Differential regulation and distribution of Acridone Synthase in Ruta graveolens in honour of Professor G. H. Neil Towers 75th birthday

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Pergamon PII] S9920Ð8311"87#99093Ð5

Phytochemistry\ Vol[ 38\ No[ 1\ pp[ 392Ð300\ 0887 Þ 0887 Elsevier Science Ltd[ All rights reserved Printed in Great Britain 9920Ð8311:87:,Ðsee front matter

DIFFERENTIAL REGULATION AND DISTRIBUTION OF ACRIDONE SYNTHASE IN RUTA GRAVEOLENS IN HONOUR OF PROFESSOR G[ H[ NEIL TOWERS 64TH BIRTHDAY KAY T[ JUNGHANNS\$ RICHARD E[ KNEUSEL\% DETLEF GROÝGER& and ULRICH MATERN $Biologisches Institut II\ Lehrstuhl fur Biochemie der P~anzen\ Albert!Ludwigs!Universitat\ Schanzlestrasse 0\ 68093 Freiburg\ Germany %QIAGEN GmbH\ Max!Vollmer!Strasse 3\ 39613 Hilden\ Germany &Institut fur P~anzenbiochemie\ Weinberg 2\ 95019 Halle:Saale\ Germany 'Institut fur Pharmazeutische Biologie\ Philipps!Universitat Marburg\ Deutschhausstrasse 06 A\ 24926 Marburg\ Germany "Received 09 September 0886#

Key Word Index*Ruta `raveolens L[^ Rutaceae^ acridone alkaloids^ acridone synthase^ elicitor induction^ plant disease resistance^ preformed defense[

Abstract*Cell suspension cultures of Ruta `raveolens L[ accumulate polyketide metabolites such as acridone alkaloids and ~avonoid pigments[ Whereas ~avonoid synthesis is induced by light\ the production of alkaloids can be enhanced in dark!cultured cells by treatment with fungal elicitors[ Acridone synthase "ACS# catalyzes the committed condensing reaction of acridone biosynthesis yielding 0\2!dihydroxy!N!methylacridone from N!methylanthraniloyl! and malonyl!CoAs[ The reaction proceeds in a manner analogous to that of chalcone synthase "CHS# which catalyzes the _rst committed step in ~avonoid biosynthesis and cDNA and protein sequences of Ruta ACS possess a high degree of sequence homology to heterologous CHSs[ ACS transcript abundance and speci_c activity were monitored in cultured R[ `raveolens cells irradiated either continuously with white light or treated with fungal elicitor over a period of 13 h and found to increase transiently upon elicitor treatment and to decrease upon light irradiation[ Immunodetection with a rabbit polyclonal ACS antiserum revealed that the amounts of ACS polypeptide decreased slightly in light!irradiated cells but increased in elicitor!treated Ruta cells[ Fluorescence microscopy and tissue print hybridizations were employed to aid in localizing the sites of storage and biosynthesis of acridone alkaloids in Ruta plants[ Yellow ~uorescing alkaloids were detected particularly in root tissue adjacent to the rhizodermis\ but also in the endodermis and vascular tissue of the hypocotyl[ ACS transcript abundance in situ followed the same spatial pattern\ indicating that the synthesis of acridones likely proceeds at all sites of deposition rather than exclusively in the root[ Expression in planta and the induction response of ACS suggest that the alkaloids serve as phytoanticipins or phytoalexins in the defense of Ruta particularly to soil!borne pathogens or as feeding deterrents[ Þ 0887 Elsevier Science Ltd[ All rights reserved

INTRODUCTION

The common rue "Ruta `raveolens L[# has been the object of numerous studies on the biosynthesis of sec! ondary metabolites abundant in the Rutaceae[ Among these are linear furanocoumarins and furoquinolines ð0Ł as well as acridone alkaloids\ a class of products that appears to be unique to the Rutaceae family ð1Ð 3Ł[ Acridones accumulate in specialized cells\ the idioblasts\ and their distribution as bright!yellow ~u! orescing droplets in vacuoles can be easily monitored

Author to whom correspondence should be sent[ 392

by ~uorescence microscopy[ Examination of indi! vidual Ruta organs indicated that the loaded idioblasts are distributed mostly in the root and root hairs and\ to a lesser extent\ in the stems and leaves ð4Ł[ This suggested that the root is the primary site of acridone synthesis\ as was proposed for other alkaloids ð5\ 6Ł\ with subsequent translocation to the shoot[ The for! mation of idioblasts\ however\ is independent of the stage of tissue di}erentiation since an identical pattern of deposition of ~uorescent acridone droplets was also observed in Ruta cell suspension cultures ð7Ł[ Acridone alkaloids have attracted attention because of their mutagenic ð8Ł\ antiviral ð09Ł and anti! plasmodial activities ð00Ł[ Their mode of action is

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Fig[ 0[ Biosynthesis of acridone phytoalexins in Ruta `raveolens L[ The committed condensing reaction is catalyzed by acridone synthase[

likely based on DNA intercalation\ which makes these compounds too toxic for therapeutic use[ In plants\ however\ acridones may act as feeding deterrents\ phy! toalexins or phytoanticipins ð01Ł and may signi_cantly enhance resistance to microbial invasion[ Con! siderable accumulation of acridone alkaloids in the root might be exploited to _ght soil!borne pathogens which are di.cult to control by other means[ The biosynthesis of acridones has been extensively studied in Ruta cell cultures and these cells have been important sources for the preparative isolation of the relevant enzymes "Fig[ 0# ð02Ð05Ł[ The pivotal reaction of acridone formation is catalyzed by acridone syn! thase "ACS#\ a polyketide synthase which condenses N!methylanthraniloyl!CoA with three malonyl!CoAs to yield 0\2!dihydroxy!N!methylacridone "Fig[ 0#[ Dark!cultured Ruta cells contain signi_cant ACS activity\ which can be enhanced by treatment of the cell cultures with a fungal elicitor causing an increased accumulation of acridones ð06Ł[ ACS cDNA was cloned from elicited Ruta cell cultures and the expression in E[ coli yielded a catalytically highly active enzyme ð07Ł[ Although the sequences of DNA and translated polypeptide showed high homologies to those of heterologous chalcone "CHSs# and stilbene synthases "STSs#\ the Ruta ACS expressed in E[ coli did not accept 3!coumaroyl!CoA as a substrate ð07Ł

nor was N!methylanthraniloyl!CoA accepted by het! erologous CHSs or STSs "J[ Schroder\ Universitat Freiburg\ personal communication#[ Furthermore\ in contrast to CHSs and STSs\ which exist as cata! lytically active homodimers ð08Ł\ ACS appeared to be active as a monomer[ Thus\ subtle di}erences in the primary polypeptide sequences are responsible for the diverse secondary structures and substrate speci_cities of known polyketide synthases[ Furthermore\ the transcriptional regulation of these three\ closely related classes of polyketide synthases di}ers sig! ni_cantly\ e[g[ CHS is predominantly expressed during ~ower development and induced upon irradiation[ The close sequential interrelationship of taxo! nomically restricted ACS ð3\ 5Ł with the more abun! dant CHSs and STSs ð19\ 10Ł\ as well as its rather divergent substrate speci_city and regulation of expression make ACS very attractive for molecular investigations[ CHS and STS from a single plant have been extensively studied ð08\ 11\ 12Ł and the com! parison of ACS with the Ruta endogeneous CHS is a particularly appealing research goal[ We describe in this report the di}erential induction characteristics of ACS and CHS in cultured Ruta cells as well as the localization of acridones and ACS transcripts in R[ `raveolens plants[

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Fig[ 1[ Northern blot hybridizations of acridone synthase transcripts from elicitor!treated or light!irradiated Ruta cells[ Poly"A#¦ RNA "1 mg:lane# from dark!cultured Ruta cells that had been induced for various time intervals with "A# Pm` elicitor or "B# white light irradiation was separated on a 0[1) agarose gel\ blotted onto nylon _lters and the blots were hybridized with 21P! labeled ACS cDNA[ The transient induction of ACS transcript amounts "0[5 kb# in Ruta cells had been recorded previously upon the addition of elicitor ð07Ł[ The autoradiographic signal intensities were compared by densitometric evaluation and equal loading of the gel was con_rmed by rehybridisation of the blots with 21P!labeled ubiquitin cDNA "1[9 kb#[

RESULTS

Transcriptional re`ulation of ACS upon irradiation or elicitor treatment The accumulation of acridone alkaloids in dark! grown Ruta `raveolens cell cultures is stimulated upon elicitation and the ACS cDNA had been cloned from the cells treated for 5 h with Pm` elicitor ð07Ł[ This cDNA did not hybridize with CHS transcripts under stringent conditions ð07Ł[ The kinetics of ACS transcript abundance in Ruta cell cultures was monitored in response to continuous irradiation with white light or treatment with Pm` elicitor by Northern hybridization of the poly"A#¦ RNA from cells harvested at various intervals of treatment over a period of 13 h "Fig[ 1#[ Calibrated equivalent amounts of mRNA were used in these studies and the blots were rehybridized with a ubiquitin cDNA probe for additional standardization[ Hybridization and densitometric scanning revealed that ACS mRNA "0[5 kb# was present in non!treated cells and reached a transient maximum of approximately 2!fold intensity within 1Ð3 h of elicitation ðFig[ 1"A#Ł[ Thereafter\ the speci_c mRNA decreased rapidly to the background level[ When Ruta cultures were irradiated with continuous white light for 13 h\ the response di}ered signi_cantly[ Under these conditions\ the amounts of mRNA decreased rapidly to a minimum at 7 h after onset of the irradiation and increased again to the initial level after 01 to 04 h ðFig[ 1"B#Ł[ ACS protein levels after irradiation or elicitation The polypeptide sequence translated from the ACS cDNA ð07Ł revealed a high degree of homology to heterologous CHSs and STSs and polyclonal CHS antibodies crosshybridized with the Ruta ACS poly! peptide ð07Ł[ Conceivably\ extensive homology to the

Ruta indigenous CHS is also to be expected[ A stretch of nine amino acids with negligible sequence hom! ology to CHSs and STSs was thus chosen from the ACS polypeptide[ This nonapeptide was synthesized\ coupled to ovalbumin and an ACS!speci_c polyclonal antiserum was generated in rabbit[ The crude anti! serum showed minor cross!reactivities with a few additional proteins in E[ coli and plant extracts\ and\ therefore\ the ACS!speci_c IgG fraction was isolated using a blot!a.nity procedure ð13Ł[ The puri_ed anti! bodies showed narrow speci_city for ACS\ which was documented by Western blotting experiments employ! ing crude and puri_ed ACS from Ruta\ CHS from Scotch pine "G[ Schroder\ Freiburg# or crude extracts from parsley cells containing CHS "data not shown#[ The speci_city was furthermore con_rmed by immu! notitration experiments in which ACS and CHS activities were quanti_ed "Fig[ 2#[ The puri_ed antibodies were used to measure the relative amounts of ACS protein in crude cell extracts of cultured Ruta cells after exposure to either con! tinuous irradiation or elicitor treatment[ Signi_cant amounts of ACS enzyme are present in non!induced Ruta cells\ which nevertheless increased continuously from 5 to 13 h following the addition of elicitor ðFig[ 3"A#Ł[ In contrast\ continuous light irradiation of the cells induced a very subtle decrease in the amount of ACS protein over the entire duration of the experi! ment ðFig[ 3"B#Ł[ The divergent induction patterns were veri_ed in these experiments by densitometric evaluation of the _lm signal intensities produced by the ECLTM detection system[ Re`ulation of ACS activity upon irradiation or elici! tation The ACS activity in crude extracts of elicitor!tre! ated Ruta cells followed the observed time course of

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Fig[ 2[ Immunotitration of ACS and CHS synthase activities with anti!ACS IgGs[ The enzyme assays were carried out with crude extracts from Ruta cells that had been treated with elicitor "ACS activity ,\ Ž# or irradiated with white light for 04 h "CHS activity \ #[ The extracts "49 mg pro! tein# were incubated for 0 h in the presence of anti!ACS "\ Ž# or preimmune IgGs "\ ,# "099 ml total volume#\ and the activities were determined from the supernatant following the centrifugation[ The immunotitrations were carried out in triplicate and the relative enzyme activity of 099) cor! responded to 2[6 mkat:kg[

ACS enzyme protein levels[ Signi_cant activity was present in non!induced cells\ which\ after a lag of about 4 h\ increased continuously to an approximately 2!fold level within 13 h following the addition of elici! tor "Fig[ 4#[ The opposite e}ect was observed on light irradiation\ which suppressed the ACS activity to minimal values of roughly 49) after 7 to 09 h but the activity returned to a near!normal level within 13 h "Fig[ 4#[ ACS expression and distribution of acridones in Ruta plant tissues The detrimental e}ect of light on acridone biosyn! thesis might suggest that acridone alkaloids are syn!

Fig[ 4[ ACS activity of crude extracts from elicitor! or light! treated Ruta cells[ The extracts were prepared from dark! cultured Ruta cells that had been induced for various time intervals with ",# Pm` elicitor or "Ž# white light irradiation[ Values are the average of _ve independent induction assays[

thesized exclusively in the roots of Ruta plants\ from where they are translocated to the shoot[ This would be compatible with reports which ascribed the highest storage of Ruta acridones to the root ð4Ł[ Acridone alkaloids emit strong yellow ~uorescence upon exci! tation at 229Ð499 nm and are readily detected in tissue sections by ~uorescence microscopy ð4Ł[ Their spatial distribution can be correlated with the probable sites of acridone biosynthesis\ if the ACS mRNAs are spot! ted in the same sections by tissue print hybridization employing an antisense RNA probe ð14Ł[ The expression of ACS mRNAs was observed in the root as well as in the hypocotyl and in the seed coat of Ruta plants[ In the root\ strong hybridization signals corresponded to the cell layers bordering the rhizodermis ðFig[ 5"A#Ł and high ACS transcript abun! dance prevailed also in the hypodermis and vascular tissue of the hypocotyl ðFig[ 5"B#Ł as well as in the seed coat ðFig[ 5"C#Ł[ The observed patterns faithfully

Fig[ 3[ Evaluation of ACS protein amounts by Western blotting employing anti!ACS IgGs[ Crude extracts "09 mg protein# were prepared from dark!cultured Ruta cells that had been induced for various time intervals with "A# Pm` elicitor or "B# white light irradiation[ The proteins were separated by 01) SDS!PAGE and blotted subsequently to nitrocellulose _lters for crossreaction with the anti!ACS!IgGs[ The crossreacting band was visualized by the ECLTM detection system\ the Mr was compared to standard protein size markers and the relative signal intensities were evaluated by densitometric scanning[

Ruta `raveolens acridone synthase

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Fig[ 5[ Tissue!speci_c expression of ACS in Ruta `raveolens L[ plants[ Hand!cut sections from the root\ stem and fruit were printed onto nylon membranes and the spatial relative abundance of ACS transcripts was monitored by hybridization with the 24S!labeled ACS antisense RNA probe using the tissue print hybridization technique ð14Ł[ Control hybridizations with labeled ACS sense RNA yielded scattered\ weak background signals only "data not shown#[ Left column] unstained tissue sections of "A# the root\ "B# the hypocotyl and "C# the fruit of R[ `raveolens[ Right column] the spatial distribution of ACS mRNAs in the tissue sections detected by tissue print hybridizations[

represented the ACS mRNA distribution\ since con! trol hybridizations with the sense RNA probe failed to reveal discrete signals "data not shown#[ The ~u! orescence of acridone alkaloids was most prominent in the root tissue\ where it was primarily present in idioblasts and in the parenchymatic cells of the pro! tective tissue "kalyptra and rhizodermis or hypo! dermis# ðFig[ 6"A#Ł[ In the hypocotyl and leaf base\ acridones accumulated in the vascular tissue as well as directly beneath the epidermis ðFig[ 6"B# and "C#Ł[

DISCUSSION

ACS catalyzes a polyketide condensing reaction very similar to those of CHS or STS but requiring an aminobenzoyl! rather than "hydroxy#cinnamoyl!CoA starter substrates "Fig[ 0#[ The marginal di}erences in the substrate speci_cities of CHS and STS have long caught the attention and stimulated numerous mutational studies ð08\ 11\ 12Ł\ whereas ACS was cloned only recently and shown to share a high degree of homology at both the DNA and polypeptide level

397

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Fig[ 6[ Tissue distribution of acridone alkaloids in Ruta `raveolens L[ plants[ The acridones were recognized under the microscope by their yellow ~uorescence "229 nm excitation\ 499 nm cut!o} _lter# and the sites of acridone deposition were denoted by arrows in hand!cut sections of "A# the root\ "B# the hypocotyl and "C# the leaf base "magni_cation 074!fold#[

with heterologous CHSs and STSs ð07Ł[ Nevertheless\ ACS cDNA did not hybridize with CHS cDNAs from Pinus sylvestris "J[ Schroder\ Freiburg# or Sinapis alba "A[ Batschauer\ Freiburg# under stringent conditions\ which was the basis for selective de novo induction studies[ The cloning of Ruta endogenous CHS is now in progress and signi_cant sequence di}erences to

ACS are to be expected in the 3!coumaroyl!CoA bind! ing site ð08Ł and other DNA regions to account for the lack of cross!hybridization^ these di}erences might reveal further information on the reaction mechanism[ Analogous to other enzymes of the plants| defense ð15\ 16Ł\ ACS mRNA abundance was transiently induced upon the elicitation of Ruta cells ðFig[ 1"A#Ł\

Ruta `raveolens acridone synthase

whereas the transcript level decreased upon light irradiation to a transient minimum at 5Ð7 h ðFig[ 1"B#Ł and the di}erential mode of induction was basically con_rmed by Western blotting experiments which revealed a steady 13 h!increase of ACS polypeptide levels in the elicited Ruta cells ðFig[ 3"A#Ł while the amounts decreased slightly upon irradiation ðFig[ 3"B#Ł[ Such a pattern was to be expected\ if the half!life of the enzyme exceeds 04 to 19 h[ The determination of ACS activities again con_rmed the signi_cantly di}erent e}ects of the elicitation vs light irradiation of Ruta cell cultures "Fig[ 4#[ CHS activity is known to be stimulated by light irradiation in various plant cell cultures ð12\ 16Ł and\ for comparison\ the CHS activity of the Ruta cells\ which remained negligible in dark!grown control cells\ also reached a transient maximum at 01Ð04 h under these conditions "unpub! lished#[ In contrast to the de novo irradiation induction ð16\ 17Ł\ light!induced down!regulation has been reported in only a few instances ð18Ð24Ł and may be accompanied by light!dependent degradation of enzyme proteins as has been shown for NADPH! dependent protochlorophyllide oxidoreductase dur! ing chloroplast development ð18\ 23Ł[ The transient down!regulation of ACS may aid in channeling mal! onyl!CoA into the accumulation of UV!_ltering ~a! vonoid pigments ð25Ł\ a phenomenon which has been shown to be induced by light treatment in many plants ð16\ 24Ł[ In this sense\ the expression of Ruta ACS and CHS is mutually competitive and is conceivably under the control of diverse and tissue!speci_c promoters ð12Ł[ CHS is distributed ubiquitously in higher plants\ whereas ACS occurs exclusively in the evolutionary advanced family of the Rutaceae[ Cladistic analysis revealed that ACS does not group readily with het! erologous CHSs or STSs "unpublished# and the evol! ution of Ruta ACS may have branched early from common ancestral genes[ Thus\ the Rutaceae appear to have managed to exploit CHS homologous genes for an entirely di}erent purpose\ although the phylo! genetic relationship of CHS and ACS remains to be more closely examined[ The accumulation of acridones in distinct plant tis! sues can result from localized gene expression and:or product translocation processes^ and the large amount of acridones in the root might suggest that root!to! shoot translocation operates in Ruta as has been shown for tropane alkaloids in solanaceous plants ð26Ł or nicotine and pyrrolizidine alkaloids in Senecio vul`aris ð6Ł[ Furthermore\ whereas an accumulation of alkaloids in the peripheral stem and leaf tissues is a common phenomenon "cf[ð6Ł# Ruta acridone alkaloids accumulate in idioblasts ðFig[ 6"A#Ł as well as in the vascular tissue ðFig[ 6"B# and "C#Ł[ The signals observed in tissue print hybridizations ðFig[ 5"A#Ð"C#Ł correlated closely with all the microscopic sites of acridone deposition\ including the vascular tissue\ which clearly demonstrates that the acridones are most likely synthesized at the site of storage and that

398

translocation is not required in Ruta plants[ Fur! thermore\ the hybridization signals from hypocotyl sections did not correspond solely to idioblasts but rather spread evenly through the hypodermal cells and were also present to some extent in the vascular tissue ðFig[ 5"B#Ł[ Since the autoradiographic resolution was su.ciently high\ these results indicate that the acri! dones may be transported into the idioblasts from neighboring cell layers[ Acridones such as rutacridone and hydroxyrutacridone epoxide "Fig[ 0# possess potent antimicrobial activities\ and their con! centration may increase up to 099!fold two days after fungal elicitation ð27Ł[ The tendency of acridones to accumulate predominantly in the peripheral cell layers of the root and the fact that ACS expression responds to fungal elicitation may thus be exploited for the transformation of other plants with the ACS gene"s# to confer resistance against soil!borne pathogens such as Phytophthora spp[ EXPERIMENTAL

Enzymes and materials Biochemicals\ vectors\ E[ coli host strains\ restric! tion enzymes and DNA and RNA modifying enzymes were purchased from Boehringer!Mannheim\ Phar! macia\ BRL\ Stratagene and USB[ All reagents were of analytical grade[ð24SŁUTPaS "approximately 26 TBq = mmol−0# and ð1!03CŁmalonyl!CoA −0 "0[78 GBq = mmol # were from Amersham!Buchler and malonyl!CoA was purchased from Sigma[ 3! Coumaroyl!CoA\ N!methylanthraniloyl!CoA and 0\2!dihydroxy!N!methyl!acridone had been syn! thesized previously ð05\ 07Ł[ Cell cultures Cell suspension cultures of the common rue "Ruta `raveolens L[ strain R!19# ð05Ł^ were propagated in the dark ð05Ł and the cells were induced by either the addition of crude elicitor "4 mg:39 ml culture# from the cell wall of Phytophthora me`asperma f[ sp[ `ly! cinea "Pm` elicitor# ð28Ł syn[ Phytophthora sojae or by irradiation with white light "07[3 W:m1#[ The elicitor was routinely dissolved in hot fresh cell culture med! ium which by itself had no inducing e}ect[ Crude enzyme extracts and enzyme assays Cell!free extracts for enzyme determination and mRNA isolation were prepared from cells harvested after various time intervals of induction\ frozen immediately in liquid nitrogen and stored at −69>C until use[ The frozen cells were extracted by grinding for 4 min with an equal volume of 099 mM sodium phosphate bu}er pH 6[4\ containing 19 mM sodium ascorbate\ 0 mM DTT\ 9[4 mM EDTA and 09) "v:v# glycerol[ The extract was cleared by centrifugation "01\999×`# and the supernatant was used immedi!

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ately for enzyme assays or SDS!PAGE[ ACS and CHS activity assays were carried out as described ð05\ 39Ł[ Polyclonal antisera The peptide AÐLÐFÐSÐAÐNÐIÐDÐT with negligible homology to heterologous CHSs was chosen as a hapten from the translated ACS polypeptide sequence[ The peptide was synthesized and coupled with BDB to ovalbumin "16 mol peptide:mol oval! bumin# "Neosystems#[ The conjugate "149 mg# in Freund|s adjuvans was injected s[c[ into a New Zea! land White rabbit followed by four booster injections "499 mg each# at 3 week intervals[ The serum used in this report was collected two weeks following the second booster injection[ The ACS!speci_c IgG frac! tion was puri_ed from the crude antiserum by blot! a.nity puri_cation ð13Ł[ Brie~y\ homogeneous ACS polypeptide was separated on a 01) SDS!PAGE gel and electroblotted to nitrocellulose[ The protein band was visualized with Ponceau S\ cut from the _lter and blocked overnight with 0) BSA in TBS "49 mM Tris = Cl pH 7[0\ 049 mM NaCl#[ The strips were then incubated overnight with the crude antiserum and washed subsequently 4 times with TBS containing 9[94) Nonidet P!39[ Bound IgG was eluted from the _lter strips in 0 ml 49 mM glycine pH 2[9 and immediately neutralized with 1 M TrisÐHCl pH 8[9[ Preimmune serum IgG was puri_ed by chro! matography on a Protein A Sepharose column "0[5×09 cm# "Pharmacia#[ The serum "4 ml# was applied to the column which had been equilibrated with 099 mM TrisÐHCl bu}er pH 6[1[ The column was washed further with this bu}er until the absorption of the eluate returned to baseline[ The IgG fraction was eluted with 099 mM glycineÐHCl bu}er pH 2[9 and the eluate was immediately adjusted to pH 7 with 0 M TrisÐHCl bu}er pH 8[ The IgG fraction was concentrated by ultra_ltration[ Immunotitration and Western blottin` Crude extract "09 mg protein in approximately 09 ml for Western blotting^ 49 mg protein in 39 ml for immu! notitration# from Ruta cells that had been induced for 04 h by treatment with elicitor or by irradiation with white light were incubated with either ACS or pre! immune IgGs[ After 0 h at room temperature\ 19 ml of Pansorbin "Calbiochem# was added and the incu! bation was continued for an additional 29 min at room temperature[ The Pansorbin precipitate was pelleted in a microfuge "04\999×` for 04 min at 3>C# and ACS ð05Ł and CHS ð39Ł activities in the supernatant were determined[ SDS!PAGE and Western blotting "equi! valent protein amounts:lane# was carried out using 01) separation and 4) stacking gels[ Fixing and staining was accomplished in 49) methanol\ 09) acetic acid\ 9[0) Serva Blue R "Serva\ Heidelberg# and destaining in the same solution without Serva Blue R[ For Western blotting\ proteins were trans!

ferred to cellulose nitrate _lters "9[34 mm\ Schleicher and Schuell\ Dassel# in transfer bu}er "14 mM Tris\ 089 mM glycine\ 19) methanol# and blocked with 0) BSA in TBS "49 mM TrisÐHCl bu}er pH 7[0\ containing 049 mM NaCl#[ The blots were treated with diluted ACS IgGs in TBS "0]299\ 0[2 ng:ml# and crossreacting proteins were visualized with the ECLTM Western blot detection system "Amersham!Buchler#[ The semi!quantitative determination of ACS poly! peptide in the range of 9[0 to 4 mg had been con_rmed with this detection system employing puri_ed ACS expressed in E[ coli ð07Ł[ Developed _lms were quant! i_ed by densitometric scanning "_ve scans per lane# using an LKB 1191 Ultroscan Laser Densitometer[ Isolation of mRNA and Northern blottin` Total RNA was isolated from deep!frozen cells using an RNeasy Midi Kit "QIAGEN# with a modi_ed protocol[ Frozen cells "09 g# were homogenized with quartz sand "0 g# in 09 ml warm "54>C# Bu}er RLT and 09 ml 1!mercaptoethanol for 4 min using a mortar and pestle[ The homogenate was _ltered through nylon mesh "099 mm# and centrifuged "04\999×` at 19>C for 09 min#[ The supernatant was passed 4 times through a 17!gauge needle and centrifuged "04\999×` at 19>C for 09 min#[ Ethanol was added to the super! natant "_nal concentration\ 31)#\ and the standard RNeasy procedure was continued[ Poly "A#¦ RNA was isolated from total RNA using Oligotex mRNA Midi Kit "QIAGEN#[ Equivalent amounts of the mRNA "1 mg:lane# were separated by agarose gel elec! trophoresis ð30Ł and blotted on Hybond!N¦ mem! branes for hybridization ð31Ł[ Blots were pre! hybridized overnight at 31>C with herring sperm DNA "09 mg:ml# in 5!fold SSC and 4!fold Denhardt|s solution ð31Ł containing 9[4) SDS[ Hybridization was continued for an additional 13 h at 51>C after the addition of the labeled ACS cDNA ð07Ł or ubiquitin probe "A[ Batschauer\ Universitat Freiburg#[ The _l! ters were washed twice in 5!fold SSC containing 9[0) SDS at room temperature for 19 min followed by one wash at 61>C for 4 min and the dried _lters were sub! jected to autoradiography ð31Ł[ The relative ACS tran! script abundance was compared to the amount of poly"A#¦ RNA determined spectroscopically "1 mg:lane# and equal loading of the gel was con_rmed on the basis of the ubiquitin mRNA abundance moni! tored by rehybridization of the _lters after washing in 9[0 SSPE[ The _lm signal intensities were quanti_ed by densitometric scanning "_ve scans per lane# using an LKB 1191 Ultroscan Laser Densitometer[ Tissue print hybridization and microscopy Stems and roots of 7!month!old Ruta plants were hand!sectioned\ printed onto Hybond!N membranes "Amersham# and cross!linked as described ð14Ł[ Tissue sections were photographed under a dissecting micro! scope[ pBluescript SK containing the ACS cDNA ð07Ł

Ruta `raveolens acridone synthase

was used for the synthesis of 24S!labeled sense or anti! sense RNA probes ð31Ł[ The blotted _lters were hybridized and washed using the conditions described for the Northern blotting experiments[ Fluorescence microscopy and photography was carried out using an Axiophot microscope "Zeiss#[ The intensive yellow ~uorescence upon excitation at 229 nm ð7Ł identi_ed the acridone alkaloids in the tissue sections[

19[ 10[

11[ 12[

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