methyl jasmonate induces traumatic resin ducts, terpenoid resin

Methyl Jasmonate Induces Traumatic Resin Ducts,Terpenoid Resin Biosynthesis, and TerpenoidAccumulation in Developing Xylem of NorwaySpruce Stems1

Diane Martin, Dorothea Tholl, Jonathan Gershenzon, and Jorg Bohlmann*

Max Planck Institute for Chemical Ecology, Carl Zeiss Promenade 10, 07745 Jena, Germany (D.M., D.T., J.G.,J.B.); and Biotechnology Laboratory (D.M., J.B.), Department of Botany (D.M., J.B.), and Department of ForestSciences (J.B.), University of British Columbia, 6174 University Boulevard, Vancouver, Canada V6T 1Z3

Norway spruce (Picea abies L. Karst) produces an oleoresin characterized by a diverse array of terpenoids, monoterpenoids,sesquiterpenoids, and diterpene resin acids that can protect conifers against potential herbivores and pathogens. Oleoresinaccumulates constitutively in resin ducts in the cortex and phloem (bark) of Norway spruce stems. De novo formation oftraumatic resin ducts (TDs) is observed in the developing secondary xylem (wood) after insect attack, fungal elicitation, andmechanical wounding. Here, we characterize the methyl jasmonate-induced formation of TDs in Norway spruce bymicroscopy, chemical analyses of resin composition, and assays of terpenoid biosynthetic enzymes. The response involvestissue-specific differentiation of TDs, terpenoid accumulation, and induction of enzyme activities of both prenyltransferasesand terpene synthases in the developing xylem, a tissue that constitutively lacks axial resin ducts in spruce. The inductionof a complex defense response in Norway spruce by methyl jasmonate application provides new avenues to evaluate the roleof resin defenses for protection of conifers against destructive pests such as white pine weevils (Pissodes strobi), bark beetles(Coleoptera, Scolytidae), and insect-associated tree pathogens.

Conifers produce extensive terpenoid-based resinsthat have long been studied for their industrial im-portance and role in defense against herbivores andpathogens (Bohlmann and Croteau, 1999; Phillipsand Croteau, 1999; Trapp and Croteau, 2001). Com-posed of approximately equal molar amounts ofmonoterpenes (10 carbon atoms) and diterpenes (20carbon atoms), conifer resin also contains a smallerproportion of sesquiterpenes (15 carbon atoms; Fig.1). The monoterpenes and sesquiterpenes constitutethe volatile turpentine fraction of conifer oleoresin,whereas the diterpene resin acids form the rosin.Conifers have specialized anatomical structures foraccumulation of resin terpenes, which can be as sim-ple as the resin blisters found in species of true fir(Abies spp.), or more complex such as the resin-filledcanals of spruce (Picea spp.) and pine (Pinus spp.)that are interconnected in a three-dimensional retic-ulate system (Bannan, 1936; Fahn, 1979).

The study of plant terpenoid biosynthesis hasmade rapid progress in recent years (Chappell, 1995;

McGarvey and Croteau, 1995; Gershenzon and Kreis,1999; Bohlmann et al., 2000; Fig. 2). Two pathwaysexist for the formation of the five-carbon biosyntheticbuilding block, IPP. The mevalonate pathway isfound in the cytosol/endoplasmic reticulum and the2-C-methylerythritol-4-phosphate pathway, whichproceeds via 1-deoxyxylulose-5-phosphate, occurs inplastids (Eisenreich et al., 1998; Lichtenthaler, 1999).Condensation of IPP and its isomer, DMAPP, byclass-specific prenyltransferases (PTs) supplies thethree central intermediates of the isoprenoid path-way, GPP, FPP, and GGPP (Alonso and Croteau,1993). The basic terpene skeletons are then formedfrom GPP, FPP, or GGPP by catalysis of terpenesynthases (TPS) resulting in monoterpenes (10 carbonatoms), sesquiterpenes (15 carbon atoms), and diter-penes (20 carbon atoms), respectively (Bohlmann etal., 1998b; Davis and Croteau, 2000). These enzymesfunction through the divalent metal ion-assisted gen-eration of carbocation intermediates from the prenyldiphosphate precursor and give rise to the hundredsof cyclic and acyclic parent skeletons typical of plantterpenoids. Many TPS yield only one or a few closelyrelated products, whereas some TPS form complexproduct mixtures (Bohlmann et al., 1998b; Steele etal., 1998a).

Most of the research on the biosynthesis of coniferterpenoids to date has focused on the TPS enzymesand their genetic regulation in grand fir (Abies gran-dis; Lewinsohn et al., 1991; Stofer Vogel et al., 1996;

1 This work was supported by the Natural Sciences and Engi-neering Research Council (funds to J.B.), by the Canadian Foun-dation for Innovation (funds to J.B.), by the British ColumbiaMinistry of Forests (funds to J.B.), and by the Max Planck Society(funds to J.G. and fellowships to D.M. and D.T.).

* Corresponding author; e-mail [email protected];fax 604 – 822– 6097.

Article, publication date, and citation information can be foundat www.plantphysiol.org/cgi/doi/10.1104/pp.011001.

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Bohlmann et al., 1997, 1998a, 1998b, 1999; Steele et al.,1998a, 1998b; Bohlmann and Croteau, 1999). Thesestudies revealed a wound-induced resin response instem tissues based on up-regulation of the TPS genesand enzymes. However, the possible role of PTs forregulation of induced resin formation has not been asthoroughly studied (Tholl et al., 2001). In addition,very little is known about the biochemical processes ofinduced terpenoid formation in other conifer species.

The genus Picea includes some of the economicallymost important species of forest trees. In Picea spp.,stem resin accumulates constitutively in axial resincanals in the cortex and in axial traumatic resin ducts(TDs), which appear within the developing xylemafter mechanical wounding, insect feeding, or fungalelicitation (Fig. 3). Recent microscopic work with

Norway spruce has described details of the TD for-mation (Nagy et al., 2000). The production of TDs isdue to a change in the developmental program ofcambial activity whereby some of the xylem mothercells initiate epithelial cells (which eventually cometo surround the TD lumen) in lieu of tracheids. How-ever, it is not known whether duct formation is as-sociated with de novo biosynthesis of resin terpe-noids in the xylem, or if preformed resin storedunder pressure in constitutive resin canals in the barktissue is mobilized to these new sites for resin accu-mulation. De novo formation could result in an in-crease in total resin quantity and a change in resincomposition.

Previous studies of the anatomy and resin chemis-try of TDs in spruce involved mechanical wounding

Figure 1. Representative structures of terpenoids of Norway spruce (Picea abies L. Karst). A and D, Monoterpenes (10 carbonatoms). B, Sesquiterpenes (15 carbon atoms). C, Diterpene resin acids (20 carbon atoms). Monoterpenes are numberedcorresponding to peak numbers in Figure 9.

Martin et al.

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or fungal inoculation of trees as a means to induceTD formation (Alfaro, 1995; Tomlin et al., 1998, 2000;Franceschi et al., 2000). In this study, we developed anoninvasive procedure for TD induction in Norwayspruce based on topical application of methyljas-monate (MeJA). This treatment enabled a detailedchemical and biochemical analysis of the traumaticresin response.

RESULTS

Induced Formation of TDs

Formation of TDs in spruce is elicited by stem-boring insects and microbial pathogens as a defenseresponse that can also be induced by mechanicalwounding or by wounding and fungal inoculation oftrees (Alfaro, 1995; Tomlin et al., 1998, 2000; France-schi et al., 2000). Because wounding of trees cancause massive bleeding and volatilization of oleo-resin and disruption of the tissues that are possiblyinvolved in de novo resin formation, it was impor-tant to develop a noninvasive method for TD induc-tion to enable a detailed chemical and biochemicalanalysis of the traumatic resin response. To deter-mine if MeJA induces the formation of TDs, 2-year-old Norway spruce saplings were sprayed with

MeJA in aqueous solution and stem samples wereexamined by light microscopy over a period of 2months after treatment. Copper acetate staining wasused to visualize the terpenes accumulating in theconstitutive resin ducts and TDs (Fig. 3).

In unsprayed saplings, axial resin ducts werelargely restricted to the bark (all tissues outside of thecambium, i.e. phloem, cortex, and periderm). How-ever, after MeJA treatment, striking morphologicalchanges became apparent as early as 6 to 9 d afterapplication. As seen in Figure 3B, some new xylemcells immediately adjacent to the cambium haddenser cytoplasm and thinner walls than surround-ing xylem cells and appeared to constitute the epi-thelial cells of nascent TDs which surround theterpene-rich lumen. Within 15 d after treatment, lu-mens of the developing ducts were clearly discern-ible in a ring within the youngest portion of thexylem. Starting at this time, the lumen began fillingwith resin, presumed to be secreted from the epithe-lial cells, that were still visible at d 25 (Fig. 3C). Twomonths after induction, the lumen had enlarged fur-ther and the epithelial cells had disappeared or di-minished in size considerably (Fig. 3, D and F). Dur-ing the same period, resin ducts in the bark were notvisibly affected.

A range of concentrations of MeJA (1–100 mm)applied as a surface spray was shown to induce TDformation in spruce stems. Response at 10 mm wasgreater than that at 1 mm, but at 100 mm MeJA xylemdevelopment was minimal after TD initiation andseveral of the treated saplings shed needles and suf-fered severely reduced growth. Thus, 10 mm wasroutinely employed in further experiments. How-ever, the MeJA concentration that is effective in theresponding tissues is probably much lower than thatof the applied surface spray because this elicitor isunlikely to easily penetrate the thick cortex of sprucestems. This anatomical feature precludes any directcomparison of effective MeJA concentration to thatpreviously used in studies of herbaceous angio-sperms or plant cell suspension cultures. A full TDresponse was also induced by much lower concen-trations of MeJA (100–500 �m MeJA) when 0.1%(v/v) Tween 20 was added to the spray solution(data not shown).

Induced Accumulation of Monoterpenes and Diterpenes

The initiation of TD formation in the developingxylem by MeJA suggested that increased accumula-tion of resin terpenoids would also be observed. Totest this possibility, the effect of MeJA on resin ter-penoid composition was evaluated in both wood andin bark. Saplings were treated with 1, 10, or 100 mmMeJA as above and the saplings were harvested forresin analysis 2 months later when TDs were fullydeveloped.

The three MeJA treatments significantly increasedthe total accumulation of monoterpenes (Table I) and

Figure 2. Scheme of the pathways of terpenoid biosynthesis in co-nifers. The five-carbon precursors, isopentenyl diphosphate (IPP) anddimethylallyl diphosphate (DMAPP), are formed via two pathways,the mevalonate pathway in the cytosol/endoplasmic reticulum andthe 2-C-methylerythritol-4-phosphate pathway (via 1-deoxyxylulose-5-phosphate) in plastids. Prenyltransferases (PTs) catalyze (1�-4)head-to-tail condensations of DMAPP with one, two, or three mol-ecules of IPP to form geranyl diphosphate (GPP; GPP synthase),farnesyl diphosphate (FPP; FPP synthase), and geranylgeranyl diphos-phate (GGPP; GGPP synthase), respectively. Terpene synthases (TPS;cyclases) of three classes (mono-TPS, sesqui-TPS, and di-TPS) convertthe three prenyl diphosphate intermediates into the hundreds ofcyclic and acyclic terpenoids characteristic of conifers.

Induced Terpenoid Defenses of Norway Spruce

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diterpenes (Table III) within the wood tissue of sap-ling stems (Fig. 4), but sesquiterpene (Table II) con-centrations were unchanged. Monoterpenes showeda 5-fold increase at 1 mm MeJA, a 12-fold increase at10 mm MeJA, and a 7-fold increase at 100 mm MeJAcompared with control saplings. Diterpene accumu-lations in the wood reflected the same trend with an11-fold increase at 1 mm, a 38-fold increase at 10 mm,and a 20-fold increase at 100 mm. The lower accumu-lation levels in the saplings treated with 100 mmMeJA as compared with 10 mm MeJA may reflect thenegative effect this high concentration had on growthand development as described above. In bark tissue,there was only a minor relative increase in monoter-pene accumulation (Fig. 4C; Table I), and no consis-tent change in diterpene levels (Fig. 4D; Table III). Itshould be mentioned that clonal trees of Norwayspruce with low constitutive amounts of monoter-penes and diterpenes in the xylem (clone 1015-903,

Fig. 4) revealed a stronger relative induction thanclonal trees with higher constitutive amounts ofmonoterpenes and diterpenes (clone 3166-728; Fig. 5).However, the absolute amounts of induced monoter-penes and diterpenes were similar in these clones.

Time Courses of Induced Monoterpenoid andDiterpenoid Accumulation

The temporal pattern of monoterpenoid, sesquiter-penoid, and diterpenoid accumulation in wood andbark tissue was analyzed over a 5-week period aftertreatment with 10 mm MeJA (Fig. 5). There was asignificant increase in the accumulation of bothmonoterpenoids and diterpenoids in the wood com-pared with untreated controls starting 10 to 15 d aftertreatment (Fig. 5). Monoterpene concentrationspeaked (1.7 mg g�1 tissue dry weight) at 18 d post-treatment (Fig. 5A) and then fell slightly over the

Figure 3. Light microscopy of induced TD dif-ferentiation in cross sections of Norway sprucestems. A and E, Unsprayed controls show theabsence of resin ducts in the constitutive xylem(X) and phloem (P), but large constitutive resinducts (CD) are present in the cortex of controltrees. B, Nine days after 10 mM methyl jas-monate (MeJA) treatment, early stage of induceddevelopment of TDs in the xylem next to thevascular cambium. C, Twenty-five days afterMeJA treatment, showing accumulation of resinin the lumen of the fully differentiated TD visu-alized by staining with copper acetate. D, Twomonths after treatment, lumen (L) of TD withresin droplet (blue) and remainder of epithelialresin duct cells (E). F, Two months after treat-ment, showing a ring of fully formed traumaticducts in the newly developed xylem.

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remainder of the 35-d time course, although theyremained greater than those seen in the controls. Asimilar pattern was seen for wood diterpenes, withthe first significant increase over controls becomingevident at d 15 (Fig. 5B). The maximum diterpeneconcentration was observed at d 25 (4 mg g�1 tissuedry weight), slightly later than the peak for monoter-pene accumulation. Thus, unlike other JA-inducedplant defense responses, the increase in resin terpe-noids after MeJA treatment persists over an extendedperiod, consistent with the persistence of the TDs.

In contrast, the bark tissue, already rich in resinfrom constitutive ducts, exhibited much smaller rel-ative changes in terpene concentrations over the 35-dtime course after MeJA treatment. Monoterpenesreached a maximum concentration (12 mg g�1 tissuedry weight) at d 15 after MeJA treatment (Fig. 5C),and decreased to approximately the same levels asthe controls by d 35. Significant increases were al-ready seen by d 6, indicating that the bark respondsmore rapidly to MeJA treatment than the wood, pos-sibly because the response in the bark does not re-quire de novo differentiation of resin producing cellsas with TDs in the xylem. For bark diterpenes, MeJAtreatment had no significant effects over the period ofthis time course (Fig. 5D).

For the third class of resin terpenoids, the sesquit-erpenoids, MeJA treatment had no effect on theiraccumulation in wood tissue and resulted in a weakresponse in the bark (less than 2-fold) over the mon-itored time course with maximum increase in accu-mulation between d 10 and 25 (Fig. 6). The timecourse of induction of sesquiterpenoid accumulationin the bark lacks a clear peak and appears moretransient than that seen for the monoterpenoid and

diterpenoid resin components, which likely explainswhy sesquiterpenoid induction was not observedwhen resin accumulation was monitored at the endof the 2-month experiment with the 1, 10, and 100 mmMeJA-treated saplings.

Composition of Constitutive and InducedResin Terpenoids

Analyses of resin from MeJA-treated and controlsaplings 18 d after treatment revealed a number ofdifferences in composition. The seven most abundantmonoterpenoids in both bark and wood tissues were,in order of decreasing abundance in induced tissues,�-pinene, �-pinene, �-phellandrene, limonene, myr-cene, �3-carene, and camphene (Table I; Fig. 1). In thebark, most of these abundant compounds increased1.4- to 2-fold upon MeJA treatment with a higherincrease (4-fold) for limonene and a lower increase(1.13-fold) for �3-carene accumulation in inducedbark tissue.

In the wood, the site of TD formation, changes inindividual monoterpene concentrations were morepronounced. Whereas �-pinene and camphene in-creased by only 2.5- and 1.5-fold, respectively, theconcentrations of most of the remaining major mono-terpenes (�-pinene, myrcene, limonene, and�-phellandrene) increased by 3.7- to 4.3-fold. �3-Carene was not detectable before treatment. The twomajor monoterpene resin constituents in the wood,�-pinene and �-pinene, were differentially affectedby MeJA. These two compounds are found in thewood in nearly equal amounts in control saplings,but the proportion of �-pinene to �-pinene increasesupon MeJA treatment reaching a ratio of 2:1 at d 15.In the bark, this proportion is 3:1 regardless of treat-ment. Several of the oxygenated monoterpenoids andminor hydrocarbons also exhibited altered concen-trations upon MeJA application, including 1,8-cineole (not present in the control), �-fenchone (1.4-fold increase), and bornyl acetate (1.7-fold increase).In the wood, increases of bornyl acetate (8-fold) and�-terpinolene (4.3-fold) were found. Chiral analysisof wood extracts showed an increase in the relativeamounts of (�)-�-pinene and (�)-�-pinene in rela-tion to their respective (�)-enantiomers (Fig. 1D)(data not shown), indicating the existence of at leasttwo different pinene synthases in the stem tissue ofNorway spruce, as in grand fir (Bohlmann et al.,1997, 1999) and in Pinus taeda (Phillips et al., 1999).

In both bark and wood, most of the individualsesquiterpenes increased by 1.4-fold after MeJA treat-ment (Table II). However, wood extracts showed a4.2-fold increase in �-caryophyllene and a 2- to 3-foldincrease in (Z)-�-farnesene, �-humulene, and (E)-�-farnesene (Fig. 1).

Although diterpene concentrations did not differbetween control and MeJA-treated bark extracts,there was an overall 2.6-fold induction in the wood

Table I. Monoterpene composition of constitutive and inducedresin in wood and bark of Norway spruce

MonoterpeneControl

BarkMeJABark

ControlWood

MeJAWood

�g g�1 dry wt

Santene 19.2 19.0 nda ndTricyclene 14.9 21.4 6.0 5.8�-Pinene 1,609.3 2,744.8 212.6 522.6Camphene 68.0 127.7 15.9 22.0�-Pinene 4,586.3 6,388.1 225.6 836.8Sabinene 22.5 34.0 1.7 7.0�3-Carene 109.8 123.2 nd 4.3�-Phellandrene 11.8 15.3 nd ndMyrcene 240.8 337.8 15.7 56.1Limonene 100.5 463.1 10.0 35.6�-Phellandrene 623.5 845.9 38.3 142.61,8-Cineole nd 31.1 3.4 1.8�-Terpinolene 31.5 41.0 1.5 6.4�-Fenchone 31.6 44.9 3.1 2.1Bornyl acetate 37.1 62.3 0.8 6.6Other 37.5 54.4 1.4 1.5

Total 7,544.3 11,354.0 536.0 1,651.2a nd, Not detected.




(Table III). Concentrations of levopimaric acid (Fig. 1)showed the highest increase (5.2-fold), whereas neoa-bietic acid, pimaric acid, and three unidentifiedpeaks exhibited approximately 2.5-fold increases intreated over control saplings. Noted also in this anal-ysis was the constant concentration of abienol, themajor diterpene alcohol found in this tissue in in-duced and control trees. Another interesting obser-vation is the decrease in dehydroabietic acid (1.5-fold) seen in treated versus control trees.

Effect of Induction on PT Activities

Accumulation of resin terpenoids in developingxylem after MeJA treatment may reflect de novosynthesis or mobilization of resin from sites of con-stitutive accumulation in the bark. To test for in-

duced de novo biosynthesis, protein extracts frombark and wood of saplings were assayed in vitro foractivities of PTs and TPS, two principal steps interpene biosynthesis (Fig. 2).

Three different types of PTs were measured, GPPsynthase, FPP synthase, and GGPP synthase, whichprovide the precursors for all monoterpene, sesquit-erpene, and diterpene formation, respectively. Allthree activities were detectable in bark extracts butwere unaffected by treatment with 10 mm MeJA overthe time course of 35 d (Fig. 7). In the xylem, theactivities of GPP synthase and FPP synthase also didnot differ between MeJA-treated and control trees(Fig. 7, A and B). However, GGPP synthase activity,which was barely detectable in constitutive woodextracts, was strongly induced by treatment withMeJA starting 3 d posttreatment and reaching a max-

Figure 4. Tissue-specific and dose-dependent changes in monoterpene and diterpene accumulation in wood and bark aftertreatment of trees with MeJA. A, Monoterpenoids, wood. B, Diterpenoids, wood. C, Monoterpenoids, bark. D, Diterpenoids,bark. Each concentration was tested on four trees. Analysis was performed in duplicate and results are presented as the meanwith SE. Wood denotes the entire xylem tissue of a stem section. Bark denotes all tissues outside the vascular cambium,including phloem, cortex, and periderm. Lower concentrations of MeJA of 100 to 500 �M induced chemical changes ofwood resin terpenoids similar to those induced with 10 mM MeJA when Tween 20 was added at 0.1% (v/v) to the surfacespray.

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imum specific activity (10 pmol �g protein�1 h�1) at d15 (Fig. 7C). At d 18 and 25, the activity declined, butremained at higher levels relative to the control. Theinduction of GGPP synthase in the xylem reflects thetissue specific response to MeJA previously observedat the microscopic structural level and by terpenoidanalysis.

Effect of Induction on TPS Activities

Two classes of TPS, mono-TPS, and di-TPS, weremeasured. In unsprayed saplings, these activitieswere at best barely detectable in wood extracts, butrevealed strong activity in the bark (Fig. 8). Treat-ment with MeJA did not affect the activity of barkmono-TPS over a time course of 35 d. In contrast,mono-TPS activity rose rapidly in wood with in-creased specific activity detectable 3 d after treatmentwith 10 mm MeJA. The peak at 9 d (28 pmol �gprotein�1 h�1; Fig. 8A) corresponded to a 28-foldincrease in activity over the control, and was 5-foldhigher than the specific mono-TPS activities in bark,the site of constitutive resin formation and resin ac-cumulation. By d 18 and 25, the activity in inducedwood had dropped but remained approximately 12times that of the control until d 35, when similarlevels of activity were found in treated and controltrees.

This transient increase of enzyme activity afterMeJA application was also observed for di-TPS inwood, which exhibited the same rapid induction de-tectable 3 d posttreatment (Fig. 8B). Induced di-TPSactivity reached a maximum at d 15 with a 22-foldincrease (66 pmol �g protein�1 h�1) in activity overthe control. This activity exceeds the specific activityof di-TPS from bark tissue at the same time point bymore than 10-fold. After the maximum at 15 d, di-TPS activity remained at an elevated level over theentire 35-d time course. This transient induction of

enzyme activities in terpene-accumulating wood tis-sue coupled with the appearance of TDs is highlysuggestive of the occurrence of MeJA-induced denovo synthesis.

Products of the Induced TPS Activities

Product analysis of the mono-TPS assays of inducedwood tissue by radio-gas chromatography (GC; Fig. 9)revealed the presence of the six major monoterpenecomponents that are all found in xylem resin:�-pinene (peak 1), camphene (peak 2), �-pinene (peak3), myrcene (peak 5) limonene (peak 6), and�-phellandrene (peak 7). The individual componentswere found in approximately the same ratios as theyoccur in the xylem resin (Table I). In addition, anincrease of �-pinene relative to �-pinene paralleledthat observed in the resin extracts from inducedxylem.

Products of the di-TPS assays analyzed byradio-GC and GC-mass spectroscopy (MS) revealedthe presence of a single peak (Fig. 10), identified asabietadiene by comparing the mass spectrum withthat of an authentic standard. The result is consistentwith the activity of the monospecific abietadiene-producing grand fir di-TPS described previously(LaFever et al., 1994). Peters et al. (2000) recentlyfound that under modified conditions, the clonedgrand fir abietadiene synthase actually produces abi-etadiene, levopimaradiene, and neoabietadiene innearly equal ratios as well as three other minor prod-ucts. The Norway spruce enzyme might also produceadditional products under similar conditions, whichwill be tested in future work once the correspondingspruce di-TPS gene has been cloned.

DISCUSSION

Conifers include some of the longest living of allorganisms, which, during their extended lifetimes,

Table III. Diterpene composition of constitutive and induced resinin wood and bark of Norway spruce

DiterpeneControl

BarkMeJABark

ControlWood

MeJAWood

�g g�1 dry wt

Manoyl oxide 460.1 1,004.0 nd ndAbienol 4,674.3 4,528.4 301.3 365.1Pimaric acid 373.5 374.5 66.3 160.8Sandaracopimarate 377.7 506.2 83.7 171.7Dehydroabietinal 145.7 145.1 nd ndIsopimaric acid 1,712.7 1,918.2 195.4 208.2Palustic acid 447.7 400.3 83.1 43.6Levopimaric acid 3,623.7 5,046.9 443.0 2,309.4Dehydroabietate 703.3 970.0 72.7 47.5Abietic acid 2,837.2 2,713.5 119.8 241.8Neoabietic acid 1,881.1 2,127.0 74.0 181.9Other 993.2 1,042.0 66.3 160.8

Total 18,230.2 20,776.1 1,505.6 3,890.8a nd, Not detected.

Table II. Sesquiterpene composition of constitutive and inducedresin in wood and bark of Norway spruce

SesquiterpeneControl

BarkMeJABark

ControlWood

MeJAWood

�g g�1 dry wt

Longipinene 14.8 25.3 0.9 0.4Longifolene 38.4 52.5 2.5 1.2�-Cedrene 14.4 17.5 1.2 0.0�-Caryophyllene 134.1 189.5 1.3 5.5(Z)-�-Farnesene 23.2 23.6 0.7 1.5�-Humulene 25.9 41.5 0.5 1.5(Z,E)-�-Farnesene 12.6 17.7 0.3 0.8(E)-�-Farnesene 5.7 8.0 nd nd(Z)-�-Bisabolene 3.9 3.7 2.2 3.4(E)-�-Bisabolene 4.9 5.8 1.3 0.9Other 20.0 25.5 4.5 7.0

Total 297.9 410.6 15.4 22.2a nd, Not detected.




must survive countless challenges by a diverse arrayof herbivore and pathogen species (Seybold et al.,2000). Constitutive and inducible resin terpenoids arecharacteristic defense compounds of many conifergenera of the pine family (Bohlmann and Croteau,1999; Phillips and Croteau, 1999; Trapp and Croteau,2001). Species of Picea, like other conifers, producecopious amounts of resin terpenoids in the bark as aconstitutive outer defense barrier, but do not accu-mulate significant amounts of resin in the constitu-tive wood and developing xylem in contrast to someother conifers. However, induced resin terpenoidsaccumulate in newly initiated axial resin ducts (TDs)in the developing xylem of Picea spp. (spruce; Nagyet al., 2000). Induced terpenoid defense responses inPicea spp. are activated by stem-boring insects, suchas coniferophagous bark beetles (Scolytidae) and thewhite pine weevil (Pissodes strobi), which are amongthe most destructive insect pests of conifer forestsworldwide, as well as by insect-associated fungi,such as the blue stain fungus Ceratocystis polonica(Alfaro, 1995; Tomlin et al., 1998; Franceschi et al.,

Figure 5. Time course of total monoterpenoid and diterpenoid content in wood and bark after treatment with MeJA. A,Monoterpenoids, wood. B, Diterpenoids, wood. C, Monoterpenoids, bark. D, Diterpenoids, bark. Data are presented as themeans with SE of duplicate or triplicate assays of extracts from treated (F) and control (E) trees.

Figure 6. Time course of total sesquiterpenoid content in bark aftertreatment with MeJA. Data are presented as the means with SE ofduplicate or triplicate assays of extracts from treated (F) and control(E) trees.

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2000). De novo formation of resin ducts in the devel-oping xylem has been described as a possible resis-tance mechanism against insects and pathogens inwhite spruce (Picea glauca), Sitka spruce (Picea sitch-ensis), and Norway spruce (Alfaro, 1995; Tomlin etal., 1998; Franceschi et al., 2000).

In an effort to characterize the induced changes interpene chemistry as well as changes in terpenoidbiosynthetic activities during the development oftraumatic resinosis in Norway spruce, we first testedthe effect of MeJA to establish a nondestructivemethod of inducing the resin defense response.Whereas the effect of exogenous MeJA and the role ofendogenous octadecanoids have been well character-ized in herbaceous angiosperm species (Farmer andRyan, 1992; Creelman and Mullet, 1997; Baldwin,1999), the effect of jasmonates has been demonstratedin only a few conifers, mainly in cell cultures. MeJAinduces the transformation of E-�-bisabolene intotodomatuic acid, a precursor of juvabione type insectjuvenile hormone analogs, in suspension culture ofgrand fir (Bohlmann et al., 1998a). In cell cultures ofTaxus canadensis, MeJA induces production of pacli-taxel (taxol) and other taxoids with the concomitant

increase in GGPP synthase and acetyl-CoA:taxadienol-O-acetyl transferase enzyme activities,both of which are involved in taxol biosynthesis(Yukimune et al., 1996; Hefner et al., 1998; Ketchumet al., 1999). Spruce cell cultures treated with MeJAshow transcript accumulations of chalcone synthaseand a 14-3-3 protein (Lapointe et al., 2001). In Doug-las fir, MeJA was found to induce the expression oftwo low-Mr heat shock proteins in dormant seeds(Kaukinen et al., 1996). Other work with jasmonatesin conifers has focused on jasmonate-induced effectson interactions with fungi. Jasmonates have beenimplicated in both symbiotic and antagonistic rela-tionships of seedlings with fungi. Apparently, MeJAapplication assists ectomycorrhizal colonization ofNorway spruce roots (Regvar et al., 1997). MeJAapplication to Norway spruce seedlings also en-hances their survival rate when they were challengedby Pythium ultimun (Kozlowski et al., 1999).

Compared with the numerous effects of jasmonatesreported for angiosperms and the effects previouslydescribed for conifers, the induction of terpenoiddefenses in Norway spruce is probably among themost complex responses. Similar to a stem-boring

Figure 7. Time course of PT activity in wood and bark after treatment with MeJA. A, GPP synthase activity, wood. B, FPPsynthase activity, wood. C, GGPP synthase activity, wood. D, GPP synthase activity, bark. E, FPP synthase activity, bark. F,GGPP synthase activity, bark. Values are the means of duplicate or triplicate assays of extracts from treated (F) and control(E) trees. A rapid increase of enzyme activity was found only for GGPP synthase in induced wood samples. The apparentincrease in specific activities of GPP synthase and FPP synthase at d 35 reflect on a decrease of total protein in these samples.Ranges of duplicate assays were normally 1% to 25% of the mean but were 45% to 60% of the mean in control d 35 in Aand B and in MeJA d 25 and 35 in B.




insect attack, treatment with MeJA alters the devel-opmental program of xylem mother cells within thevascular cambium switching differentiation from tra-cheids to that of TD cells, two cell types with enor-mous contrasts in structure and function (Esau,1977). In addition, induced de novo differentiation ofresin ducts in the xylem is also associated with resinterpenoid accumulation and de novo resin biosynthe-sis. Interestingly, this effect is strictly tissue specificbecause existing resin canals in the phloem and cor-tex are not affected in the young trees of this study.Unlike older trees, young Norway spruce trees donot accommodate significant radial resin duct forma-tion in the phloem. Because the phloem of older treesis the primary site of attack by bark beetles, thepossibility that radial ducts in the phloem of oldertrees could also be activated by MeJA will be testedin future work.

Differential effects of MeJA in Norway spruce stemtissues are further observed at the level of terpenoidbiosynthesis and terpenoid accumulation. Inducedaccumulation of resin terpenoids is most apparent inthe wood and barely detectable in the bark, wherehigh constitutive resin levels already exist. Amongthe resin terpenoids, levels of the two major classes ofresin components, monoterpenes and diterpenes, arestrongly increased, whereas the sesquiterpenes, theleast abundant class of resin terpenoids, are onlyweakly affected in the induced xylem. The formationof traumatic resin occurs on a much longer time scalethan that of most other induced defenses. However,it has been demonstrated that the site of TD devel-opment not only corresponds to the area in whichbark beetle or weevil larvae could potentially de-velop, but that the time of TD development is syn-chronized with the time during which these insects

Figure 8. Time course of monoterpenoid synthase activity and diterpenoid synthase activity in wood and bark aftertreatment with MeJA. A, Monoterpenoid synthase activity, wood. B, Monoterpenoid synthase activity, bark. C, Diterpenoidsynthase activity, wood. D, Diterpenoid synthase activity, bark. Data are the means of duplicate or triplicate assays ofextracts from treated (F) and control (E) trees.

Martin et al.



would be emerging from their eggs, thereby floodingthe brood with toxic and immobilizing resin constit-uents (Alfaro, 1995).

In measuring terpenoid biosynthetic enzymes, weobserved a dramatic response in both monoterpenoidand diterpenoid pathway activities in wood tissue,whereas sesquiterpene biosynthesis was only weaklyand transiently affected in the bark. Of the three PTsof resin terpenoid biosynthesis (Fig. 2), only GGPPsynthase was induced, but not GPP synthase and FPPsynthase. The lack of FPP synthase activation reflectsthe weak effect of MeJA on sesquiterpene accumula-tion associated with TD development. Similarly,sesqui-TPS activities in these tissues are below detec-tion limits (data not shown). However, despite astrong induction of monoterpene accumulation andmono-TPS induction in developing xylem, GPP syn-

thase is not induced, suggesting that GPP synthaseactivity is not limiting for induced monoterpene for-mation and accumulation. Similar results were ob-tained from grand fir saplings, which showed noincrease in GPP synthase activity over a 20-d periodafter mechanical wounding of the stem (Tholl et al.,2001), even though mono-TPS enzyme activity andgene expression increased strongly during this sameperiod (Bohlmann et al., 1997; Steele et al., 1998b). Incontrast, GGPP synthase is active at a very low levelin the constitutive xylem and induced accumulationof diterpene resin acids is preceded by coordinatelyincreased activities of both GGPP synthase and di-TPS in xylem of MeJA-treated spruce trees. Theseresults suggest that unlike monoterpene accumula-tion, the induction of diterpenes is controlled notonly by enzyme activity at the level of TPS but also at

Figure 9. Analysis of products formed in vitroby constitutive and induced monoterpenoidsynthase activity from wood tissue. A, Radio-GCtraces for monoterpenoid synthase assay prod-ucts from wood of control saplings. B, Radio-GCtrace for monoterpenoid synthase assay prod-ucts from wood of saplings treated with 10 mM

MeJA. C, Thermal conductivity detector (TCD)trace for monoterpene standards. Standard for�-phellandrene not shown. Peak 1, �-Pinene; 2,camphene; 3, �-pinene; 4, �3-carene; 5, myr-cene; 6, limonene; 7, �-phellandrene.




the level of the PT that yields the 20-carbon precur-sor. The differential enzymatic regulation of the threeclasses of resin terpenoids (monoterpenoids, sesquit-erpenoids, and diterpenoids) has important implica-tions for attempts to alter resin composition in

spruce. For instance, increased monoterpenoid for-mation may not require altering expression of GPPsynthase, whereas altering GGPP synthase togetherwith di-TPS may be necessary to alter diterpeneformation.

Figure 10. Analysis of products formed in vitro by induced diterpenoid synthase activity from wood tissue. A, Radio-GCtrace for di-TPS assay products. B, GC-MS fragmentation pattern for major di-TPS assay product. C, GC-MS fragmentationpattern for authentic abietadiene standard.

Martin et al.



This is the first study, to our knowledge, that dem-onstrates the constitutive and induced activity ofterpenoid synthases at a tissue-specific level in agymnosperm system. Enzymatic and molecular stud-ies of terpenoids at the tissue-specific level have pre-viously been reported only in some angiosperm sys-tems, the glandular trichomes of Mentha � piperita(McConkey et al., 2000), the trichomes of Nicotianaglutinosa (Guo and Wagner, 1995), or the scent-producing floral tissues of Clarkia breweri (Dudareva etal., 1996). The results described here justify futureanalysis of MeJA-induced TD differentiation and reg-ulation of terpenoid biosynthesis in spruce at the cel-lular and subcellular level to elucidate the controlmechanisms responsible for such localized expression.

This study did not intend to investigate the role ofjasmonates as endogenous signal molecules in coni-fers. Treatment of trees with MeJA was tested anddeveloped solely as a procedure to study in detail thechemical, biochemical, and anatomical processes oftraumatic resin defense in spruce. Nevertheless, ourresults could imply that octadecanoids are involvedin insect-induced defense in conifers, a possibilitywhich will be tested in future work. Because this isthe first demonstration of TD formation by a signalmolecule, to our knowledge, we will also evaluatethe effect of other signal molecules to elucidate theendogenous signal cascade active in xylem-specificresinosis and TD development in response to insects,pathogens, or wounding. The noninvasive and dose-dependent activation of defenses in spruce by MeJAtreatment provides new opportunities to evaluate thesignificance of induced responses in conifers in bio-assays with insects and pathogens, as recently dem-onstrated for agricultural crops (Thaler, 1999). Con-sidering restrictions on use of pesticides for insectand pathogen control in forest systems, pretreatmentof trees with elicitors can be explored as a strategy fortree protection. Furthermore, nondestructive induc-tion of traumatic resinosis in spruce provides a su-perb system for discovery of genes associated withresin canal differentiation in the developing xylemand genes of induced terpenoid formation and accu-mulation, a paramount characteristic of conifers.

MATERIALS AND METHODS

Plant Materials

Norway spruce (Picea abies L. Karst) trees of clonal lines 3166-728 and1015-903 were propagated from lateral branches of current and previousyear growth at the Niedersachsische Forstliche Versuchsanstalt (Escherode,Germany). Fully regenerated, 2-year-old, rooted saplings were grown in 2-Lpots in a 2:3 (v/v) ratio of peat:universal planting mix for at least 6 monthsbefore experiments. Trees were fertilized with Osmocoat and maintained ingrowth chambers under illumination with high-pressure sodium vaporlamps. The photoperiod and ambient temperature cycled from 1 h at 220�mol m�2 s�1 (20°C), 4 h at 440 �mol m�2 s�1 (20°C), 3 h at 660 �mol m�2

s�1 (22°C for 2 h and 24°C for 1h), 7 h at 440 �mol m�2 s�1 (24°C for 1 h,22°C for 2 h, and 20°C for 4 h), and 1 h at 220 �mol m�2 s�1 (18°C). This wasfollowed by 8 h of darkness (18°C). The relative humidity was maintainedat 50% throughout the entire cycle. Saplings were introduced to these

growth conditions 4 weeks before use in experiments to ensure a completebreak of dormancy.

Substrates, Standards, and Reagents

Chemical reagents were from Sigma-Aldrich (Steinheim, Germany) orRoth (Karlsruhe, Germany). Terpene standards were from Sigma-Aldrich,Roth, Bedoukian Research (Danbury, CT), and Helix Biotech (Richmond,BC) and were of the highest purity available. All solvents were GC grade.The substrates, [1-14C] IPP (54 Ci mol�1), [1-3H]GPP (20 Ci mol�1), andall-trans-[1-3H]GGPP (58 Ci mol�1) were from Biotrend (Koln, Germany).[1-3H]FPP (125 Ci mol�1) was the gift of Rodney Croteau (Washington StateUniversity, Pullman). Unlabeled DMAPP, GPP, and FPP were from EchelonResearch Laboratories Inc. (Salt Lake City). Diazomethane was made freshfrom Diazald (Aldrich, Milwaukee, WI) by a standard procedure (Aldrichtechnical information bulletin no. 180).

MeJA Treatment and Harvest of Tissues

To test dose-dependent effects of MeJA, saplings (clone 1015-903) weresprayed with 1, 10, or 100 mm solutions of MeJA (95% [w/w] pure, Sigma-Aldrich) dissolved in distilled water. Time course experiments were donewith saplings (clone no. 3166-728) sprayed with 10 mm MeJA dissolved indistilled water. Saplings were placed in a ventilated fume hood and eachtree was sprayed with 150 mL of MeJA solution over a period of 30 min toobtain a complete and even coating. Saplings were kept in fume hoods for1 h after treatment to allow evaporation of excess MeJA solution beforetransferring to growth chambers. Control saplings (four for dose-dependentexperiment and four for each time point for the time course) were sprayedwith water. Control and MeJA-treated saplings were kept in separategrowth chambers. To determine dose-dependent effects of MeJA, trees wereharvested after 42 d by cutting the stem above the ground and just below theupper internode and freezing the entire section in liquid nitrogen. Needleswere removed and the tissue was stored at �80°C. The lowest 3 cm of thestem section was discarded. The next 3 to 4 cm of stem tissue was preparedfor resin extractions. This section was cut into two pieces of equal length.The bark of these stem sections was sliced longitudinally with a razor blade,and while still frozen, was peeled away from the wood. Bark and woodwere extracted separately for resin analysis. An additional 2 cm of the stemwas used for light microscopy analysis of TD development. Saplings foreach of the time points were harvested in the same manner for microscopyand resin analysis. An extra 7 cm of the harvested stem was used for enzymepreparations. For enzyme extracts, bark and wood tissues were separated asindicated and the similar tissues of four saplings for each time point werecombined into one protein extract.

Light Microscopy

Samples were prepared for cryosectioning by soaking small sections (2–3cm long) of the stems in a solution of 4% (w/v) formaldehyde and 100 mmK2HPO4 (pH 7.5) for 4 h. The samples were then washed with distilled waterand submerged in saturated (aqueous) copper acetate overnight. Beforesectioning, the samples were removed from the copper acetate solution andfrozen at �20°C. Cryosectioning of the samples took place at �20°C andeach section was sliced into 18-�m cross sections. A polyvinyl alcohol-basedglue was used to attach the coverslip immediately after sectioning. Aftervisualizing by light microscopy (Axiophot, Zeiss GmbH, Jena, Germany),digital images were taken of the samples.

Extraction of Resin Terpenes

Extraction of terpene constituents was modified from Lewinsohn et al.(1993). All steps of this procedure were carried out in 2-mL vials (glass witha teflon-coated screw cap, Hewlett-Packard, Palo Alto, CA). Bark and woodtissue samples of approximately 1 cm to 1.5 cm in length were submergedseparately into 1.5 mL of tert-butyl methyl ether in a 2-mL vial containing150 �g mL�1 isobutylbenzene and 200 �g mL�1 dichlorodehydroabieticacid as internal standards. The tissue samples were extracted over 14 h withconstant shaking at room temperature. To purify extracted terpenes fromother small organic acids, the ethereal extract (approximately 1.5 mL) was




transferred to a fresh vial and washed with 0.3 mL of 0.1 m (NH4)2CO3 (pH8.0). Diterpene acids were then methylated by adding 0.4 mL of the washedetheral extract to 0.16 mL of methanol and 0.15 mL of diazomethane in aseparate vial, which was then capped and left at room temperature for 30min to allow the methylation reaction to go to completion. Then, the solventwas evaporated under nitrogen, leaving the residual diterpene fraction. Themonoterpenes, sesquiterpenes, and diterpenes were then recombined bydissolving the methylated diterpene residue in 0.6 mL of the washed etheralextract. The extract was prepared for capillary GC or GC-MS analysis byfiltering through a Pasteur pipette column filled with 0.3 g of silica gel(Sigma 60 Å) overlaid with 0.2 g of anhydrous MgSO4. The column wasfurther eluted with 1 mL of diethyl ether to release bound oxygenatedterpenes and both eluants were collected in a fresh vial. Finally, the samplewas evaporated to an approximate volume of 100 �L that was stored at�20°C. The dry weights of each extracted tissue were determined afterdrying at 70°C for 20 h to calculate terpene constituent concentrations on amg g�1 dry weight basis. ses were calculated from eight independentextracts per treatment.

Analysis of Monoterpenes,Sesquiterpenes, and Diterpenes

For monoterpene and sesquiterpene analysis, a Hewlett-Packard 6890 GCwas equipped with a flame ionization detector (FID) fitted with a DB-WAXcolumn (0.25 mm � 0.25 �m � 30 m, J&W Scientific, Folsom, CA). The flowrate was 2 mL H2 min�1 and the FID was operated at 300°C. One microliterof extract was introduced into the injection port at 220°C and was split ineither a 10:1 ratio for the bark extracts or a 5:1 ratio for the wood extracts.The GC was programmed with an initial oven temperature of 40°C (3-minhold), and temperature increased at a rate of 3°C min�1 until 80°C, followedby 5°C min�1 until 180°C and then 15°C min�1 up to 240°C (5-min hold).GC-MS analysis was accomplished with a Hewlett-Packard 6890 GC-MSDsystem (70 eV), using a DB-WAX column as described above. Split injections(1-�L etheral extract) were made at a ratio of 5:1 (bark extracts) or 3:1 (woodextracts) with an injector temperature of 220°C. The instrument was pro-grammed from initial temperature of 40°C (3-min hold) and increased at arate of 1.5°C min�1 until 45°C, then increasing at 3°C min�1 up to 80°C, 5°Cmin�1 until 180°C, followed by an additional ramp of 10°C min�1 up to240°C (5-min hold). Helium was used at a constant flow of 1 mL min�1.Chiral analysis of monoterpene constituents utilized the same GC-FIDequipped with a Cyclodex-B (0.25 mm � 0.25 �m � 30 m, J&W Scientific).The same etheral samples were injected in a split ratio of 20:1 (220°Cinjection port). The oven was programmed initially at 40°C, increasing at1°C min�1 until 45°C, then at 5°C min�1 until 65°C, followed by 20°C min�1

until 230°C (2-min hold). All other conditions were identical to those usedin analysis with the DB-Wax column mentioned above.

Analysis of diterpene constituents was performed on the same GC-FIDand GC-MS instruments fitted with an HP-5 column (0.25 mm � 0.25 �m �30 m, Hewlett-Packard). Injections were 1 �L of the etheral extracts. ForGC-FID analysis, the split ratios were 40:1 or 20:1 for bark and woodextracts, respectively. The injection port was operated at 250°C and the FIDwas maintained at 300°C. The oven was programmed from an initial tem-perature of 120°C to 150°C at a rate of 1°C min�1 followed by 5°C min�1

until 280°C (5-min hold). GC-MS split ratios were 20:1 (bark extracts) or 10:1(wood extracts) with an injector temperature of 220°C. The instrument wasprogrammed from an initial temperature of 120°C and increased at a rate of1°C min�1 until 150°C, followed by 5°C min�1 up to 280°C (6-min hold).Helium was at a constant flow of 1 mL min�1.

GC-FID-generated peaks were integrated using Hewlett-Packard Chem-station software. Terpene concentrations were calculated by comparing theintegrated peak area to that of the internal standard. Isobutylbenzene wasused as the internal standard for both monoterpenes and sesquiterpenes.Methylated dichlorodehydroabietic acid was employed as an internal stan-dard to calculate diterpene concentrations. Identification of terpenes wasbased on comparison of retention times and mass spectra with authenticstandards or with mass spectra in the Wiley or National Institute of Stan-dards and Technology libraries.

Protein Extraction

Tissue samples (bark or wood) from each of the four saplings per timepoint were combined into one protein preparation and extracted as previ-

ously described (Lewinsohn et al., 1991). Using an analytical grinding mill(A10, IKA WORKS, Cincinnati), the tissue was ground to a fine powder inliquid nitrogen and combined with extraction buffer {50 mm MOPSO [3-(N-morpholino)-2-hydroxypropanesulfonic acid], pH 6.8; 5 mm ascorbic acid; 5mm sodium bisulfite; 5 mm dithiothreitol (DTT); 10 mm MgCl2; 1 mm EDTA;10% [v/v] glycerol; 1% [w/v] polyvinylpyrrolidone [Mr 10,000]; 4% [w/v]polyvinylpolypyrrolidone, 4% [w/v] amberlite XAD-4; and 0.1% [v/v]Tween 20) in a ratio of 1:10 (g tissue:mL buffer). The preparations wereallowed to shake at 4°C for 30 min and were then centrifuged at 10,000g for30 min. The supernatant was then filtered through two layers of no. 1 filterpaper (Whatman, Kent, UK), divided into 4-mL aliquots, frozen in liquidnitrogen, and kept at �80°C. Extracts were thawed only once before enzymeassay. Total protein concentration of each protein extract was determinedusing the Coomassie reagent and protocol (Bio-Rad Laboratories, Hercules,CA).

PT Enzyme Assays

Wood and bark protein extracts were desalted into a buffer containing 20mm MOPSO, pH 7.0, 10 mm MgCl2, 10% (v/v) glycerol, and 2 mm DTT.Assays were carried out in duplicate or triplicate in a final volume of 500 �Lcontaining 40 �m [1-14C]IPP (54 Ci mol�1) and 40 �m DMAPP. To reducecompeting IPP isomerase activity, 5 mm iodoacetamide was added. Afterthe reaction was initiated by addition of the enzyme preparation, the assaymixture was immediately overlaid with 1 mL of pentane and incubated for1 h at 30°C. To stop the assay and hydrolyze all diphosphate esters (bothunreacted substrate as well as products), a 500-�L solution of calf intestinealkaline phosphatase (Sigma, 4 units) and potato apyrase (Sigma, 4 units) in0.2 m Tris-HCl, pH 9.5, was added to each assay and incubated at 30°C for8 to 12 h. After enzymatic hydrolysis, the resulting prenyl alcohols wereextracted into 2 mL of diethyl ether and, after addition of a mixture ofterpene standards, the organic extract was prepared for radio-GC as de-scribed previously (Burke et al., 1999). Radio-GC analysis was performed ona Hewlett-Packard HP6890 gas chromatograph (injector at 220°C and TCDat 250°C) in combination with a Raga radio detector (Raytest, Giessen,Germany). The concentrated organic phase (1–2 �L) was injected on aDB-wax capillary column (30 m � 0.25 mm with 0.25-�m phase coating;J&W Scientific). Separation was achieved under a He flow rate of 2 mLmin�1 with a temperature program of 3 min at 40°C, a ramp to 70°C at 3°Cmin�1 (1-min hold), and a second ramp from 70°C to 240°C at 6°C min�1

(30-min hold). Both mass and radioactivity traces were monitored simulta-neously. Products were identified by comparison of retention times withthose of co-injected authentic standards.

Preliminary enzyme assays had shown that 3H-labeled GPP and FPPwere not incorporated into longer C-15 or C-20 prenyl diphosphate prod-ucts, when GPP or FPP concentrations of 2 to 5 m were used in the presenceof 40 m IPP and 40 m DMAPP. Thus, individual PT activities could be clearlydistinguished.

TPS Enzyme Assays

TPS activities were determined by published procedures (Lewinsohn etal., 1991; LaFever et al., 1994; Bohlmann et al., 1997) with minor modifica-tions. Before assaying enzyme activity, the frozen protein extracts wereplaced at 37°C until just thawed. The protein extracts were desalted inBio-Rad Econo PacI0DG sizing columns pre-equilibrated with appropriateassay buffers: mono-TPS buffer (25 mm HEPES, pH 7.5; 5 mm DTT; 10%[v/v] glycerol; 1 mm MnCl2; and 100 mm KCl), sesqui-TPS buffer (25 mmHEPES, pH 7.3; 10 mm MgCl2; 10 mm DTT; and 10% [v/v] glycerol), ordi-TPS buffer (30 mm HEPES, pH 7.2; 7.5 mm MgCl2; 20 �m MnCl2; 5% [v/v]glycerol; and 5 mm DTT). Enzyme activity was assessed with 1 mL of thedesalted extracts with the addition of 10 �m GPP (with 1 �Ci 3H-GPP) formono-TPS activities, or 10 �m GGPP (0.5 �Ci 3H-GGPP) as substrate fordi-TPS assays. All enzyme assays were done in duplicate, overlaid with 1mL of pentane to collect released volatiles, and incubated at 30°C for 1.5 h.To stop all enzyme activity, the extracts were immediately frozen. Afterthawing, the aqueous assay fraction was rapidly extracted with the pentanefraction by vortexing, and separation of the aqueous and organic fractionswas achieved by centrifugation at 2,500g for 2 min. The 1-mL pentaneoverlay was removed and filtered through a Pasteur pipette filled with 0.4 gof silica gel (Sigma 60 Å) overlaid with 0.6 g of MgSO4 to remove nonspecific

Martin et al.



substrate hydrolysis products and to dry the pentane extract. Each enzymeassay was extracted with an additional two portions of pentane, vortexed,and centrifuged as before. These sequential extractions were also passedover the same column and pooled with the initial column eluent. Subse-quently, the column was washed with pentane (2 � 1 mL) and the totalvolume was determined. The extracts were analyzed by liquid scintillationcounting, 0.1 mL in 0.3 mL of Lipoluma (J.T. Baker, Deventer, The Nether-lands) and GC.

The conditions for all enzyme assays, including pH optimum, incubationtime, substrate concentration, and temperature optimum, were optimizedfor this system such that maximum activity was achieved in a linear rangeof product generation. In addition, the possibility that enzyme activities ininduced tissues might have been inhibited by additional resin or phenolicsubstances was ruled out by experiments in which extracts from differentstages of the time course were mixed together. In all cases, the resultingenzyme activity was additive, implying that additional compounds found ininduced tissues had no effect on enzyme activity.

Analysis of TPS Assay Products

The extracts of duplicate assays were combined and evaporated on ice to50 to 100 �L. From this, 2 �L was analyzed by radio-GC coupled with aTCD. The radio detector enabled the detection of radioactive substances andthe TCD provided a nondestructive method to determine retention times ofco-injected, unlabeled standards. For the mono-TPS assays, the Hewlett-Packard 6890 GC was fitted with a DB-WAX column (described above). Thecolumn flow rate was 2 mL H2 min�1, and the TCD was kept at 250°C withan additional 7 mL H2 min�1 (make-up gas). Parameters for the RAGAradio-detector (Radiomatic, Giessen, Germany) were as follows: The plati-num catalyst was operated at 740°C, the total H2 flow rate including themake-up gas was 20 mL min�1, and methane quench gas flowed through a2-mL counting tube at 5 mL min�1. The samples were introduced to theinjection port at 220°C and were split in either a 5:1 ratio for the bark assayextractions or a 3:1 ratio for the wood assay extractions. The oven wasprogrammed with an initial temperature of 40°C (3-min hold), and in-creased at a rate of 1.5°C min�1 until 70°C, followed by an increase of 15°Cmin�1 until 240°C (3-min hold). Monoterpenes were identified by compar-ing retentions times with those of known standards.

Extracts of di-TPS assays were analyzed by GC-MS employing a DB-WAX column (described above). Extracts from the bark and wood assayswere split 10:1 or 5:1, respectively. The injection port was operated at 250°Cand the column flow and the radio detector were maintained as for themonoterpene analysis. The oven was programmed from an initial temper-ature of 180°C to 240°C at a rate of 4°C min�1, which was followed by a6-min hold. The extracts were further analyzed by GC-MS. For this analysis,the same DB-WAX column was used. A splitless injector was kept at 220°Cand a flow rate of 1 mL He min�1 was maintained. The initial temperaturewas 50°C and the rate increased at a constant 10°C min�1 up to 280°C (3-minhold). All other parameters were as described for the GC-MS diterpene resinanalysis. For peak identification, the 70-eV mass spectra generated werecompared with an authentic abietadiene standard (Rodney Croteau). Allanalysis was performed with Hewlett-Packard Chemstation software.

ACKNOWLEDGMENTS

We thank Juergen Schmidt for trees, Rodney Croteau for substrates andthe abietadiene standard, and Tina Letsch and Nadine Gallitschke for ex-cellent technical assistance.

Received November 5, 2001; returned for revision January 23, 2002; acceptedFebruary 27, 2002.

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Martin et al.



methyl jasmonate induces traumatic resin ducts, terpenoid resin

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