Running Title: Alkaloids in tobacco BY-2 cells Corresponding author

28 Pages·2008·0.65 MB·English

PlantCellPhysiol.49(8):1209–1216(2008)
doi:10.1093/pcp/pcn096,availableonlineatwww.pcp.oxfordjournals.org
(cid:1)TheAuthor2008.PublishedbyOxfordUniversityPressonbehalfofJapaneseSocietyofPlantPhysiologists.
Allrightsreserved.Forpermissions,pleaseemail:[email protected]
Why does Anatabine, But not Nicotine, Accumulate in Jasmonate-Elicited
Cultured Tobacco BY-2 Cells?
Tsubasa Shoji and Takashi Hashimoto *
GraduateSchoolofBiologicalSciences,NaraInstituteofScienceandTechnology,8916-5Takayama,Ikoma,Nara,630-0192Japan
Suspension-cultured cells of Nicotiana tabacum cv. a Japanese researcher at Hatano Tobacco Experimental
BrightYellow-2(BY-2)growrapidlyinahighlyhomogenous Station in 1968, and was distributed to many plant
D
populationandstillexhibitthegeneralbehaviorofplantcells, laboratories worldwide for basic research (Nagata et al. o
w
and thus are often used as model systems in several areas of 1992, Nagata et al. 2004). Tobacco BY-2 cells grow very n
lo
plant molecular and cellular biology, including secondary rapidlyinculture;thetypicaldoublingtimeisabout13h.In ad
e
mniectoatbinoeliasmsa. mWahjiolreatlhkealpoaidr,enthtaelctuoltbuarcecdotovbaaricectoycseyllnsthmeasiinzelys ceealclhsuostpheenr,siaonndcautltmuoresst,oBnYly-2focremllssmaraellwaeglglrseegpaatreastoedfafrfoemw d from
produce a related alkaloid anatabine, instead of nicotine, cells.ThecellcycleofBY-2cellscanbesynchronizedeasily h
when elicited with jasmonates. We report here that cultured andefficiently.Becauseoftheirhighhomogeneityandrapid ttps
BY-2 cells scarcely express N-methylputrescine oxidase growth, tobacco BY-2 cells have been used as model ://a
c
(MPO) genes even after jasmonate elicitation. MPO is the systemsforhigherplants,whicharecomparablewithHeLa ad
e
second enzyme in the biosynthetic pathway that supplies the cellsinhumanresearch,andareespeciallyusefulforstudies m
ic
pyrrolidine moiety of nicotine and nornicotine, but is of cell division, cytoskeletons, plant hormone signaling, .o
u
predicted to be dispensable for the biosynthesis of anatabine, p
intracellular trafficking, organelle differentiation, and pri- .c
anabasineandanatalline,whichdonotcontainthepyrrolidine mary and secondary metabolism. om
moiety.WhenMPOwasoverexpressedintobaccoBY-2cells, /p
TobaccoBY-2cellsretaintheirresponsestoexogenous c
p
nicotinesynthesiswasdramaticallyenhancedwhileanatabine applicationoffungalelicitorsandjasmonates.Elicitationof /a
formation was effectively suppressed. As a complementary BY-2 cells with methyl jasmonate induces large transcript- rticle
approach, we suppressed MPO expression by RNA inter- omic changes and initiates denovo synthesis of phenylpro- -a
b
ference in tobacco hairy roots that normally accumulate s
tnaicbointien,ea.nIanbathsieneManPdOa-nsuaptaplrleinsese,dasrwooeltlsa,sthNe-mcoenthteynlptsutorfesacninae- p20a0n3o,idGsaa´lnisdeatlakla.l2o0id0s6)(.ImThanesisehjiaesmtaoln.a1t9e9-8in,dGuociobslseensesceotnadl-. tract/4
and putrescine, markedly increased to compensate for sup- adreyfenmseestabooflitetsobaarcecothopulgahnttsto(Sbteeppinuvhonlvedet inal.che2m00ic4a)l. 9/8/1
pressed formation of nicotine and nornicotine. These results 20
Interestingly, jasmonate-elicited BY-2 cells accumulate 9
identify the transcriptional regulation of MPO as a critical /1
mainly anatabine, an analog of nicotine; nicotine and 8
rate-limitingstepthatrestricts nicotine formationin cultured 26
other tobacco alkaloids are produced in relatively minor 1
tobaccoBY-2cells. 8
amounts (Goossens et al. 2003). Since the original Bright 1 b
Yellow variety of tobacco accumulates predominantly y g
Keywords: Alkaloids — Anatabine — Nicotine — u
nicotine (Ha¨kkinen et al. 2004), there should be one or e
s
N-methylputrescine — Tobacco. morestepsdeficientinthebiosyntheticpathwayofnicotine t o
n
Abbreviations: CaMV, cauliflower mosaic virus; in otherwise jasmonate-competent BY-2 cells. 05
MPO, N-methylputrescine oxidase; PCA, perchloric acid; The pyrrolidine moiety of nicotine is derived from a Ap
PMT, putrescine N-methyltransferase; RNAi, RNA inter- symmetric diamine putrescine (Fig. 1). Putrescine is first ril 2
ference; RT–PCR, reverse transcription–PCR; QPT, qui- N-methylated by putrescine N-methyltransferase (PMT), 01
9
nolinate phosphoribosyltransferase. and the product N-methylputrescine is then oxidatively
deaminated by N-methylputrescine oxidase (MPO) to
4-aminobutanal, which spontaneously cyclizes to give
N-methylpyrrolinium cation. MPO belongs to a subclass
of diamine oxidases that require copper and topaquinone
Introduction for activity (Heim et al. 2007, Katoh et al. 2007). The
N-methylpyrrolinium cation is then coupled with a
ThetobaccocelllineBY-2wasoriginallyinducedfrom hypothetical nicotinic acid-derived intermediate to form
thepithofNicotianatabacumL.cv.BrightYellowNo.2by nicotine (Leete 1983, Hashimoto and Yamada 1994).
*Correspondingauthor:E-mail,[email protected];Fax,þ81-743-72-5529.
1209
1210 AlkaloidsintobaccoBY-2cells
Nicotinic acid is formed in the salvage pathway of suggested by feeding studies (Watson et al. 1990, and
nicotinamideadeninedinucleotide(NAD),whichisderived references therein). Although recombinant tobacco MPOs
fromasparticacidinplants(Katohetal.2006).Quinolinate show a weak but measurable activity that converts
phosphoribosyltransferase (QPT) connects the aspartate- cadaverine to 5-aminopentanal (Heim et al. 2007, Katoh
derived pathway to the pyridine nucleotide cycle (Sinclair etal.2007),itisnotknownwhetherMPOisinvolvedinthe
et al. 2000). The structural genes for nicotine biosynthesis anabasine biosynthesis. The biosynthetic route of anatal-
are generally under the control of the specific regulatory line, which contains two pyridyl rings and a central
loci, NIC1 and NIC2 (Hibi et al. 1994, Reed and Jelesko saturated heterocyclic ring, is still unresolved.
2004,Caneetal.2005,Heimetal.2007,Katohetal.2007; A very high ratio of anatabine to nicotine in
our unpublished results). jasmonate-treated BY-2 cells indicates that one or more
D
Other tobacco alkaloids are heterocyclic compounds step(s) in the pyrrolidine moiety formation of nicotine is o
w
composedofonenicotinicacid-derivedpyridylringandone blocked. Indeed, when PMT expression was down- nlo
or two 5- or 6-membered N-containing ring(s) (Fig. 1). regulated in tobacco hairy roots and regenerated plants, ad
e
A main route to nornicotine is direct N-demethylation of anatabine accumulated to high levels while nicotine levels d
nicotine,whichiscatalyzedbyaparticularsubclassofP450 decreased (Chintapakorn and Hamill 2003). In this study, from
monooxygenases (Siminszky et al. 2005, Gavilano and we found that MPO, but not PMT, is responsible for the h
ttp
Siminszky 2007). Feeding of labeled nicotinic acid to high anatabine to nicotine ratio in jasmonate-elicited BY-2 s
N. tabacum and N. glutinosa plants suggested that both cells.ExceptfortheidentifiedmisregulationofMPOgenes, ://a
c
a
rings of anatabine are derived from the dimerization of a tobacco BY-2 cells apparently induce all other enzyme d
e
metabolite of nicotinic acid (Leete and Slattery 1976). genes necessary for nicotine biosynthesis in response to m
ic
Anabasine,amajoralkaloidinN.glauca,ismainlyformed methyl jasmonate treatment, and are useful for metabolic .o
u
from lysine via a symmetric diamine cadaverine, as studies of tobacco alkaloids. p.c
o
m
/p
c
putrescine p
PMT NH2 NH2 /artic
aspartate le
-a
N-methylputrescine H bs
lysine NH NH2 2HN N NH2 trac
quinolinate CH3 spermidine t/4
MPO 9
QPT /8
M5P-aOcma?dinaovpeerinnteanal NAD nminacoicocnitodointnianuticceleotide PNCyuyrccildleeinoetide CNHH3Cs4Hp-Ooanmtainnoebouutasn2aHlN HNsperHNmineNH2 /1209/18261
cyclization 8
1
∆1-piperidine b
N 1-methyl-∆1-pyrrolinium cation y g
u
CH3 es
anabasine t on
0
N NH N NH N NCH3 nicotine 5 April 2
0
anatabine 1
9
N nornicotine
N
anatalline H
N
N
H
N
Fig. 1 Biosynthesis of tobacco alkaloids. Nicotine and nornicotine are synthesized by condensation of the 1-methyl-(cid:1)1-pyrrolinium
cation and a nicotinic acid-derived precursor, whereas anatabine and possibly anatalline are derived from nicotinic acid only. Lysine-
derived(cid:1)1-piperidineisincorporatedintothepiperidinemoietyofanabasine.PMTandMPOcatalyzetheinitialtworeactionsleadingto
formationofthe1-methyl-(cid:1)1-pyrroliniumcationfromputrescine.QPTisinvolvedinthesynthesisofNAD,whichisconvertedtonicotinic
acid in the pyridine nucleotide cycle. MPO, N-methylputrescine oxidase; PMT, putrescine N-methyltransferase; QPT, quinolinate
phosphoribosyltransferase.Brokenlinesindicatemorethanonereactionorundefinedreactions.
AlkaloidsintobaccoBY-2cells 1211
Results cultured tobacco cells (data not shown). Apparently,
tobaccoBY-2cellsareparticularlyresponsivetojasmonate
MPOishardlyexpressedinanatabine-accumulatingtobacco treatment, and are capable of metabolic reprogramming to
BY-2 cells synthesize tobacco alkaloids.
We first compared alkaloid profiles of tobacco BY-2 Next, expression levels of three structural genes for
cells, which had been treated with methyl jasmonate at nicotinebiosynthesis(MPO,PMTandQPT)wereanalyzed
100mMfor3d,withthoseoftobaccohairyrootsthatwere byRNAgelblots(Fig.2B).PMTandQPTwereexpressed
derived fromthecultivar SR1(Fig.2A).Methyljasmonate inhighandcomparablelevelsinhairyrootsandjasmonate-
treatment does not significantly change the alkaloid treated BY-2 cells. In contrast, whereas considerable MPO
composition in tobacco hairy roots (Shoji et al. 2008). In expressionwasdetectableinhairyroots,wecouldnotdetect
D
this study, tobacco hairy roots were untreated with methyl significant accumulation of MPO mRNA in the elicited ow
jasmonate. The total amounts of tobacco alkaloids were BY-2 cells. In addition, we found that MPO transcript nlo
a
comparable in two culture systems; the alkaloid levels far levels were reduced by only 34% in nic1nic2 mutant roots, d
e
enxiccoeetidneedw1asmtghge(cid:1)m1aDjoWr alokfalcoeildls(.(cid:2)I6n9%toboafctcootahlaailrkyalrooiodtss),, coofmPpMarTedawndithQwPiTld-wtyepreersoevoetrse,lwyhreerdeuacsetdhebtyra8n8scarnipdtl6e6v%els, d from
followed by nornicotine (27%), anabasine (3%), anatabine respectively, in the mutant roots (Fig. 2C). These results http
(B1Y%-2) acnedllsanpartoadlluinceed(0.m1%os)t.lyInancoantatrbainste, j(a8s3m%o)n,atwe-itthrealtoewd pcrriotimcapltefdacutosrtotheaxtplcoaruesweshetthheerhMighPOaneaxtparbeisnseiotnomnaiycobtienae s://ac
a
levels of accumulation of nicotine (5%), anabasine (4%) ratio in jasmonate-treated BY-2 cells. de
m
and anatalline (8%). Nornicotine was not detected in our ic
culture conditions. These alkaloid compositions were Overexpression of MPO restores nicotine formation in .ou
p
similar to and confirm those reported for tobacco hairy tobacco BY-2 cells .c
o
roots(e.g.Shojietal.2000)andforjasmonate-treatedBY-2 If very low expression of MPO is the primary cause m
/p
cells (e.g. Goossens et al. 2003). for the failure to synthesize nicotine efficiently in tobacco cp
Since the tobacco BY-2 cell line was selected for its BY-2 cells, overexpression of MPO should boost nicotine /artic
high growth rate and has been subcultured for several formationattheexpenseofanatabineaccumulation.Totest le
decades, it was of interest to determine whether the high thispossibility,tobaccoMPO1cDNAwasexpressedunder -ab
s
anatabine to nicotine ratio is also found in other tobacco the control of the cauliflower mosaic virus (CaMV) 35S tra
c
cell lines. We elicited with methyl jasmonate two other promoter in tobacco BY-2 cells. By screening more than t/4
9
tobacco cell lines derived from cultivars Samsum NN and a dozen transgenic cell lines, we found three independent /8
XanthiNCunderthesameconditions.However,onlytrace lines (OX4, OX6 and OX14) that showed highly increased /12
0
amounts of tobacco alkaloids (50.004mgg(cid:1)1 DW of cells; MPO mRNA accumulation when examined in the 9
/1
mainly consisting of nicotine) were detected in these jasmonate-elicited condition (Fig. 3A). Cultured tobacco 8
2
6
1
8
1
b
A B Hroaoitry BY-2 C WilPd-ltaypnet rnoico1ntsic2 y gue
s
W) 5 HBYa-ir2y root MPO MPO t on 05
D 4 A
nt (mg/g 3 PQMPTT PQMPTT pril 2019
e
nt
o 2
C
d TUB TUB
oi
al 1
k
Al
rRNA rRNA
0
Nicotine NornicotineAnabasine Anatabine Anatalline
Fig. 2 Profiles of tobacco alkaloids and gene expression. Tobacco BY-2 cells were treated with 100mM methyl jasmonate for 3d for
alkaloidanalysis(A)orfor1dforRNAgelblotanalysis(B).TobaccohairyrootsofthecultivarSR1(A,B)andtherootsofwild-typeandthe
nic1nic2mutanttobaccoplantsofthecultivarBurley21(C)werenottreatedwithmethyljasmonate.(A)AlkaloidcontentsintobaccoBY-2
cells(blackbars)andtobaccohairyroots(whitebars).Dataaremeans(cid:3)SDofmorethanfourreplications.(B,C)RNAgelblotanalyses.
1212 AlkaloidsintobaccoBY-2cells
A Northern Blot Analysis C 3
nicotine nornicotineanabasine anatabine anatalline
OX lines
C 4 6 14
MPO1
W)
D 2
rRNA g
g/
m
ent ( D
B nt ow
ein) d Co nloa
prot aloi 1 ded
mg 200 Alk fro
vity (pkat/ 100 m https://a
MPO Acti 0 NCD 4 6 14 0 C 4 614 NCD4N6D14 C 4 614 C 4 614 C 4 614 cademic
OX lines OX OX OX OX OX .ou
p
.c
Fig.3 OverexpressionofMPO1intobaccoBY-2cells.Culturedtobaccocellsweretreatedwith100mMmethyljasmonatefor1dfor om
RNAgelblotanalysis(A)andMPOenzymeassay(B),andfor3dforalkaloidanalysis(C).Dataaremeans(cid:3)SDofmorethanfoursamples. /p
c
ND,notdetected. p
/a
rtic
le
cells of these cell lines also showed high enzymatic activity much more strongly than MPO2 in tobacco hairy roots -ab
s
of MPO, in contrast to the negligible MPO activity in the (Supplementary Fig. S1). Therefore, we used an MPO1 tra
c
control wild-type cells (Fig. 3B). cDNAfragmenttosuppressexpressionofbothMPO1and t/4
9
AlkaloidsintobaccoBY-2cellswereanalyzedafterthe MPO2.Afterscreening11independenthairyrootlines,we /8
treatment with methyl jasmonate. The three MPO-over- identified three lines (R10, R11 and R39) in which the /12
0
expressing cells accumulated nicotine at the level of transcriptlevelsofMPOgenesweresignificantlydecreased 9
/1
1.5–2.5mgg(cid:1)1 DW of cells, which was about a 14- to (Fig.4A).IndividualRT–PCRamplificationofMPO1and 82
6
24-fold increase compared with the wild-type BY-2 cells MPO2transcriptsbyusingspecificprimersetsshowedthat 1
8
(Fig. 3C). Small amounts of nornicotine, which is unde- expressionofbothMPOgeneswaseffectivelysuppressed.In 1 b
y
tectableinwild-typecells,werefoundintheOX4andOX14 contrast,expressionlevelsoftwoothernon-targetednicotine g
u
cell lines. In the MPO-overexpressing lines, the cellular biosynthetic genes (PMT and QPT) and of tubulin genes e
s
levels of anatabine, anabasine and anatalline decreased by were indistinguishable among wild-type hairy roots, a t o
n
(cid:4)50%, compared with the wild-type cells. Therefore, vector-transformed line and MPO RNAi lines. Consistent 0
5
tobacco alkaloids containing the pyrrolidine ring (nicotine withthereducedRNAlevel,theMPOenzymeactivitieswere A
p
and nornicotine) increased while non-pyrrolidine alkaloids significantly lower in these three lines than those in the ril 2
decreased by enhanced MPO activity, and the alkaloid controlroots(Fig.4B). 01
9
profilesofMPO-overexpressingBY-2cellsresembledthose We analyzed alkaloid contents in the MPO down-
of tobacco hairy roots (see Fig. 2A). regulated lines. In all three lines, the contents of nicotine
and nornicotine markedly decreased to 1–7 and 3–7%,
Down-regulation of MPO in tobacco hairy roots respectively,ofthecontrols,whiletherespectivecontentsof
As a complementary approach, we used an RNA anatabine,anabasineandanatallineincreased5-to10-fold,
interference(RNAi)technologytodown-regulatetheMPO 1.5- to 6-fold and 5- to 7-fold compared with the controls,
genesintobaccohairyroots.Thetobaccogenomecontains respectively (Fig. 4C). Accordingly, anatabine now con-
two MPO genes, MPO1 and MPO2 (Katoh et al. 2007). stituted the major alkaloid (80% of total alkaloids)
Analysisof MPOtranscriptsbyreversetranscription–PCR accumulating in the hairy roots, and the alkaloid profiles
(RT–PCR), followed by discriminative digestion with intheMPO-suppressedhairyrootsbecamesimilartothose
restriction enzymes, showed that MPO1 was expressed of BY-2 cells (see Fig. 2A).
AlkaloidsintobaccoBY-2cells 1213
A RNAi lines B
C VC R10R11R39 n)
ei
MPO1/2 ot 160
pr
MPO1 g
m 120
MPO2 at/
k
PMT y (p 80
vit
QPT cti 40
A
TUB O
P 0
M C VC R10 R11 R39 D
o
w
n
lo
C 4 a
nicotine nornicotine anabasine anatabine anatalline de
d
fro
m
W) 3 h
g D ttps
mg/ ://a
c
ontent ( 2 ademic
C .o
kaloid 1 up.com
Al /p
c
p
/a
0 rticle
C VCR10R11R39 C VCR10R11R39 C VCR10R11R39 C VCR10R11R39 C VCR10R11R39 -a
b
Fstirga.in4(CD)oorwonf-aresgtrualiantiohnarboofMrinPgOaninetmobpatyccvoechtaoirry(VrCoo)otsr.aToMbPaOcc-oRNhaAiriyverocototsr(wRe).re(Ao)bRtTai–nPeCdRaaftneraliynsfiesc.tEioxnproefssthioenwoifldtw-toypMePAO.rgheiznoegsewneass strac
analyzedtogether(MPO1/2)orseparately(MPO1andMPO2).(B)MPOenzymeactivity.(C)Alkaloidcontent.Dataaremeans(cid:3)SDof t/4
9
morethanfoursamples. /8
/1
2
0
9
/1
8
2
We extended our metabolic analysis to cellular poly- Discussion 61
8
amines that are related to the MPO reaction (Fig. 1). 1 b
Polyamineswerefirstseparatedintotwofractionsbasedon MPO is the rate-limiting enzyme for nicotine biosynthesis in y g
u
their solubility in perchloric acid (PCA), and then tobacco BY-2 cells e
s
polyamines conjugated to phenylpropanoids (Ga´lis et al. A high anatabine to nicotine ratio in elicited tobacco t o
n
2006) and other compounds were analyzed after acid BY-2 cells indicates that one or more steps in the subpath- 0
5
hydrolysis, along with non-conjugated free polyamines way that supplies the pyrrolidine moiety of nicotine is A
p
(Fig. 5). In tobacco hairy roots, diamines (putrescine and blocked. PMT and MPO are two known enzymes that are ril 2
N-methylputrescine) were more abundant than higher specifically involved in this subpathway. Indeed, when 01
9
polyamines (spermidine and spermine), and the conjugated eitherPMT(ChintapakornandHamill2003)orMPO(this
forms constituted considerable proportions of total poly- study) was suppressed in hairy roots or whole plants of
amines. In the MPO-suppressed lines R11 and R39, the tobacco, anatabine formation greatly increased while
contents of free and conjugated forms of diamines nicotine formation was reduced to very low levels. These
increased; the increase was higher in N-methylputrescine studies suggest that when the cellular supply of 1-methyl-
(2.4- to 3.8-fold of the control) than putrescine (1.4- to (cid:1)1-pyrrolinium cation is limited, an activated derivative of
1.9-fold). In contrast, the contents of spermidine and nicotinic acid condenses with another molecule of the
sperminewerenotmarkedlyaffected.Overall,themetabolic nicotinate metabolite to form anatabine. Anatabine con-
impact of suppressed MPO activity in tobacco hairy roots tentsvaryfrom2to(cid:4)20%ofthetotalalkaloidsintheroots
was restricted to its substrate and, to a lesser extent, to the of Nicotiana species (Saito et al. 1985, Sisson and Severson
immediate precursors, among the compounds tested. 1990). The natural variation of anatabine might be caused
1214 AlkaloidsintobaccoBY-2cells
60 in vivo evidence that MPO is not involved in the
N-methyl biosynthesis of anabasine.
putrescine spermidine spermine
-putrescine
50 Regulation of MPO expression
PCA-insoluble conjugate form Structural genes encoding biosynthetic enzymes of a
PCA-soluble conjugate form
particular secondary metabolic pathway are often coordi-
PCA-soluble free form
40 nately regulated (Koes et al. 2005, Saito et al. 2007). In
W)
nicotinebiosynthesis,tobaccogenesforPMT,QPTandan
D
g A622 oxidoreductase are preferentially expressed in the
mol/ 30 root, up-regulated by jasmonate treatment or wounding, D
µ o
nt ( andareunderthecontrolofNICregulatoryloci(Hibietal. wn
e 1994, Shoji et al. 2000, Shoji et al. 2002, Goossens et al. lo
Cont 20 2003, Reed and Jelesko 2004, Sinclair et al. 2004, Cane aded
et al. 2005; our unpublished results). Although MPO is fro
regulated similarly by these factors (Heim et al. 2007, m
h
Katohetal.2007),theNiclociregulateMPOexpressionless ttp
10 teingzhytmlyatthicanacetixvpitryesosifonMoPfOPiMs nTotanredduQcPedTa(sFimg.uc2hC)i.nTthhee s://ac
a
roots of nic mutants, compared with the PMT activity de
m
0 VC R11R39 VCR11R39 VCR11R39 VC R11R39 (nSoatusntdriecrtslyacnodoBrduisnhat1e9d7w9)i.thAepxpparreesnstiolynMofPoOtheexrpsrtersusciotunraisl ic.ou
Fig. 5 Polyamine contents in MPO-suppressed tobacco hairy genes for nicotine biosynthesis. Poor jasmonate-induced p.c
roots. Free polyamines and their conjugate forms in the PCA- o
activation of MPO in cultured BY-2 cells might be m
solubleor-insolublefractionwereanalyzed.Dataaremeans(cid:3)SD /p
mechanistically linked to its loose control by the NIC c
ofmorethanfoursamples. p
regulatorygenes.PromoteranalysisoftobaccoMPOgenes /a
may provide further information on their unique control rticle
when gene expression or the catalytic activity of PMT or patterns. -a
b
s
MPO is uncoordinated with other enzymes in the nicotine tra
biosynthetic pathway. Materials and Methods ct/4
Jasmonate-elicitedBY-2cellsexpressedverylowlevels 9/8
of MPO transcripts, compared with tobacco hairy roots, Plantmaterialsandtransformation /12
0
whereas PMT was expressed strongly in both culture BinaryvectorswereintroducedintoAgrobacteriumrhizogenes 9
systems.AccumulationofN-methylputrescineintheelicited strain ATCC15834 and A. tumefaciens strain EHA105 by /18
2
electroporation.SterileplantsoftheN.tabacumcv.PetitHavana 6
BY-2 cells (Goossens et al. 2003) is consistent with a much 1
lineSR1weregrownonhalf-strengthB5medium(Gamborgetal. 8
lower MPO enzymatic activity than the PMT activity. In 1968) containing 1.5% sucrose and 0.3% gellan gum under 1 b
thisstudy,wedemonstratedthatoverexpressionofMPOis continuous illumination at 268C. To generate the transgenic y g
u
sufficient to restore efficient formation of nicotine to com- hairy roots, leaf discs were co-inoculated with A. rhizogenes e
s
pensate for suppressed formation of anatabine. Therefore, harboringpBI-MPO-RIasdescribed(Kanegaeetal.1994).After t o
in jasmonate-treated BY-2 cells, MPO is the major rate- selection and disinfection on solid MS medium containing n 0
limiting enzyme for biosynthesis of the pyrrolidine moiety 50mgl(cid:1)1 kanamicin and 250mgl(cid:1)1 carbenicillin, the hairy roots 5 A
were subcultured in liquid MS medium (Murashige and Skoog p
ofnicotine,andtheexpressionlevelofMPOdeterminesthe 1962)with3%sucroseevery2weeks. ril 2
ratio of pyrrolidine-type and other tobacco alkaloids. Tobacco BY-2 cells (N. tabacum cv. Bright Yellow-2) were 01
9
Thepiperidineringofanabasineisderivedfromlysine subcultured in liquid MS medium supplemented with 20mgl(cid:1)1
by way of cadaverine (Watson et al. 1990). Cadaverine is KH2PO4, 0.5gl(cid:1)1 MES and 0.2mgl(cid:1)1 2,4-D every week.
TransgenictobaccoBY-2cellsweregeneratedwithA.tumefaciens
convertedto5-aminobutanalbyadiamineoxidase(Walton
strain EHA105 with the pBI-MPO as described (An 1985). After
et al. 1988), followed by a spontaneous cyclization to form
4-day-oldBY-2cellswerefirstrinsedwithandtransferredtofresh
(cid:1)1-piperidine (Fig. 1). The diamine oxidase MPO prefers auxin-free MS medium with methyl jasmonate at 100mM, they
N-methylated diamines for substrates and is much less were cultured for an additional 1d for the RNA and protein
activetowardnon-N-methylatedamines,suchascadaverine analyses,orfor3dforalkaloidmeasurement.
Tobaccocelllinesderivedfromcv.SamsumNNandXanthi
(Heim et al. 2007, Katoh et al. 2007). In our transgenic
NCwereculturedinliquidMSmediumwith0.2mgl(cid:1)12,4-D,and
studies with overexpression and suppression of MPO,
liquid B5 medium with 0.22mgl(cid:1)1 2,4-D and 8.6mgl(cid:1)1 kinetin,
anabasine contents changed in concert with anatabine respectively. One-week-old cells were treated with 100mM methyl
contentsandinaninversewaytonicotinecontents,offering jasmonatefor3dintheauxin-andcytokine-freemedia.
AlkaloidsintobaccoBY-2cells 1215
Plasmidconstruction centrifuged at 15,000r.p.m. for 30min. The pellet fraction
For RNAi experiments, a portion of MPO1 cDNA (þ1,530 containing PCA-insoluble polyamines was suspended in 1ml of
to þ2,050bp) was amplified by PCR, subcloned into 1N NaOH. To liberate free polyamines from conjugated forms,
pHANNIBAL (Wesley et al. 2001), and then inserted into the supernatant and the resuspended pellet were combined with
pBI121 (Clontech, Mountain View, CA, USA) to provide pBI- equalvolumesof12NHClandincubatedat1608Cfor17hinglass
MPO-RI. For MPO overexpression, the full-length MPO1 ORF vials. After removinginsoluble debris by centrifugation, 100mlof
was amplified from MPO1 cDNA with a pair of appropriate the samples were dried up at 708C and dissolved in 100ml of 5%
primersandplacedbetweentheBamHIandSacIsitesofpBI121. PCA.A100mlaliquotwasmixedwith100mlofsaturatedsodium
High-fidelityKOD-plusDNApolymerase(Toyobo,Osaka,Japan) carbonate and 400ml of 7.5% (w/v) dansylchloride acetate
was used for all PCR amplifications and the PCR products were solution, and kept at 608C for 1h. To the reaction mixture,
sequenced to verify that no unintentional mutations were 100ml of 10% (w/v) proline was added and incubated at room
introduced. temperaturefor30mininthedark.Thesamplewasextractedwith D
500ml of toluene and dried at 378C for 20min in an evaporator. ow
RT–PCRandRNAgelblotanalyses The dried residues were dissolved in acetonitrile and analyzed by nlo
Total RNA was isolated by using an RNeasy Kit (Qiagen) HPLC (LC-10 system; Shimadzu) equipped with a YMC-Pack ade
fwroasmsypnlathnetstiizsesduebsyfrSouzpeenrbSycrliipqtuIiIdrneivterorsgeetnr,aannsdcrfipirtsats-setr(IannvditcrDogNenA, OweDreCe-AluMtedcofrloummnth(eYcMolCu,mKnywoittoh,aJappraong)r.aDmamnesydlasoteldvepnotlgyraamdiinenest d fro
m
of 2% phosphate triethylamine buffer (pH 5.2): acetinitrile (v/v)
Camarplslibfiaedd, bCyAP,CURSAw).ithAlaiqpuporotsproiaftethperifmirestr-sst(rbaontdhcMDNPOA1waenrde from60to90%in30minataflowrateof1.0mlmim(cid:1)1at408C, http
MTTPGOC2A,5T0-ACTCCTCTATTGTGTATCCCCCATTTTTGAGT-C30T;GMCPTOG1C,5-30-0CaAndGC50T-GTGTTCCT aemndisswioernewdaevteecletendgtahtothfe51e0xcnimta.tionwavelengthof365nmandthe s://ac
a
TCCTAGCTAAGC-30 and 50-CTCTCTGTCCTGAATACAAG d
e
TGG-30;MPO2,50-TGGAAGTTTGCCTGTTGTGTGTC-30 and m
50-AAGCTGGTACCTGGTCCAAACTG-30; PMT, 50-CACTTT Enzymeassay ic.o
CGAACATCGGAACGG-30 and 50-GCCTGTGTGCATACAA Fresh tissues were homogenized in 200mM potassium up
CTCCTCC-30; QPT, 50-TCACTGCTACAGTGCATCCT-30 and phosphate buffer (pH 7.4) by using a Polytron (Kinematica, .co
50-TTAGAGCTTTGCCGACACCT-30;a-TUBLIN,50-AGTTGG Lucerne, Switzerland). After the homogenate was centrifuged at m
AGGAGGTGATGATGTATGATG-30 and 50-TATGTGGGTC 15,000r.p.m. at 48C, the cleared supernatant was desalted by /pc
p
GCTCAATGTC-30). To avoid saturated amplification, template passing it through a NAP-5 gel filtration column (Amersham /a
amounts and cycle numbers were adjusted in individual Pharmatia Biotech). Protein concentration was determined by a rtic
experiments. Coomassie Protein Assay Reagent Kit (Pierce). The enzyme le-a
Total RNA (10mg) was separated by electrophoresis on a reaction was carried out in 200mM potassium phosphate bs
1.2% formaldehyde agarose gel, and transferred onto a Hybond- (pH 7.4) containing 50mM N-methylputrescine, 24kUl(cid:1)1 gluta- tra
Nþnylonmembrane(AmershamPharmaciaBiotech,Piscataway, mate dehydrogenase from beef liver (Oriental Yeast, Tokyo, ct/4
NJ, USA). Equal loading was confirmed by ethidium bromide Japan), 5.7mM 2-oxoglutarate and 0.19mM NADH, at 308C. 9/8
staining. The blots were hybridized in 50% (v/v) formamide, 5(cid:5) Ammonia formed by the MPO reaction was measured enzymati- /1
SSPE, 2(cid:5) Denhardt’s solution and 0.1% (w/v) SDS at 428C for callywithglutamatedehydrogenasebymonitoringthedecreasein 20
9
16h with probes labeled with 32P by using a Bcabest labeling kit NADHat339nm(KuscheandLorenz1983). /1
8
(Takara, Kyoto, Japan). Washing was performed three times in 2
6
0.2(cid:5)SSPEand0.1%(w/v)SDSat658C.Theblotswereexposed 1
8
toaBASImagingPlate(Fujifilm,Tokyo,Japan)forquantification Supplementary data 1 b
ortoanX-rayfilmforimageacquisition. y g
u
Supplementary data mentioned in the article is e
s
Metaboliteanalysis available at Plant and Cell Physiology online. t o
n
To extract tobacco alkaloids, samples (50mg DW) were 0
lyophilized,homogenizedandthensoakedin4mlof0.1NH2SO4. 5 A
T3,h1e00hro.pm.mog.efonrat1e5mwains.Tsohneicsuatpeedrnfaotran6t0wmasinneauntrdalcizeendtrbifyuagdeddinagt Funding pril 2
0
E0.x4tmrelluot-f125co%lumNnH4(OMHer.ckT)heanmdixetluurteed(1wmitlh) w6amsllooafdcehdlooronftoorman. Ministry of Education, Sports, Science and 19
Theelutantwasdriedupat378C.Thedryresiduesweredissolved Technology, Japan Global COE Program (Frontier
inethanolcontaining0.1%dodecaneasaninternalstandardand Biosciences: strategies for survival and adaptation in a
analyzed by gas–liquid chromatography (GC-14B; Shimadzu, changing global environment).
Kyoto, Japan) equipped with an Rtx-5 Amine capillary column
(Shimadzu). The column temperature was held at 1008C for
10min, then increased from 100 to 2508C in 35min. Nicotine,
Acknowledgments
nornicotine,anatabineandanabasinewerepurchasedfromWako
(Kyoto, Japan). Authentic anatalline was a kind gift from
Dr.Kirsi-MarjaOksman-Caldentey(Ha¨kkinenetal.2004). WethankDr.Kirsi-MarjaOksman-Caldentyfortheauthen-
To extract polyamines and N-methylputrescine, samples ticsampleofanatalline,RIKENBRCforthetobaccoXanthiNC
(37.5mg DW) were lyophilized, homogenized and then soaked in cellline,Drs.IchiroMitsuharaandYukoOhashiforthetobacco
1mlof5%(v/v)PCA.Thehomogenatewaskeptat48Cfor2hand SamsumNNcellline,andYukoOnofortechnicalassistance.
1216 AlkaloidsintobaccoBY-2cells
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(ReceivedMay11,2008;AcceptedJune17,2008)