cid on growth and stomatal movements of (Fukami et al, 2002). AbSAMTI might be involved , Vicia faba L.: evidence fot salieylic acid metabo- in these responses, because it induced by the was lization. J. Cbcm. Ecol.. 18: 1525 1539. addition of abundant into the culture medium. SA Metraux, J. P.. Signer, H., Rynls, J., Ward, E., Wyss Benz, other also contribute this However, enzymes may to M., Gaudin, J.. R~schdQrf, K.. Schmid, E.. Biurn, W., catal- phenomenon, because AbSAMTI could not Inve~ardi, B., 1990, Incre,'~se in s~licylic acid at the yze the conversion from MSA to MMB, which is onset of systemic acquired tesistance in eucu~nbcr. clearly observed in this response. Purification and Science, 250: 1004 lO06. characterization of the methyl transferases that Nakanishi, F., Sasaki, K., Shimornura, K., 1998. Isolatton identification littorinc contribute to these responses in transformed roots of and of from hairy roots ofAtropa A. belladonna, especially the methyl transferase of bella:donna. Plant Cell Rep., 18: 249 251. Pierpoint, W. S., 1994. Saiicylic acid and iis derivatives in MSA, are in progress in our laboratory, plants: medicines, metabolites and messenger moie- Acknowledgements cuies. Adv. Bot. Res.* 20: 163 235. Schulaev, V., Silvern2~~~, P., Raskin* l., 1997. Airborne thank Dr. Tomoyoshi Akashi and Professor We signaling by methyl salicylate in plant pathogen re- Shinich Ayabe of Nihon University for technical sistance. Natu~e, 385: 718-- 721 , assistance and valuable discussion. We also thank Tanaka, S., Hayakawa, K., U~nctani, Y., Tabata, M., 1990. Profess_or Hiroshi Ashihara of Ochanomizu Univer- Glucosylation of isomeric hydroxybenzoic acids by c-ell sity for valuable discussion. This work was sup- suspension cultwes of Mallotus japonicus. Phytoche- ported in part by the Science Research Fund mistry, 29: 1555 155 8.