THE JOURNAL 0 1991 hy The American Society for Biochemistry and Molecular Biolow, Inc

OF BIOLOGICAL CHEMISTRY Val. 266, No. 7, Issue of March 5, pp. 4648-4653, 1991 Printed in U. S. A.

Hyoscyamine GB-Hydroxylase, an Enzyme Involved in Tropane Alkaloid Biosynthesis, Is Localized at the Pericycle of the Root*

(Received for publication, August 31, 1990)

Takashi Hashimoto$$, Asuka Hayashi$, Yasuhiro AmanoS, Junko Kohno$ll, Hiroko IwanariII , Sadakazu Usudall , and Yasuyuki Yamada$ From the $Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan and the lllnstitute of Immunology, Shimoishibashi 170, Ishibashi-cho, Shimobuga-gun, Tochigi 329-05, Japan

Hyoscyamine GB-hydroxylase (H6H; EC 1.14.11.1 1) catalyzes the first reaction in the biosynthetic pathway from hyoscyamine to scopolamine in several solana- ceous plants. Four monoclonal antibodies were raised against H6H purified from cultured roots of Hyoscy- amus niger. The IgGl antibody mAb5 inhibited H6H activities present in cell-free extracts of H. niger roots and specifically recognized 38-40-kDa proteins from six different scopolamine-producing plant species in Western blot analysis after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The other three monoclonal antibodies all recognized SDS-dena- tured H6H protein from Hyoscyamus species, but did not bind to native H6H. Western blot analysis of pro- tein extracts from various tissues of H. niger using these antibodies showed that H6H is abundant in cul- tured roots, present in plant roots, but absent in leaf, stem, calyx, cultured cells, and cultured shoots. Im- munohistochemical studies using monoclonal antibody and immunogold-silver enhancement detected H6H only in the pericycle cells of the young root in several scopolamine-producing plants. Mature roots that underwent secondary growth and lacked the pericycle did not react with the antibody. This pericycle-specific localization of scopolamine biosynthesis provides an anatomical explanation for the tissue-specific biosyn- thesis of tropane alkaloids and may be important for translocation of tropane alkaloids from the root to the aerial parts.

Hyoscyamine and scopolamine are the two most common tropane alkaloids found in the Solanaceae, and many plants containing these alkaloids have been used for their medicinal, hallucinogenic, and poisonous properties. Classical grafting experiments in which scions from tropane alkaloid-producing species are grafted onto root stock from nonproducing species result in plants that do not accumulate alkaloids, whereas grafting in the reciprocal combination produces plants that accumulate tropane alkaloids (reviewed by Waller and No- wacki, 1978). These experiments first demonstrated that the

* This work was supported by the Ministry of Education, Science, and Culture of Japan through a grant “Molecular Mechanisms of Metabolite Accumulation in Plants” (to T. H.). A preliminary report of some of this work was presented at the Seventh International Congress of Plant Tissue Culture, Amsterdam, The Netherlands, June 1990, Extended Abstract, pp. 775-780. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

8 To whom correspondence should be addressed. 7l Current address: Plantech Research Inst., Kamoshida-cho 1000,

Midori-ku, Yokohama, Kanagawa 227, Japan.

main site of tropane alkaloid biosynthesis is the root and that the tropane alkaloids are translocated from the root to the aerial parts of the plants. More recently, activities of two enzymes of tropane alkaloid biosynthesis, putrescine N-meth- yltransferase (Hashimoto et al., 1989) and hyoscyamine 6p- hydroxylase (Hashimoto and Yamada, 1986), have been shown to be high in cultured roots but very low in cultured cells and cultured shoots.

The latter enzyme, H6H’ (EC 1.14.11.11), catalyzes the first oxidative reaction in the biosynthetic pathway leading from hyoscyamine to scopolamine (Fig. 1) and requires alka- loid substrate, 2-oxoglutarate, ferrous ion, ascorbate, and mo- lecular oxygen for activity and thus belongs to the 2-oxoglu- tarate-dependent dioxygenases (Hashimoto and Yamada, 1986). H6H not only hydroxylates various hyoscyamine deriv- atives but also epoxidizes 6,7-dehydrohyoscyamine, a syn- thetic alkaloid, to scopolamine (Hashimoto and Yamada, 1987; Yamada and Hashimoto, 1989). Although 6,7-dehydro- hyoscyamine was transformed to scopolamine when fed to plants (Fodor et al., 1959; Hashimoto et al., 1987), scopolamine biosynthesis does not proceed through the unsaturated alka- loid (Hashimoto et al., 1987) (Fig. 1).

Recently we succeeded in purifying H6H to homogeneity from cultured roots of Hyoscyamus niger L. (Yamada et al., 1990). In the present report, the purified H6H protein was used to immunize mice for the production of monoclonal antibodies against H6H. Immunohistochemical studies dem- onstrated the tissue- and cell-specific expression of H6H in various scopolamine-producing plants.

MATERIALS AND METHODS’

RESULTS

Characterization of Monoclonal Antibodies-Two different preparations of highly purified H6H were obtained from cul- tured roots of H. niger and used to immunize mice. For the first fusion, the homogeneous immunogen was obtained by a series of chromatographic steps; whereas, for the second fu- sion, the immunogen was purified by two of the column chromatographic steps used for the first immunogen, followed by preparative sodium dodecyl sulfate-polyacrylamide gel

The abbreviations used are: H6H, hyoscyamine 6P-hydroxylase; PBS, phosphtate-buffered saline; ELISA, enzyme-linked immunosor- bent assay; BSA, bovine serum albumin; TBS, Tris-buffered saline; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; PAGE, polyacryl- amide gel electrophoresis; mAb, monoclonal antibody.

* Portions of this paper (including “Materials and Methods” and Figs. 2 and 4) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

4648

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Hyoscyamine 6P-Hydrc

electrophoresis (SDS-PAGE) to give a semi-homogeneous preparation. After hybridomas were screened twice with an enzyme-linked immunosorbent assay, one stable antibody- secreting hybridoma cell line (mAb5) was established from the first fusion and three cell lines (mAb55, mAh67, and mAb 94) from the second fusion. Subclass analyses showed that mAb5 and mAbfi7 were IgG, antibodies and mAb55 and mAb94 IgM antibodies.

These monoclonal antibodies were tested for their ability to inhibit the HfiH activities in a partially purified enzyme preparation. The mAb5 antibody inhibited the two oxidation reactions catalyzed by HfiH (see Fig. 1) to a similar degree: the hydroxylase activity and the epoxidase activity that forms scopolamine from fi,7-dehydrohyoscyamine (Fig. 2; Mini- print). The other three antibodies (mAb55, mAbfi7, and mAb94) (data not shown) and nonspecific mouse immuno- globulins IgG (Fig. 2; Miniprint) and IgM (data not shown) did not inhibit the enzyme activities. An immunoprecipitation assay using anti-mouse immunoglobulins adsorbed to Staph- ylococcus aureus cells indicated that these three antibodies did not bind to the native enzymatically active form of HfiH (data not shown).

Each of the monoclonal antibodies recognized the SDS- denatured enzyme following electrophoretic transfer to ImmobilonTM (Western blot). Crude enzyme extracts from cultured roots of four Hyoscyamus and four other species were separated by SDS-PAGE and analyzed by Western blotting (Fig. 3). The analyzed extracts contained the following levels

HO " ..' 60-hydroxy- \ hyoscyamine

-0

N-Me ...OR

hyoscyamine q&H scopolamine H

R=-COd-Ph kn,on

6,7-dehydro- (s)-tropyl- hyoscyamine

Frc.. 1. Biosynthetic pathway from hyoscyamine to scopol- amine. The pathway hy which 6,7-dehydrohyoscvamine, a non- natural alkaloid, is converted to scopolamine is also shown. H6H catalyzes hot h the hydroxylation of hyoscyamine and the epoxidat ion of 6,7-dehydrohyoscyamine.

-43

5 - 50

55

87

94

FIG. 3. Immunoblotting of crude extracts from various cul- tured roots. SI)S-I'A(;I< was performed in a 12.500 polyacrylamide gel with 10 pg o f protein applied per lane. The hlots were incuhated with monoclonal antihodies mAh5 (top gel) , mAh55, mAh67 (rniddlr p l s ) , or mAh94 (hotforn gel). Only the part of the hlots ( M , 43.000- :10,000) are shown for mAh55, mAh67, and mAh94. IAne I, A. hrlla- donna; lane 2, H . carnpestris; lane 3 , D. lrichhardtii; lane 4, H. alhus; lane Fi, H. g-yorfii; lane 6, H. muticus; lane 7, H . nigrc lane 8, N . tahacum.

wylace in Root Pericycle 4649

of hydroxylase activities; 0.03 picokatal in Atropa belladonna (lane I ) , 0 picokatal in Brassica campstris (lane 2), 0.18 picokatal in Duboisia leichhardlii (lane 9). 0.03 picokatal in Hyoscyamus alhus (lane 4 ) . 0.02 picokatal in Hyoscyamus gyorffi (lane .5 ), 0.Ofi picokatal in Hyoscyamus muticus (lane fi), 0.26 picokatal in H. niger (lane 7 ) . and 0 picokatal in Nicotiana tabacum (lane 8). The antihodv mAh5 recognized bands of M, 38,000-40,000 in cell extracts from all scopola- mine-producing root cultures (lanes 1 , 3. and 4 - 7 ) and the intensities of the bands were well correlated with the hvdrox- ylase activities in the extracts. The strongest signal was obtained in H. niger, whereas no signals were detected in non- scopolamine-producing cultures of H . camprstris and N . /a- bacum (lanes 2 and 8). The other three antihodies also reacted strongly with the 38-kDa hand of H. nigcr (lanr> 7 , Fig. 3) . The purified HfiH of H. niger (Yamada rt 01.. 1990) also had the same M, of 38.000. There were differences, however, in the specificities of the antihodies. The antihodv mAh55 ap- peared to recognize almost exclusively the :18-kI)a protein of H. niger (lane 7, Fig. 3) , and mAh94 was specific to Hyoscy- amus species (lanes 4 " ) , whereas mAhfi7 also recognized a 39-kDa protein of Duboisia (lane *?I.

Immunoaffinity Purification--Purified mAh5 antihodv was covalently attached to Sepharose C L 4 R resin hv cyanogen bromide activation. A partially purified H6H preparation from cultured H. niger roots was applied to the antihodv affinity column. Nonspecifically hound protein was washed from the column with a phosphate huffer and specific elution of HfiH was accomplished with 10 mM horate huffer, pH 10.5. The eluted enzyme of 38 kDa appeared to he nearlv homoge- neous, as judged hv SDS-PAGE and silver staining (Fig. 4; Miniprint). This purified HfiH protein was recognized hv all the four monoclonal antibodies on Western hlots (data not shown). Enzyme activity was not detected in the unhound fraction, hut 5-1006 of the applied activitv was recovered in the hound fraction under the elution conditions used. T-ypi- cally HfiH was purified to near homogeneity hv precipitation with ammonium sulfate, followed hy chromatography on the hutyl-hydrophobic column and the mAh5 affinitv column. About 220 pg of purified H6H was ohtained after starting with 750 g of cultured roots (1.3 g total protein). The specific activity of the purified enzvme was 930 picokatals/mg protein.

Because IgM antihodies are not suitahle for affinitv chro- matography (Arvieux and Williams, 1988). similar immu- noaffinity resin was prepared only with the other IgG antihodv mAhfi7 as the coupling antihodv. Enzymaticallv active HfiH that had heen partiallv purified hy precipitation with ammo- nium sulfate and the hutyl-hvdrophohic column was first denatured hy treatment with SDS and then loaded on the mAbfi7 column (after removal of excess SDS), the denatured HfiH protein was specifically retained on the column. Borate huffer of pH 10.5 eluted a homogeneous (although enzvmati- cally inactive) HfiH protein which was recognized hv all four monoclonal antibodies on Western hlot (data not shown).

Tissue- and Cd-specific Imxdizathn-The relative ahun- dance of HBH protein in various tissues of H. niger was determined hv immunohlotting with mAh5 after separation of proteins hv SDS-PAGE (Fig. 5 ) . No protein reacting with mAh5 was detected in the crude cell extracts from petals, calyxes, leaves, stems, cukured cells, or cultured shoots. whereas the HfiH protein of 38 kDa was present in extracts from plant roots and cultured roots. The amount of the HfiH protein in the cultured roots was considerahlv higher than that in the mature plant roots. Similar immunohlotting anal- yses of various H. niger tissues using the other antihodies

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4650 Hyoscyamine 6/3-Hydroxylase in Root Pericycle

FIG. 5. Immunoblotting of crude extracts from various tis- sues of H. niger. Proteins (10 pg per lane) were separated by SDS, 12.5% PAGE, and the blot was incubated with mAh5. The arrowhead indicates the position of H6H protein (38 kDa). Lane 1, calyx; lane 2, leaf; lane 3, petal; lane 4, root; lane .5, stem; lane 6, cultured cells; lane 7, cultured roots; lane 8, cultured shoots.

I

FIG. 6. Immunolocdizntion of f I 6 H in various cultured roots. Paraffin-emt)r,tltlc.tl transections were treated with either mAh5 ( A , C , E, and C;) or nonspecific I d ; (H, D, F, and H ) . A and A, H. gyorffc C and I ) , H . muticus; E and F, H. nifier; G and H , N . lahacum. c, cortex; up, vascular parenchyma; x, xylem. Bar = 0.1 mm.

(mAb55, mAb67, and mAb94) gave similar results (data not shown). In situ localization of H6H in plant tissues by immunogold-

silver enhancement staining was done, the mAb5 antibody being used as the primary antibody because it binds to both the native and the SDS-denatured forms of the enzyme and recognizes H6H proteins from various scopolamine-producing plants (Fig. 3). Cultured roots were embedded in paraffin, and 10 pm transverse sections were prepared. Significant disinte- gration of cortical cells and some loss of antigenicity occurred as a result of the embedding process. Immunohistochemical localization of H6H in the transections revealed that H6H is present only in a layer of cells near the vascular cylinder in the scopolamine-producing cultured roots of H. gyorffi (Fig. 6A) , H. niger (Fig. 6C), and H. muticus (Fig. 6E). No signal was detected in the sections from cultured tobacco roots (which produce no tropane alkaloids) (Fig. 6G), nor in any sections when a nonspecific mouse IgG was used as the primary antibody (Fig. 6, R , D F, and H ) . When cultured roots of A. belladonna and H. albw were analyzed in the same way by using mAb5, the same cell layer sometimes reacted weakly (data not shown) in accorda&e with the low hydrox- ylase activities in these cultured roots (Hashimoto and Ya- mada, 1986).

To determine whether the H6H near the vascular cylinder is in the endodermis or the pericycle of the root and also to check whether this localized expression of H6H is specific to the root, we prepared by a cryomicrotome 20-pm transverse or longitudinal sections from young tissues of Dubokia my- oporoides. The leaf and stem tissues were obtained from a shoot culture, and the root tissue was from young primary

roots that had developed from the shoot culture. A strong hlack signal was clearly seen at the pericycle of the root; no other types of root cells were stained (Fig. '7, E and G). The pericycle in these root cross-sections was identified hy its location relative to the endodermis cells, which showed clear differentiation of the Casparian strip at higher magnification (data not shown). In the slender sessile leaf hlade of Duhoisia, vascular parenchyma cells reacted slightly (Fig. 7 A ) , hut in the stem transverse sections no difference was found hetween the mAh5-treated sample and the nonspecific antihody con- trol (Fig. 7 , C and D). In this Duboisia species, low levels of HfiH activity have been detected in the cultured shoots (Hash- imoto and Yamada, 1986).

Greenhouse-grown H. nigw plants at their flowering stage were analyzed for the cell-specific localization of HfiH protein. Some nonspecific silver deposits were ohserved in differen- tiated xylem cells. Cryosections of the leaf and stem tissues did not give any immunohistochemical signals (Fig. 8, A and R ) . The primary roots and the main hranch roots showed considerable secondary vascular growth and did not have a distinct cell type corresponding to the pericycle, and no H6H signals were detected in cryosections from these roots (Fig. 8C). In contrast, sections of small hranch roots with little secondary growth reacted strongly at the pericycle (Fig. 8D). Although it is sometiems difficult to delineate the pericycle (and the endodermis) unamhiguously using light microscopy, identity of the HGH-expressing cells with the pericycle cells is further supported by our ohservations with cryosectioned samples of the H. niger small hranch roots (Fig. 81)) and cultured D. leichhardtii roots (data not shown). Most cortical cells in these samples were often disintegrated, thus leaving only the endodermal cells attached to the vascular cylinder in the immunogold-silver stained transverse sections. The H6H protein was detected just heneath the periphery of the vascular cylinder (i .e. the second outermost cell layer in these samples).

DISCUSSION

Four monoclonal antihodies against H6H from H. n i g u were obtained in this study. Specificity of the antibody mAh.5 was confirmed by inhihition of enzyme activities and hv immunoaffinity purification of enzymatically active, nearly homogeneous protein. Inhihition of enzyme activities suggests that mAb5 interacts with the enzyme near the act.ive site. Attempts to prevent the inhihition by prior incuhation of the enzyme with its cofactors in combinations, however, did not give clear results.:' Reaction of the enzymes from different plant species with mAh5 on Western blots (Fig. 3) suggests that the epitope is well conserved in many H6Hs from various sources. This may reflect the importance of the amino acid sequence recognized by mAh5 for enzyme catalysis.

The other three antibodies (mAh55, mAhfi7, and mAh94) appear to recognize only the enzymatically inactive protein that has been denatured hy SDS. The variahle affinities of these antibodies to H6Hs from different species also indicate that their epitopes are huried inside the folded enzyme and are not necessarily conserved in the H6Hs from different sources. The H6H protein used in the second immunization experiment was recovered from preparative SDS-PAGE as the final purification step and the nature of the immunogen may be responsible for the failure of the antibodies to recog- nize the native enzyme. The screening method used to ohtain HGH-specific antihodies (enzyme-linked immunosorhent as- say) may also have favored the detection and recovery of clones producing antibodies that hind to the denatured en- zyme.

T. Hashimoto and Y. Yamada. unpuhlished results.

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FIG. 7. Immunolocalization of H6H in various tissues of Duboisia myoporoidee.Cryosections were treated with eitheE mAb5 (A, C, E, and C ) or nonspecific IgG (B, D, F, and H). A and B, cross-sections of young leaf blades; C and D, cross-sections of young stems; E a d F, cross-sections of young roots, which have just regenerated from a shoot culture; G and H, longitudinal sections of young roots. c, cortex; en endodermis; ep, epidemnis, p pith; pc, pericycle; rc, root cap; ue, vascular cyl- inder; up, vascular parenchyma; x, xy- Iem. Bar = 0.1 mm.

Hyoscyamine 6fl-Hydroxylase in Root Pericycle 4651

Our immunohistochemical studies localized H6H to the pericycle of cultured roots and of relatively young roots. The pericycle is commonly one layer of cells in thickness, is located at the periphery of the vascular cylinder (stele) of the root, and consists mainly of thin-walled parenchyma cells (Esau, 1977). The organization of the vascular tissue in the roots is quite different from the organization in the stem and leaf veins of Hyoscyamus and other seed plants. These aerial tissues lack the pericycle and the endodermis which in the root separate the vascular region from the cortex. In light of the pericycle-specific localization of H6H, the absence of the pericycle in the stem and the leaf provides an anatomical explanation for the very low expression of H6H in these tissues. In addition, the primary root and the main branch roots of many mature dicotyledonous plants (including Hyos- cyamus and Duboisia species used in this study) typically display secondary growth to various degrees, represented by thickening of the root caused by the activity of the vascular cambium with concomitant transformation of the pericycle cells to vascular cambium of phellogen (Esau, 1977). Cultured roots, on the other hand, are characterized in most cases by

the absence of secondary growth (Hashimoto and Yamada, 1991). The more uniform distribution and greater abundance of pericycle cells in cultured roots is probably responsible for the much higher amount of H6H protein in cultured roots than in mature roots of H. niger (Fig. 5). Northern blotting analysis using an H6H cDNA as a probe also detected consid- erably higher amounts of H6H mRNA in cultured roots than in mature roots of H. niger and suggested that the pericycle- specific expression of H6H is primarily controlled at the transcriptional leve1.4

The pericycle is also known as the site where lateral roots originate most frequently. When a lateral root is initiated, several contiguous pericyclic cells acquire dense cytoplasm and divide first periclinally, then both periclinally and anti- clinally, to form the root primordium (Esau, 1977). In cultured roots of many scopolamine-producing plants, addition of auxin to the culture medium induces abundant root primordia (Hashimoto et aL, 1986), and this induction is accompanied by a concomitant decrease in H6H activity and in scopolamine

J. Matsuda, S. Okabe, T. Hashimoto, and Y. Yamada, submitted for publication.

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4652 Hyoscyamine 6/3-Hydroxylase in Root Pericycle

FIG. 8. Immnnolocalization of HBH in various tissues of H. niger plant. Cryosections were treated with mAb5. A, cross-section of a leaf; B, cross- section of stem; C, cross-section of a primary root; D, cross-section of a small branch root. sx, secondaw xylem; vc, vas- cular cambium. Other abbreviations are the same as in Fig. 7. Bar = 0.1 m..

content (Hashimoto and Yamada, 1986). Our results suggest that this suppression of H6H activity in cultured roots by auxin may be mediated by,$hysiological and morphological changes in the pericycle cells.

The mechanism of alkaloid translocation from the root to the aerial part is still a matter of controversy. Alkaloid trans- location apparently does not require specialized transport cells because scions from non-alkaloid-producing plants translocate and accumulate tropane alkaloids produced in root stocks (Waller and Nowacki, 1978). Although root-to-shoot translocation of alkaloids is generally believed to proceed through the xylem (Waller and Nowacki, 1978), phloem trans- port of alkaloids has been suggested for root-to-shoot trans- location of pyrrolizidine alkaloid N-oxides, based on inhibi- tion of alkaloid translocation by steam-girdling (Hartmann et al., 1989). Whether translocation of tropane alkaloids pro- ceeds through the xylem or the phloem, biosynthesis of these alkaloids within the vascular cylinder in the root would be of obvious advantage for their translocation.

Finally, it should be mentioned that not all the enzymes in the scopolamine biosynthetic pathway are necessarily ex- pressed in the pericycle cells only. For example, suspension- cultured cells and cultured shoots of Hyoscyamus species have considerable activities of diamine oxidase that functions in the early biosynthetic pathway of hyoscyamine (Hashimoto et al., 1990). It might be possible that precursors of scopola- mine (e.g. hyoscyamine) are synthesized in root cells other than the pericycle and then moved to the pericycle to be converted to scopolamine. Further histochemical studies and microanalysis of alkaloids in different types of cells will lead to better understanding of the cell-specific expression of al- kaloid biosynthesis, and of alkaloid translocation.

Acknowledgments-We thank Dr. Minoru Fujita and Dr. Masateru

Shinozaki for help with preparation of cryosections and paraffin sections, respectively, and Dr. John Fitchen for helpful discussions and correction of our English.

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T Hashimoto, A Hayashi, Y Amano, J Kohno, H Iwanari, S Usuda and Y Yamadabiosynthesis, is localized at the pericycle of the root.

Hyoscyamine 6 beta-hydroxylase, an enzyme involved in tropane alkaloid

1991, 266:4648-4653.J. Biol. Chem. 

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