Capsaicin formation in p-fluorophenylalanine resistant and normal cell cultures of Capsicum frutescens and activity of phenylalanine ammonia lyase

T SUDHAKAR JOHNSON*, R SARADA and G A RAVISHANKARt Plant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore 560013, India *Present address: Dabur Research Foundation, 813, Asaf Ali Road, New Delhi 110002, India t Corresponding author (Fax, 91-821-517233; Email, [email protected]).

Callus cultures of Capsicum frutescens capable of producing a maximum of 53 ~g capsaicin/g FW were exposed to various levels of p-fluorophenyialanine (PFP) at 100, 400, 1000 and 2000~M to develop a resistant cell line that over produces capsaicin. After 15 days of culturing on media lacking PFP, cell lines resistant to 100, 400 and 1000~M registered 18%, 34.5% and 45% increase in capsaicin content over normal cell line (cells not exposed to PFP). Capsaicin accumulation was inhibited in 2000 ~M PFP resistant cell line. The profile of phenylalanine ammonia lyase (PAL), the key enzyme in pheny1propanoidpathway in resistant cell cultures was studied and compared with normal cell cultures to understandits role in capsaicin formation. Importantly increased production of capsaicin was obtained using PFP resistant cell lines. The activity profile of PAL had no correlation with capsaicin content in both control and PFP resistant cells.

1. Introduction 2. Materials and methods

Capsaicin, a major pungent principle of chilli pepper, is .2.1 Initiation and maintenance of callus derived from phenylpropanoids (Bennet and Kirby 1968). The aromatic moiety of capsaicin is derived from phenyl- Seeds of C. frutescens Mill. IHR 1203 were germinated and alanine and the fatty acid moiety from valine (Leete and maintained in a green house. Callus cultures were initiated Louden 1968). The enzyme, phenylalanine ammonia lyase from 3-week old seedlings on MS medium (Murashige (PAL) is responsible for conversion of phenylalanine to and Skoog 1962) supplemented with 2,4-D (2 mg I-I) and t-cinnarnic acid (Suzuki and Iwai 1984). The increased Kn (0.5 mg 1-1)as described earlier (Johnson et al 1990). availability of phenylalanine in p-fluorophenylanine Regular subculturing was done at 2-week interval. (PFP)-resistant cultures might alter the activity of PAL, 2.2 Development of PFP-resistant callus cultures the first enzyme in the phenylpropanoid pathway. Accumulation of capsaicin in cell cultures of Capsicum Small, uniform-sized callus masses (approx. 250 mg) has been studied (Lindsey and Yeoman 1984; Mavituna were transferred to petri dishes containing MS medium et al 1987; Johnson et al 1990, 1991; Johnson 1993; supplementedwith 2,4-D (2 mg I-I), Kn (0.5 mg I-I), 3% Johnson and Ravishankar 1996). Salgado-Garciglia and (w/v) sucrose solidified with 1% agar [designated as Ochoa-Aleio (1990) reported increased capsaicin content regular medium (RM)] with or without PFP. PFP was in PFP-resistant cell cultures of Capsicum annuum. How- filter sterilized and added to the agar medium at 37°C ever, there are no reports on activity of PAL in PFP- at 25 and 100~M. The cultures were incubated at resistant cell cultures of Capsicum. In this communication, 25 :t 2°C under a light intensity of 3000lux. Certain we report on the activity profile of PAL and its relation zones of calluses resistant to PFP grew vigorously and to capsaicin formation in PFP-resistant cell cultures of these clones were further subcultured after 21 days on Capsicumfrutescens. to 100~M PFP containing medium.

Keywords. Capsicumfrutescens; phenylalanineammonia lyase; p-fluorophenylalanine;capsaicin

J. Biosci., 23, No.3, September 1998, pp 209-212. @ Indian Academy of Sciences 209 210 T Sudhakar Johnson, R Sarada and G A Ravishankar

After 15 days of growth on 100JlM PFP containing and PFP resistant cell cultures with time. In the control medium, cultures were either transferred to 100JlM PFP maximum capsaicin was observed on the 9th day while medium or to media containing 400, 1000 and 2000JlM it was seen on the 12th day for the PFP resistant culture. concentrationsof PFP. Those cultures which were resistant Throughout the 12 day period, capsaicin content was to different concentrations of PFP were grown on MS found to be higher in the PFP resistant culture (figure 2A). medium lacking PFP (RM). Capsaicin analysis as well The activity profile of PAL expressed both in terms of as activity of PAL was done after 2 week~ of culture units per gram fresh weight and specific activity showed in callus resistant to 100JlM PFP. The PFP resistant gradual increase in both the cultures (figure 2B). The callus was maintained on RM for 90 days with regular increase in PAL activity on the 12th day is significantly subculturing at 15 day intervals. The callus thus main- higher in PFP resistant cultures than in controls. tained was subcultured on RM and the activity of PAL Similarly a time course study of the activity profile and capsaicin content was analysed at 3 day intervals of PAL and accumulation of capsaicin in cell lines for 12 days and compared with control cell cultures. resistant to 1000 11M and 2000 11M PFP is shown in figure 3A. As mentioned earlier the control culture 2.3 Extraction and quantiration of capsaicin showed maximum production of capsaicin on 9th day while PFP resistant cultures showed this on the 12th Extraction and quantitation of capsaicin in callus cultures day. Considerable difference in capsaicin content was was done by HPLC method as described earlier (Johnson not observed in 1000 11M and 2000l1M PFP resistant et at 1992). cultures as it was in 100 11M PFP resistant culture. The activity profile of PAr (both in terms of total units and 2.4 Extraction and assay of PAL (EC 4.3.1.5) specific activity) was found to be higher in PFP resistant cultures compared to the control culture (figure 3B,C). The activity of PAL was assayed according to the method described by Havir and Henson (1968). Five hundred mg of fresh tissue was sonicated in the presence of 4. Discussion polyvinyl polypyrrolidone (PVPP) for 5 min in an ice bath and extracted in 3 ml 0.04 M borate buffer (pH 8.7). Selection of cell lines resistant to PFP that overproduces The extract was centrifuged at 3500 g for 20 min and phenylalanine or phenolic compounds has been reported the supernatant was used as enzyme extract. The reaction (Palmer an~ Widholm 1975; Berlin 1990). Widholm mixture containing 1 ml 0.04 M borate buffer (pH 8.7), (1972) has shown that cell suspension cultures of tobacco 1 mg L-phenylalanine and 1 ml enzyme extract was in- developed resistance to 5-methyl tryptophan, a toxic cubated for 1 h at 37°C and the reaction was terminated analogue of tryptophan by an alteration in anthranilate with 1 ml 0.5 N HCI. The reaction mixture was centrifuged sYllthase (a key enzyme which controls tryptophan bio- at 2000 g for 5 min before reading at 290 nm. One unit of enzyme was defined as formation of 1 ~g trans-cin- namic acid per 1 h at 37°C under the conditions of - assay. The specific activity was expres~ed as units/mg :s: protein. Protein estimation was done by the method of Ll. Lowry et at (1951). rn '"rn ~ Results -+- C OJ -+- Cell cultures resistant to 100, 400, 1000 and 2000 ~M C PFP were selected and grown on MS medium tree of PFP 0 to) with 2,4-D (2 mg I-I) and Kn (0.5 mg I-I). The capsaicin c content was estimated after 15 days of growth and compared 'u with the control culture. As shown in figure 1, the culture '0 U1 that was resistant to 100 ~m PFP showed 18% increase a. 0 in capsaicin over the control, while cultures resistant to to) 400 and 1000 ~M PFP showed 34.5% and 45% increase in capsaicin, respectively. However 2000 ~M PFP resistant control 100 400 1000 2000 culture showed less content of capsaicin. PFP Concentration (~M) A time course study of the activity profile of PAL and the accumulation of capsaicin in the control and Figure 1. Profile of capsaicin content in cultures resistant to 100 ~M PFP resistant cell culture is shown in figure 2. different concentrations of PFP (analysed on 15th day). Data Capsaicin content gradually increased in both control indicate an average of 3 replicates in two repetitive experiments.

3. Capasaicin formation in Capsicumfrutescens 21

synthesis), and thereby over-produced tryptophan. control cell line. PFP resistant cell lines (100, 1000 and Salgado-Garciglia and Ochoa-Aleio (1990) reported 2000 ~M) selected were maintained on RM free of PFP increased capsaicin content in PFP resistant suspension for 90 days and were found to retain resistance for PFP cultures of C. annuutn. They showed that capsaicin (data not shown). Cell line resistant to low concentration content was 6.7% and 24.9% of the level in fruits in of PFP (100 ~M) were found to have higher content of non-selectable cell suspension and in resistant cell lines capsaicin throughout the experimental period compared respectively. However, there is no report on the activity to the control but did not exhibit high level of PAL profile of PAL in PFP-resistant cell lines of Capsicum. activity except on the 12th day. On the otherhand, cell In the present study PFP-resistant cell lines (1000 and lines resistant to high concentrations of PFP (1000 and 2000 ~M) exihibited high PAL activity (both in terms 2000 ~M) exhibited high activity of PAL with no increase of total units and specific activity) throughout the 12 in capsaicin content when compared to the control. From day period while the 100 ~m PFP resistant cell line figure I it was clear that capsaicin accumulation is showed high activity on the 12th day compared to the related to resistance to PFP concentration up to

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Figure 3. Time course study of PAL activity and capsaicin production in cultures resistant to 1000 and 2000 ~M PFP Figure 2. Time course study of PAL activity and capsaicin maintainedfor 90 days on regular medium (RM). (A) Capsaicin production in cultures resistant to 100 IJ.M PFP. (A) Capsaicin content, (B) PAL activity and (C) specific activity of PAL. content, (8) PAL activity and (C) specific activity of PAL. (- 0 -), Control cell line; (- x-), 1000~M PFP resistant cell (- 0 -), Control cell line; (- x-), 100 ~M PFP resistant cell line; (- ~ -), 2000~M PFP resistant cell line. Data represent line. Bars indicate:t SO. Data representan averageof 3 replicates. an average of 3 replicates. Bars indicate:t SO. 212 T Sudhakar Johnson; R Sarada and G A Ravishankar

1000flM. It is known that PFP resistant cells will have increased levels of secondary metabolites; in Secondary the capability to produce high level of phenylalanine products from plant tissue culture (eds) B V Charlwood and will also be insensitive to its feedback inhibition and M J C Rhodes (New York: Oxford Science Publ.) pp 117-137 through altered chorismate mutase (Berlin 1990). PFP Hall R D and Yeoman M M 1991 The influence of intracellular by itself need not be metabolizedaccounting for enhanced pools of phenylalanine derivatives upon the synthesis of capsaicin levels. However increasedendogenous phenyl- capsaicin; Planta 185 72-80 alanine will be useful to the cell for further metabolism Havir E A and Henson K A 1968 L-phenylalanine iunmonia to produce phenylalanine derived compounds such as lyase I. purification and molecular size of the enzyme from potato tubers; Biochemistry(USA) 7 1896-1903 phenylpropanoidsor capsaicin. High content of capsaicin Holland S S 1989Studies on enzymesof the capsaicinbiosynthetic in the 100flM PFP resistant cell line may be due to pathway in Capsicumfrutescens, Ph.D. thesis, University of increased utilization of phenylalanine for capsaicin syn- Edinburg, UK thesis, whereas, non correlation of capsaicin and PAL Johnson T S, RavishankarG A and VenkataramanL V 1990 activity in cell lines resistant to high concentration of In vitro capsaicinproduction by immobilisedcells and placental tissues of Capsicum annuum L. grown in liquid medium; PFP could be explained in two ways. In these cell lines Plant Sci. 70 223-229 phenylalanine might be channelized to other phenyl JohnsonT S, RavishankarG A and VenkataramanL V 1991 propanoid production through an altered PAL activity Elicitation of capsaicin production in freely suspendedcells similar to the report by Widholm (1972) for tryptophan and immobilised cell cultures of Capsicumfrutescens Mill.; production through altered anthranilate synthase or as Food Biotechnol. 5 197-205 JohnsonT S, RavishankarG A and VenkataramanL V 1992 reported by Hall and Yeoman (1991) that the activity Separationof capsaicinfrom the phenyl propanoidcompounds of the first two enzymesof the phenyl propanoidpathway by high performanceliquid chromatographyto determine the PAL and trans-cinnamate-4-hydroxylaseresponsible for the biosyntheticstatus of cells and tissues of Capsicumfrutescens conversion of phenylalanine to trans-cinnamicacid and Mill. in vivo and in vitro; J. Agric. Food Chem.40 2461-2463 subsequentlyto coumaric acid had no correlation with Johnson T S 1993 Stu(;jies on production of capsaicin in immobilized cells and placental tissues of Capsicumannum capsaicin synthesis. Holland (1989) further showed that and c. frutescens;Ph.D. thesis, Mysore University, Mysore L-arnino oxyphenylpropionicacid, the hydroxylamineana- JohnsonT S and RavishankarG A 1996 Biotransformationof logue of (-phenylalanine which is a competitive inhibitor ferulic acid and vanillylamine to capsaicin and vanillin in of PAL, had no effect on the incorporationof C14phenyl- immobilised cell cultures of Capsicumfrutescens; Plant Cell alanine into capsaicinoidsin fruits of capsicumwhen used Tiss. argo Cult. 44 117-126 Leete E and Louden C L 1968 Biosynthesis of capsaicin and at a concentrationbelow 0.7flM. Hall and Yeoman(1991) dihydrocapsaicinin Capsicumfrutescens; J. Am. Chem. Soc. concluded that PAL was not likely to be the limiting step 90 6837-6841 for the production of capsaicinoids. Lindsey K and Yeoman M M 1984 The synthetic potential of In the present study the profile of high capsaicin immobilized cells of Capsicumfrutescens Mill C.v. annuum; content in 100flM PFP resistant cell line without increase flanta 162495-501 Lowry 0 H, RosebroughN J, Farr A L and RanadallR J 1951 in PAL activity (except on the 12th day) and the profile Protein measurementwith the folin phenol reagent; J. Biol. of high PAL activity in 1000 and 2000 flM PFP resistant Chem. 193 265-278 cells without considerable increase in capsaicin content Mavituna F, Wilkinson A K, Williams P D and Park J M C (except on the 12th day) shows that PAL activity is not 1987 Production of secondarymetabolites by immobilized the limiting factor for capsaicin production. The activity plant cells in novel bioreactors; in Bioreactors and biotrans- fo/7nations (eds) Moody G W and Baker P B (London: profile of PAL has no correlation with capsaicin content Elsevier)pp 26-37 in C. frutescence cell culture in both control and PFP Murashige T and Skoog F 1962 A revised medium for rapid resistant ones. growth and bioassayswith tobacco tissue cultures; Physiol. Plant. 15 473-497 Palmer J E and Widholm J 1975 Characterisationof carrot and Acknowledgment tobacco cell cultures resistantto p-fluorophenylalanine;Plant TSJ acknowledges the receipt of a Senior Research Physiol. 56 233-238 Salgado-GarcigliaRand Ochoa-Aleio N 1990 Increasedcapsaicin Fellowship granted by the Council of Scientific and content in PFP-resistantcells of chilli pepper (Capsicum Industrial Research, New Delhi. annuum L.); Plant Cell Rep. 8 617-620 Suzuki T and Iwai I 1984 Constituentsof red pepper species: Chemistry, biochemistry,pharmacology and food sciencesof References the pungentprinciple of Capsicumspecies; The Alkaloids 61 227-299 Bennet D C and Kirby G W 1968 Constitutionand biosynthesis Widholm J M 1972 Cultured Nicotiana tabacum cells with an of capsaicin; J. Chern. Soc. (C) 442-445 altered anthranilatesynthase which is less sensitive to feed Berlin J 1990 Screeningand selectionfor variant cell lines with back inhibition; Biochim. Biophys. Acta 261 52-58'97,' MS received 8 October l~ accepted1 July 1998

Corresponding editor: SIPRA GUHA-MUKHERJEE