Plant Physiol. (1990) 94, 157-1650032-0889/90/94/0000/00/$01 .00/0

Received for publication February 21, 1990Accepted May 17, 1990

Thermoinductive Regulation of Gibberellin Metabolism inThiaspi arvense L.V

1. Metabolism of [2H]-ent-Kaurenoic Acid and [14C]Gibberellin A12-Aldehyde

Jan P. Hazebroek2 and James D. Metzger*U.S. Department of Agriculture, Agricultural Research Service, Biosciences Research Laboratory, State University

Station, Fargo, North Dakota 58105

ABSTRACT

Field pennycress (Thiaspi arvense L.) is a winter annual cruciferwith a cold requirement for stem elongation and flowering. In thepresent study, the metabolism of exogenous [2H]-ent-kaurenoicacid (KA) and [14C]-gibberellin A12-aldehyde (GA12-aldehyde) wascompared in thermo- and noninduced plants. Thermoinductiongreatly altered both quantitative and qualitative aspects of [2H]-KA metabolism in the shoot tips. The rate of disappearance ofthe parent compound was much greater in thermoinduced shoottips. Moreover, there was 47 times more endogenous KA innoninduced than in thermoinduced shoot tips as determined bycombined gas chromatography-mass spectrometry (GC-MS). Themajor metabolite of [2H]-KA in thermoinduced shoot tips was amonohydroxylated derivative of KA, while in noninduced shoottips, the glucose ester of the hydroxy KA metabolite was the mainproduct. Gibberellin A, (GA,) was the only GA in which theincorporation of deuterium was detected by GC-MS, and this wasobserved only in thermoinduced shoot tips. The amount of incor-poration was small as indicated by the large dilution by endoge-nous GA,. In contrast, thermo- and noninduced leaves metabo-lized exogenous [2H]-KA into GA2o equally well, although theamount of conversion was also limited. These results are con-sistent with the suggestion (JD Metzger [1990] Plant Physiol 94:000-000) that the conversion of KA in to GAs is under thermoin-ductive control only in the shoot tip, the site of perception forthermoinductive temperatures in field pennycress. There wereessentially no differences in the qualitative or quantitative distri-bution of metabolites formed following the application of [14C]-GA12-aldehyde to the shoot tips of thermo- or noninduced plants.Thus, the apparent thermoinductive regulation of the KA metab-olism into GAs is probably limited to the two metabolic stepsinvolved in converting KA to GA12-aldehyde.

Field pennycress (Thlaspi arvense L.) is a cruciferous winterannual that has a requirement for a period oflow temperatures(0-10°C) for the initiation of stem growth. In the accompa-nying paper (14), the efficacies of various exogenous GAs3

'Supported in part by U.S. Department of Agriculture-Competi-tive Research Grants Office Grant No. 86-CRCR-1-1967.

2 Present address: Department of Botany and Plant Pathology,Purdue University, West Lafayette, IN.

'Abbreviations: GA(X), gibberellin A(,X); EtOAc, ethyl acetate; Fr(s),

and GA-precursors to elicit stem growth in TI and NI plantswere used to formulate a hypothesis regarding the relationshipof thermoinduced stem growth to alterations in GA metabo-lism. It was proposed that the lack ofstem growth in NI plantsis due to an inability to convert KA to GAs, and thatthermoinduction results in the removal ofone or more blocksin KA metabolism such that a specific GA accumulates abovesome threshold level, leading to the initiation of stem growth(14). Because KA had a higher level of biological activity inTI plants than GA53, the first committed intermediate in theearly C-13 hydroxylation pathway leading to GA, (6), it wasalso suggested that thermoinduction preferentially directs KAmetabolism to C-1 3 desoxy GAs and that GA9 is the 'effector'GA for thermoinduced stem growth (14).

In the present paper, the results of experiments designed todirectly test aspects of this hypothesis are reported. First, bothqualitative and quantitative aspects of [2H]-KA metabolismin shoot tips of TI and NI plants were examined. Second, asimilar comparison of [2H]-KA metabolism was made forleaves. This distinction is important because the site of per-ception of thermoinductive temperatures resides in the apicaltissues of the shoot tip (13) and the regulation ofGA metab-olism may be different in other tissues. In the third aspect ofthis paper, the metabolism of '4C-GAI2 aldehyde, was inves-tigated to further pinpoint other steps in the GA metabolicpathway that may be under thermoinductive control.

MATERIALS AND METHODS

Plant Material

All experiments were performed with plants of an inbredline (CRI) of field pennycress grown under temperature andphotoperiodic conditions described before (1 1, 14). Briefly, 6-week-old plants grown at 21°C were thermoinduced by sub-jecting them to a 4-week cold treatment at 6C. The plants

fraction(s); GC-RC, gas chromatography-radio counting; glc, glucose;grad HPLC, gradient-eluted reverse phase HPLC, iso HPLC isocratic-eluted reverse phase HPLC; Hex, hexane; KA, ent-kaur-16-ene-19-oic acid; M+, molecular ion; Me(X), methyl ester of X; MeOH,methanol; NI, noninduced; OH KA, hydroxylated ent-kaur-16-ene-19-oic acid; R, retention time; TI, thermoinduced; TMS(X), trimeth-ylsilyl ether or ester of X.

157

HAZEBROEK AND METZGER

were then returned to a growth chamber at 21C; labeledcompounds were applied 10 d after the end of the coldtreatment. At this stage, stems of the plants were between 1and 2 cm, just prior to the linear phase of stem growth (1 1).NI plants used in metabolism studies were the same chrono-logical age as the TI plants when labeled compounds wereapplied. Metabolism studies comparing TI and NI plants wereconducted simultaneously.

Labeled Chemicals

KA was obtained from sunflower florets as described pre-viously (15). [2H]-KA was synthesized by first treating unla-beled KA with Os04-NaIO4 to form the norketone followedby a Wittig reaction (5) using (methyl-d3)-triphenylphosphon-ium bromide (Aldrich4). The resulting [2H]-KA was a mixtureof analogs containing zero to four 2H atoms molecule-' inthe following proportions (% of total) as determined by GC-MS: 0 atom 2H, 2.5%; 1 atom 2H, 13.2%; 2 atoms 2H, 3 1.0%;3 atoms 2H, 35.6%; 4 atoms 2H, 17.8%. [3H]-KA (specificactivity = 1.96 x 106 Bq Amol-'), prepared in analogousfashion as [2H]-KA, was a gift from Dr. T. Gianfagna, RutgersUniversity. [ 1,7,12,1 8,-'4C4]-GA12-aldehyde was prepared byincubating R-[2-14C] mevalonic acid (specific activity = 1.99x 106 Bq Amol-', Amersham) with pumpkin (Cucurbitamaxima L.) endosperm preparations (1, 7). The resulting['4C]-GA12-aldehyde had a specific activity of 7.03 x 106 BqMmol-' as determined by GC-MS (3).

Application of Labeled Compounds

Fifty microliters of a 50% (v/v) aqueous acetone solutioncontaining 0.01% (v/v) Tween 20, 50 Mg of [2H]-KA, and 830Bq of [3H]-KA were applied to each shoot tip of 50 TI and50 NI plants. After 6 h, the shoot tips (apex plus 2-3 youngleaves) were excised, frozen in liquid N2, lyophilized, andstored at -15°C until extraction. Since data from a prelimi-nary experiment indicated minimal movement of radioactiv-ity to lower leaves and roots during the 6 h period betweenapplication and harvest (data not shown), these tissues werediscarded.

In another experiment, a mixture of [2H]-KA (10 Mg) and[3H]-KA (75 Bq) dissolved in 50 AL of 50% (v/v) aqueousacetone with 0.01% (v/v) Tween 20 was applied to each offive young leaves; a total of 10 TI and 10 NI plants were used.After 6 h, the treated leaves were separated from the rest ofthe shoot, frozen with liquid N2, lyophilized, and stored at-1 soC until extraction.The metabolism of ['4C]-GA12-aldehyde by shoot tips of TI

and NI plants was compared. A solution containing 353 ng(7867 Bq) of ['4C]-GAI2-aldehyde in 50 ML of 50% (v/v)aqueous acetone with 0.01I% (v/v) Tween 20 was applied toeach of 36 TI and 36 NI plants. The shoot tips were excised6 h later, frozen in liquid N2, lyophilized, and stored at -15Cuntil extracted.

4Mention of trademark or proprietary product does not constitute aguarantee or warranty of the product by the U.S. Department ofAgriculture and does not imply its approval to the exclusion of otherproducts that may also be suitable.

Extraction and Purification Procedures

[2H,3H]-Labeled compounds were extracted from shoot tipsby homogenization with ice-cold acetone for 2.0 min using aPolytron tissue homogenizer (Brinkman). After filtration, 50mL of deionized H20 were added, and the acetone removedunder reduced pressure at 35°C. The pH of the aqueousresidue was adjusted to 2.5 with 6 N HCI and partitioned fourtimes against H20-saturated EtOAc. Insignificant amounts ofradioactivity were detected in the aqueous fractions, and werediscarded. The organic fractions were combined, dried overanhydrous Na2SO4, and the EtOAc removed. The residue wasdissolved in 1 mL of tetrahydrofuran and subjected in 100,ML lots to iso HPLC on a Whatman 9 x 250 mm MagnumC18 column. The HPLC system was identical to that describedpreviously (15). The column was eluted with a H20-MeOHmixture (1:9, v/v) at 5 mL min-'. Both solvents contained0.1% (v/v) acetic acid. After 10 min, the solvent compositionwas changed to 100% MeOH over a period of 1 min and thecolumn eluted for an additional 9 min. The Rt of authenticGA9, 7,8-OH KA, GA12, KA, and ent-kaurene were 3.25, 3.75,4.38, 8.20, and 16.17 min, respectively. Fractions were col-lected from 3 to 5 (I), 5 to 7.5 (II), and 7.5 to 12 (III) minafter injection, respectively, and each fraction assayed forradioactivity. Frs I and II were then subjected to adsorptionchromatography on 10 x 1.5 cm columns of silica (200-400mesh). The dried fractions were dissolved in a few mL ofEtOAc:CHCl3 (6:4, v/v) and applied to the silica column. Thecolumns were eluted with 100 mL of the EtOAc-CHCl3mixture. The solvent was removed and the residue chromat-ographed on grad HPLC using a convex gradient ofincreasingproportion (30-100%) ofMeOH in H20 as the eluting solventsystem (15). Fractions were collected every min and sampledfor radioactivity.

Fr III resulting from iso HPLC was also subjected to silicaadsorption chromatography. The dried residues were chro-matographed on 30 g of silica using a step gradient of 5%increments from 0 to 30% (v/v) EtOAc in Hex. One bedvolume of each increment was used. The column was theneluted with four bed volumes ofEtOAc. Eluates were sampledfor radioactivity.

Labeled metabolites were extracted from leaves and shoottips following application of [2H,3H]-KA and ['4C]-GA12-aldehyde, respectively, by homogenizing tissue with ice-coldMeOH (about 250 mL) for 2.0 min. The homogenates werefiltered, combined, and reduced in volume. Sufficient H20and MeOH was added to make a 90% (v/v) aqueous MeOHsolution with a final volume of 150 mL. The solutions werestirred for 30 min at 6°C with 10 g of Preparative C18 (55-105 M, Waters Associates). The C18 was removed by filtrationand washed with 100 mL of 90% (v/v) aqueous MeOH. Theprocess was repeated with another 10 g of clean C18. Thefiltrates and washes were combined and the MeOH removedunder reduced pressure at 35°C. After the addition of purifiedH20 to make a final volume of 50 mL, the pH was adjustedto 2.5 with 6 N HCI. The extracts were then partitioned fourtimes against equal volumes of EtOAc. The aqueous fractionscontained little radioactivity and were discarded. The organicfractions were combined, dried over anhydrous Na2SO4, re-

158 Plant Physiol. Vol. 94, 1990

REGULATION OF GA METABOLISM IN T. ARVENSE

duced in volume, and subjected to adsorption chromatogra-phy on silica. The column was eluted with 100 mL ofEtOAc:CHCl3 (6:4 v/v) and then with 100 mL of MeOH as

described before. Eluates with significant radioactivity were

reduced in volume and subjected to grad HPLC as describedbefore.

Derivatization

Fractions to be analyzed by GC-MS were dissolved in a fewmilliliters of MeOH and treated with an excess of etherealdiazomethane. The methylated samples were then dried andredissolved in 25 to 200 uL ofN-methyl-N-trimethylsilyltriflu-roacetamide. The samples were allowed to stand at roomtemperature for at least 2 h before GC-MS analysis.

GC-MS

Derivatized samples were subjected to capillary GC using aHewlett-Packard 5890 instrument containing either a 30 mx 0.32 mm i.d. fused silica column with a 0.25-rim thickmethyl silicone stationary phase (Hewlett-Packard) or a 15 mx 0.32 mm i.d. column with a 0.25 gm 5% phenyl methylsilicone stationary phase (J&W Associates). Samples were

introduced onto the column via a cool-on column injector(1-2 ,L) or via a split-splitless injector (1-5 uL). In the caseof the former, the column was 1 50°C at injection, and after a-min isothermal hold, the temperature was increased to300°C at 7° min-'. The column head pressure of the carriergas (He) was maintained at 35 kPa. The temperature programwhen injecting in the splitless mode was as follows: after a 1

min isothermal hold at 50°C, the column temperature was

ramped at 50°C min-' to 150°C, whereupon the rate oftemperature increase was slowed to 7°C min-' to a maximumof 300C. The injector purge was turned off at injection for a

period of 1 min. The column head pressure was 25 kPa.Regardless of the type of column or injector, columns were

coupled directly to the ion source of a Finnigan-MAT IonTrap mass spectrometer. Positive ions were generated with astream of electrons at 80 uA. Full scan mass spectra (100-600 amu) were recorded at a rate of 2 scans s-'. For thecalculation of ion ratios and specific activities, ion currentresponses integrated over the entire GC peak were used.

GC-RC

Some '4C-labeled metabolites were also analyzed by GC-RC. Derivatized samples were injected onto a glass column(2 mm x 2 m) packed with OV- 17 and installed in a Hewlett-Packard 5880 A instrument. At the column effluent end, thestream of carrier gas (He) was split such that approximately1% went to the flame ionization detector and the rest went toa Barber-Coleman Series 5000 proportional counter. Thetemperature at injection was 100°C, which was raised after 1

min to 300C at 10°C min-'. The flow rate of the carrier gaswas 6 mL min-'.

RESULTS

Metabolism of [2H,3H]-KA in Shoot Tips

The qualitative and quantitative distribution of radioactiv-ity following iso HPLC of extracts from shoot tips 6 h after

application of [2H,3H]-KA were similar under both TI andNI conditions. The vast majority (>90%) of the recoveredradioactivity was contained in iso HPLC Fr III, where au-

thentic KA eluted (data not shown). When Fr III was chro-matographed on silica with a step gradient of increasingproportions of EtOAc in Hex, radioactivity was widely dis-persed in many fractions (Table I). Authentic KA consistentlyeluted almost entirely from silica columns with 15% EtOAcin Hex, even under preparative conditions. However, only11.2 and 37.0% of the radioactivity recovered from the silicacolumns from TI and NI plants, respectively, was containedin this Fr. Furthermore, almost one-third of the recoveredradioactivity from both treatments eluted with 100% EtOAc.In total, these results indicate that iso HPLC Fr III containedmore than just unmetabolized KA.

This conclusion was confirmed by GC-MS analysis. Thefractions resulting from the silica adsorption column thatwere eluted with 15 to 30% EtOAc were combined, deriva-tized, and analyzed by GC-MS. The 100% EtOAc Frs were

treated similarly. Figure 1 shows the total ion current tracesfor the 15 to 30% EtOAc eluates from TI and NI plants. Mostevident was the large difference in the KA peaks (peak 1)from the two treatments. Despite having only 1.4 times moreradioactivity, the Fr from NI plants contained almost 24times more MeKA than the corresponding TI sample as

measured by the total ion current responses for the GC peaksthat had the same R, as authentic MeKA (Table II). Analysisof the mass spectra of the two peaks showed that the greatdisparity between the relative differences in total amount ofradioactivity in each sample and the total ion current re-

sponses for the MeKA peaks was due to both a large contri-bution ofendogenous, unlabeled KA to the total KA extractedfrom NI shoot tips and to a greater amount of metabolism oflabeled KA by TI shoot tips. Comparison of the ion responsefor m/z 256, an abundant ion in the mass spectrum ofMeKAbut almost totally absent in [2H]-MeKA, showed there was

approximately 47 times more endogenous KA in shoot tipsfrom NI plants than in TI tissue (Table II). Conversely,examination ofthe ion response for m/z 260, an ion prevalent

Table I. Distribution of Radioactivity (I3H) Following SilicaAdsorption Chromatography of Isocratic HPLC Fr 111 from EitherThermo- or Noninduced Shoot TipsColumns were eluted with a step gradient of increasing proportions

(5% increments) of ethyl acetate in hexane.Percent of Total Radioactivity

Percent RecoveredEthyl Acetate

Thermoinduced Noninduced

0 0 05 0 010 0 4.215 11.2 37.020 22.0 10.025 20.2 9.130 14.2 8.4100 32.3 31.3

Total (Bq x 103) 11.94 17.06

159

HAZEBROEK AND METZGER

I9B9.

Noninduced

*1 ''''1'''' 1-'--, ---506 69 700 809 S99

Scan Number

10B

I On

Thermoinduced

12

I

I,.I- ,. -.1.. 1... -,.. .. 1-. . ... Is..599 6 790 899

Scan Number980

Figure 1. Total ion chromatograms of the derivatized 10 to 30%ethyl acetate in hexane fractions resulting from silica adsorptionchromatography of an extract of noninduced or thermoinduced shoottips following application of [2H,3H]-kaurenoic acid. Peak 1, methylkaurenoate; peak 2, MeTMS derivative of a hydroxylated kaurenoicacid metabolite.

only in the mass spectrum of [2H]-MeKA, showed there wasroughly 19 times more labeled KA in the extracts from NIshoot tips (Table II).These results demonstrate that metabolic turnover for KA

is much faster in TI shoot tips. They also indicate a highproportion of the radioactivity in the Fr from TI plants isassociated with one or more metabolites rather than KA. Thiswas confirmed by inspecting the mass spectra of the peaks inthe total ion chromatograms shown in Figure 1. The Frs fromboth TI and NI plants contained a number of compoundsthat appeared from the mass spectra to be labeled with deu-terium. However, most of these compounds were minor com-ponents and were not dramatically different between the twotreatments. The compound observed at scan 746 (peak 2) wasan exception. Its mass spectrum clearly indicated the incor-poration of deuterium with ion clusters around the molecularion (m/z 406-408), the base peak (m/z 255-258), and severalother fragments that closely resembled those observed in themass spectrum of [2H]-MeKA (Fig. 2A). This compoundapparently is a hydroxylated KA, but its R, on GC and massspectrum did not match those of authentic 7a-,7fl-, 11,3,13-(steviol), or 1 5$-OH KA. Furthermore the presence of mod-erately intense and unlabeled ions at m/z 109, 121, 123, 148,and 181, which are derived from the A ring ofthe ent-kaureneskeleton following cleavage of ring B (4), indicates that the

OH group is not located on that portion of the moleculeeither. This limits the positions open for hydroxylation to C-9, -12, -14, and the a epimers at C- 1 and C-15.

Regardless, this compound does not appear to be endoge-nous to field pennycress, or occurs in minute amounts becausethe ratio of ion intensities for the molecular ion cluster werenearly identical to [2H]-MeKA. The unknown compound wasnot detected in protio form in large scale extracts of either TIor NI shoots (data not shown). However, the extent to whichthis compound accumulated was strongly dependent onthermoinduction. There was over 9 times more OH KAmetabolite in the extracts of TI shoot tips as measured by thetotal ion current responses.The 100% EtOAc eluates resulting from silica adsorption

chromatography of iso HPLC Fr III were also analyzed byGC-MS. Although the amount of radioactivity contained inthe fractions from the two treatments was not radically differ-ent (Table I), the qualitative pattern of compounds labeledwith deuterium was. There were two compounds in the Frfrom TI shoot tips with detectable incorporation of deute-rium. One had an identical R, and mass spectrum as the OHKA metabolite found earlier, while the other had a massspectrum indicating it was a diOH KA (Fig. 2B). The identityof this compound is not clear, but its retention time and massspectrum eliminates 6,B,7,B- and 7f3, 1 3-diOH KA as possibili-ties (data not shown). However, the similarities between frag-mentation patterns of the OH KA and diOH KA (Fig. 2, Aand B) suggests the two have one common hydroxylatedposition. If so, the former compound may be a precursor tothe diOH KA.

These two metabolites were not observed in the extractsfrom NI shoot tips. Instead, another 2H-labeled compoundthat was a major component of the total ion chromatogramwas detected with a mass spectrum containing 2H-enrichedion clusters (Fig. 2C). The intense ion at m/z 450, which wasnot part ofa cluster characteristic ofother ions in the spectrumlabeled with deuterium, is indicative of TMS glc conjugates(10, 18). Treatment of this fraction with KOH at 50°C for 12h, followed by rederivatization and GC-MS resulted in thecomplete disappearance of the compound from the total ionchromatogram and the concomitant appearance of another

Table II. GC-MS Analysis of Methyl Kaurenoate Extracted fromShoot Tips of Thermo- or Noninduced Field Pennycress Plantsfollowing Application of [2H,3H]-Kaurenoic Acid

Thermoinduced Noninduced

Total radioactivity (Bq x 103) 8.08 11.71Total ion current response of 232,260 5,556,550

the MeKA peakIon current response for m/z 621 29,431256a

Ion current response for m/z 10,770 208,360260a

Ratio 260/256 17.1 7.0a The values for the ion current responses of m/z ions 256 and

260 were corrected for the minor contributions made by [2H]- and['H]-methyl kaurenoate, respectively.

I--

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., $.1 e .)_

160 Plant Physiol. Vol. 94, 1990

I

2. IM -1,I

REGULATION OF GA METABOLISM IN T. ARVENSE

1K9 257

Il ilsil i2j1

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5W,.

A

497

B

315

423

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1 391K00 2K 3K 4K 5K iK

. ~~~~~~~~~~450257

17 241 359~1471 375 46

117 311111[ll.,.183 ..,,, , 1 ,, 1,I

2W 3. 4, 5u 'U

mlz

Figure 2. Mass spectra of the derivatized 2H-labeled metabolitesextracted from field pennycress shoot tips following application of[2H,3H]-kaurenoic acid. A, MeTMS derivative of a hydroxylated kau-renoic acid metabolite; B, MeTMS derivative of a dihydroxylatedmetabolite of kaurenoic acid metabolite; C, TMS derivative of theglucose ester of (A).

compound with an identical R, and mass spectrum as theMeTMS-OH KA metabolite (data not shown). It was there-fore concluded that the metabolite is the glc ester of theputative OH KA metabolite.

Iso HPLC Fr II (5-7.5 min after injection) contained verylittle radioactivity and was not analyzed further. In contrast,significantly more radioactivity was detected in iso HPLC FrI (3-5 min), where logical KA metabolites such as 7,B-OH KAand GA12 elute. When this Fr from both TI and NI plantswas subjected to grad HPLC, no significant differences in thedistribution of radioactivity following grad HPLC from eithertreatment were found (data not shown). In both cases, mostof the radioactivity eluted in grad HPLC Frs 22 and 23.Although this is where the KA metabolites, 7,8-OH kauren-

olide, 6#,713-diOH KA, and 7A3-OH KA elute in this gradientsystem (15), none (labeled or unlabeled) were detected byGC-MS. Likewise, no 2H-labeled compounds were detectedin grad HPLC fractions 24 and 25 where GA12 and GA12-aldehyde elute (15).Grad HPLC Fr 21 from either treatment contained only

small amounts of radioactivity. However, in the TI extract, alarge peak in the total ion chromatogram was observed thathad an identical retention time and mass spectrum as MeGA9.This compound was not detected in grad HPLC Fr 21 fromNI plants. Close inspection of its mass spectrum revealedsmall, but significant shifts in the ion abundance ratios forthe base peak cluster, m/z 270-274 (Table III). The magnitudeof these shifts were consistent in mass spectra obtained fromtwo additional injections of the same sample. These resultsindicate a small amount of deuterium was incorporated intoGA9.

Like GA9, GA51 and GA25 (both C-13 desoxy GAs) werefound only in grad HPLC Frs 20 and 21, respectively, fromextracts ofTI plants. However, no incorporation ofdeuteriumwas detected in either mass spectrum.

Metabolism of [2H,3H]-KA in Young Leaves

As for the shoot tips, the qualitative and quantitative dis-tribution of radioactivity following grad HPLC was nearlyidentical for leaves of TI and NI plants. Most of the radioac-tivity was contained in grad HPLC Fr 27, where authenticKA eluted (data not shown). When this Fr was derivatizedand analyzed by GC-MS, the major peak in the total ionchromatogram for the TI sample was unmetabolized [2H]-MeKA (Fig. 3, top). However, no [2H]-MeKA was observedin the total ion chromatogram of the NI sample. Instead themajor compound, which eluted about 1 min later thanMeKA, had a mass spectrum indicating that it was probablythe glc ester of [2H]-KA (Fig. 3, bottom). The base peak(m/z 331 ) is characteristic of many glc esters and arises fromthe fragmentation of the TMS-glucosyl moiety (10, 18). Theidentification of this metabolite as KA-glc ester was corrobo-rated by two additional lines of evidence. First, both base and

Table Ill. GC-MS Analysis of MeGA9 Extracted from Shoot Tips ofThermoinduced Field Pennycress Plants following Application of[2H,3H1-Kaurenoic Acid

Ion Current ResponseIon (m/z) Authentic Field pennycress

MeGA. MeGA9

Total 9158 11,425270 1597 (59.3) 1289 (54.4)271 844 (31.3) 709 (29.9)272 230 (8.5) 301 (12.7)273 21 (0.8) 56 (2.4)274 0 (0.0) 16 (0.7)

a The values represent the ion current response integrated overthe entire GC peak. Numbers in parentheses are the percentages ofthe sums of the ion current response for the ion cluster m/z 270 to274.

0-%

(Ac

U)0.ux

0)

m

z

161

180

73

1III

HAZEBROEK AND METZGER

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Thermoinduced

.I ,,,,,...389 409 420 440 460 489 599

Scan Number

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389 490 429

529 548

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int:La I 1371 451

0/Noninduced

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449 469 489 59 529 540Scan Number

Figure 3. Total ion chromatograms of the derivatized gradient-elutedHPLC Fr 27 from an extract of thermoinduced and noninduced leavesfollowing application of [2H,3H]-kaurenoic acid. Insets show the massspectra 2H-labeled methyl kaurenoate (top) and the TMS derivativeof the putative glucose ester of kaurenoic acid (bottom).

enzymatic hydrolyses led to the appearance of a 2H-labeledcompound with an identical R, and mass spectrum as authen-tic [2H]-MeKA (data not shown). Second, hydrolytic removalof the TMS groups with H20-MeOH followed by acetylationusing acetic anhydryde at 25°C for 20 h resulted in a com-pound with a R, and mass spectrum of authentic 0-acetyl-KA glc ester that was synthesized according to the methoddescribed by Hiraga et al. (8) (data not shown).Examination of the intensity ratios of ions within labeled

clusters did not indicate the presence ofunlabeled endogenousKA glc ester. However, GC-MS analysis ofa partially purifiedextract of whole shoots from 100 NI plants established thatKA-glc ester does indeed occur naturally in field pennycress,albeit in low concentrations. Interestingly, no KA-glc esterwas detected in similar extracts from TI plants (data notshown).

Small amounts of tritium were associated with other gradHPLC Frs and no significant differences were noted betweenthe two treatments. These Frs were derivatized and analyzedby GC-MS. However, only in grad HPLC Fr 12 was theincorporation of deuterium into the mass spectrum of aknown endogenous compound observed. MeTMS GA20 wasdetected in this Fr from both TI and NI plants. Analysis ofthe M+ cluster (m/z 418-421) indicated there were small, butsignificant amounts of deuterium incorporated into the mol-ecule (Table IV).

Metabolism of [14C]-GA12-Aldehyde in Shoot TipsSix h after the application of ['4C]-GA12-aldehyde to TI and

NI plants, shoot tips were harvested, extracted, and subjectedto adsorption chromatography on silica. For both treatments,approximately 75% ofthe radioactivity applied to the columneluted with the EtOAc-CHCl3 mixture, while the remaindereluted from the column with MeOH. These Frs were thensubjected to grad HPLC. Figure 4 shows a comparison of thedistribution of radioactivity following grad HPLC of theEtOAc-CHCl3 Frs from the two treatments. Although therewas slightly more total radioactivity in the Fr from TI plants,the distribution patterns of radioactivity were similar. Twomajor peaks of radioactivity were observed at Frs 17 and 24,while much smaller peaks occurred at Frs 12, 14, and 21.

All Frs containing radioactivity were derivatized and ana-lyzed by GC-MS. No residual ['4C]-GA12-aldehyde was de-tected in grad HPLC Frs 24 and 25 from either treatment.The only labeled compound observed was MeGA12. Calcula-tion of specific activities (3) showed that little dilution oc-curred, although there was more dilution in the NI sample(Table V). Analysis of grad HPLC Frs 12, 13, and 14 showedincorporation of '4C into MeTMS GA20, MeTMS GA44, andMeTMS GAI9, respectively. In contrast to MeGA,2, there wasa considerable reduction in the specific activities of thesecompounds, indicating a large contribution from the endog-enousGA pool. This reduction in specific activities was higherfor those compounds from NI plants (Table V).Grad HPLC Fr 17 from both treatments contained the

second greatest amount of radioactivity of any Fr in thechromatogram (Fig. 4). None of the known endogenous GAsin field pennycress (16) chromatograph in this Fr. Neverthe-less, a labeled compound was detected in Fr 17 from bothtreatments with a mass spectrum suggesting that it was anisomer of GA,9 (Table VI). Although neither m/z 462 (M+)nor 470 (M+ + 8) were detected, other fragments in the massspectrum support this interpretation, e.g. the loss ofCO (M+-28), HCOOCH3 (M+-60), and HCOOCH3 + CH3 or HO =Si(CH3)2 (M+-75), more easily explain the appearance ofions

Table IV. GC-MS Analysis of MeTMS-GA20 Extracted from Leavesof Thermo- (TI) and Noninduced (NI) Plants following Application of[2H,3H]-Kaurenoic Acid

Ion Current Responsea

Ion (m/z) Authentic NI field TI fieldMeTMS GA2o pennycress pennycressMeTMSGA20 MeTMS GA2o

418 10,179 (65.3) 10,012 (62.7) 8,843 (61.1)419 3,955 (25.4) 4,005 (25.1) 3,724 (25.7)420 1,227 (7.9) 1,402 (8.8) 1,330 (9.2)421 216 (1.4) 410 (2.6) 423 (2.9)422 0 (0) 140 (0.9) 144 (1.0)

a The values represent the ion current response integrated overthe entire GC peak. Numbers in parentheses are the percentages ofthe sums of the ion current responses for the ion cluster m/z 418 to422.

0-

0-a)

a}0)

CLC1)cr:

U

C0

00)

L.

L-

cr:

. I 71=i-" I . . . . --. "Ir-Is _

162 Plant Physiol. Vol. 94, 1990

REGULATION OF GA METABOLISM IN T. ARVENSE

5 7 9 11 13 15 17 19 21 23 25 27 29

Fracilon Nuw

Figure 4. Distribution of radioactivity following gradient-eluted HPLCof the ethyl acetate-chloroform fraction from silica adsorption chro-matography of an extract of shoot tips treated with ['4C]-GA,2-aldehyde. Fractions collected every minute.

442 (434, unlabeled), 410 (402), 395 (387), and 367 (359),respectively, in the mass spectrum than other possibilities. Nodilution of this compound was evident in either treatmentsince the calculated specific activities were slightly higher thanMeGA2 (Table V). This compound was not detected in protioform in large extracts of whole field pennycress shoots fromeither TI or NI plants (data not shown), and is therefore eithernot a naturally occurring compound in field pennycress, or

occurs in extremely low amounts.GC-MS analysis of grad HPLC Fr 21 from both treatments

did not reveal any compounds with mass spectra indicatingthe incorporation of 14C. TI plants did contain MeGA9, butit appeared unlabeled. As before, no MeGA9 was found ingrad HPLC Fr 21 from NI plants. These two Frs were subse-quently analyzed by GC-RC. They both contained a verysmall amount of a radioactive compound with the same R, asauthentic MeGA9. The major radioactive component cochro-matographed with MeGA,5 (Fig. 5). GA15 is a C- 13 desoxyprecursor to GA9 (6).When the MeOH eluates from the silica column were

subjected to grad HPLC, only one peak of radioactivity (Fr15) was observed in both treatments (data not shown). GC-MS analysis showed that this Fr contained one main labeledcompound with a mass spectrum very similar to the publishedspectrum of MeTMS 16,17 diOH-16,17 dihydro GAI2 (17).The calculated specific activities for this compound in theextracts from the two treatments were nearly the same asthose of ['4C]-GA12, indicating little or no dilution by endog-enous material (Table V).

DISCUSSION

Thermoinductive Regulation of KA Metabolism

In the shoot tips, thermoinduction clearly resulted in quan-titative and qualitative alterations of KA metabolism (TableII). Although these results demonstrate increased KA turnoverfollowing thermoinduction, they provide no information

about the ability to convert KA to GAs. The great increase in[2H]-KA metabolism observed in TI shoot tips was not re-

flected in a dramatically higher conversion to GAs. Indeedthe two most prevalent metabolites following application of[2H]-KA to the shoot tips were an OH-KA isomer and its glcester (Figs. 1 and 2), neither of which have been found as

endogenous compounds in field pennycress. Nevertheless,small amounts of deuterium were evident in the mass spec-

trum of MeGA9 isolated from TI shoot tips (Table III),although there was no indication for metabolism of [2H]-KAto any GAs by NI shoot tips. Thus, these results are consistentwith the hypothesis that the metabolism of KA by fieldpennycress shoot tips to GAs is under thermoinductive con-trol. However, the level of incorporation into GA9 was lowand no labeled intermediates in the metabolic pathway (6)from KA to GA9 (e.g. 7( OH-KA, GA12-aldehyde, GA12,GA15, or GA24) were detected.

In contrast to the shoot tips, no effects of thermoinductionon the conversion of KA to GAs were observed in leaves(Table IV). This is not unexpected since the site of perceptionof thermoinductive temperatures in field pennycress residesin the shoot tips, not the leaves (13).The most dramatic difference between TI and NI plants in

[2H]-KA metabolism was the tendency of both shoot tips andleaves to accumulate glc conjugates as metabolites. There waslittle metabolism of [2H]-KA when applied to TI leaves, whilein TI shoot tips the major metabolite was an OH KA isomer.In sharp contrast, little or no free [2H]-KA or [2H]-OH KAmetabolite were detected in NI leaves and shoot tips, respec-tively. These compounds were almost entirely metabolized tothe glc esters (Figs. 1 and 4). Furthermore, KA-glc ester wasdetected as an endogenous component only in large-scaleextracts of NI shoots. The physiological significance of thesedifferences in KA metabolism are unclear, but it is unlikelythey are related to thermoinductive control of stem growth.Although the KA-glc ester could be a storage form that servesas a readily mobilizable source ofKA following thermoinduc-tion, this does not appear likely since the KA-glc was detectedonly in leaves which are removed from the site of perceptionfor thermoinductive temperatures (13). In any event, thedifferences in the ability to accumulate glc esters may be an

indication that the glucosyl transferase(s) responsible for the

Table V. Specific Activities of Labeled Metabolites Resulting fromApplication of ['4C1-GA12-Aldehyde to the Shoot Tips of Thermo- (TI)and Noninduced (NI) Plants

Specific ActivityaMetabolite

NI TI

Bq x 106,SMo 1-

GA12 6.44 6.92GA44 0.40 0.74GA19 0.34 2.02GA20 0.28 0.63Putative GA19-isomer 6.74 7.0416,17 diOH-16,17 dihydro GA,2 6.37 6.97

a Specific activities determined by the method of Bowen et al. (3).

163

HAZEBROEK AND METZGER

conjugation reactions are under strong developmental con-trol, or greatly affected by the cold treatment in ways unrelatedto thermoinduction (e.g. cold adaptation).

Thermoinductive Regulation of GA12-AldehydeMetabolism

Unlike the case for KA, GA12-aldehyde metabolism isprobably not under thermoinductive control since the quali-tative and quantitative distribution of metabolite formationwas similar in TI and NI shoot tips (Figs. 4 and 5; Table V).Moreover, as judged by GC-RC analysis, both TI and NIshoot tips had similar capabilities for converting 14C-GA12aldehyde to C- 13 desoxy GAs (Fig. 5) despite the fact thatthese compounds were not detected in their unlabeled formin NI plants. This, coupled with the observation that only TIshoot tips converted [2H]-KA into GA9, indicates there areprobably at most two reactions under thermoinductive con-trol in field pennycress shoot tips: KA -. 7,3-OH KA and 713-OH KA -- GA12-aldehyde.The metabolism of ['4C]-GA12-aldehyde was extremely

rapid. No parent compound was detected by GC-MS 6 h afterapplication in either TI or NI shoot tips (Fig. 4; Table V).Rapid metabolism of ['4C]-GA12-aldehyde was observed inboth soybean and pea cotyledons as well (2, 9). The mostprevalent metabolite was [14C]-GA12. It is not certain howmuch of the formation of this metabolite is due to non-enzymatic oxidation of the C-7 aldehyde, although little oxi-dation occurred when ['4C]-GA12-aldehyde was subjected tonormal work-up procedures used in the fractionation of plantextracts (data not shown).

['4C]-GA12-aldehyde was metabolized to two other majorproducts tentatively identified as 16,17 diOH-16,17 dihydroGA12 and an isomer ofGAI9 (Table V). The former metabolitehad specific activities nearly identical to that calculated for[14C]-GA12, and those of the putative GAI9 isomer were alsosimilar, if not slightly higher (Table V). This lack of dilutionindicates extremely small or nonexistent pools of the endog-enous counterparts of these two metabolites. Thus, as ob-served from the metabolism of [2H]-KA, major metabolitesdo not appear to be endogenous. In contrast, there was farless conversion ofeither [2H]-KA or ['4C]-GA12-aldehyde intoendogenous GAs as evidenced by the extreme dilution of label(Tables III, IV, and V). This suggests that the amounts oflabeled substrates applied in these experiments exceeded thecapacity of the endogenous metabolic machinery to convertthe precursors into GAs. In this regard, the positions of theOH groups on the GA,9-isomer and the OH KA metabolite

Me-GA.N

0 Thermoinduced

A~ ~ *'

Noninduced

0 12.5 25

Retention Time (min)

Figure 5. GC-Radiochromatography of the derivatized gradient-eluted HPLC Fr 21 following application of [14C]-GA12-aldehyde tothermo- or noninduced shoot tips.

are of interest. A common position for hydroxylation wouldsuggest the existence of a hydroxylase with broad substratespecificities, a property that might be expected for an enzymesystem involved in xenobiotic metabolism.The apparent differences in the ability of shoot tips to

convert ['4Cl-GA12-aldehyde and [2H]-KA to members of theearly C-13 OH pathway may be due in part to the form oflabel used to follow metabolism. Only a relatively smallamount of [3H]-KA was applied simultaneously with the[2H]-KA to track metabolites through the extraction andpurification procedures. In contrast, nearly seven times moreradioactivity was used when examining ['4C]-GA12-aldehydemetabolism although the total mass of applied substrate wasalmost 200-fold less. GC-MS analysis was done only with

Table VI. Mass Spectral Analysis of Two 14C-Labeled Metabolites Extracted from Thermo- (TI) andNoninduced (NI) Shoot Tips following Application of 14C-GA12-Aldehyde

Compound Source Principal Ions and Relative Abundance (% base peakrPutative GA19 isomer TI 470 (M+,0) 442 (29) 410 (100) 367 (28) 240 (72)

NI 470 (M+,0) 442 (35) 410 (100) 367 (56) 240 (74)16,17 diOH-16,17 dihydro GA12 TI 546 (M+,0) 486 (2) 443 (100) 383 (71) 293 (19)

NI 546 (M+,0) 486 (1) 443 (100) 383 (49) 293 (25)a All ions shown are from the 14C-labeled analog of the metabolite.

Me-GA15

1100 cpm

164 Plant Physiol. Vol. 94, 1990

REGULATION OF GA METABOLISM IN T. ARVENSE

fractions that contained significant amounts of radioactivity.Thus, it is possible that small amounts [2H]-KA were incor-porated into C- 13 OH GAs, but went undetected becauselittle or no radioactivity chromatographed with these com-pounds.

CONCLUSIONS

In the accompanying paper, a hypothesis based on com-parative biological activities of exogenous GAs and GA pre-cursors was presented for the biochemical basis of thermoin-duced stem growth in field pennycress (14). It was proposedthat the conversion of KA to GAs is regulated in such a waythat a specific GA accumulates to levels sufficient for theinitiation of stem growth only in TI plants. It was alsosuggested that GA9 is responsible for controlling thermoin-duced stem growth in field pennycress (14). The followinglines of evidence reported here support this hypothesis.

Evidence for thermoinductive regulation ofKA metabolismto GAs was observed in the shoot tips, but not the leaves(Tables II and IV). This is reasonable if one compares thephysiological characteristics of stem and leaf growth in fieldpennycress. Although, like stem growth, petiole elongationhas a GA-dependent component, growth rates of petioles arethe same in both TI and NI plants (12). In addition, the siteof perception of low temperatures for thermoinduced stemgrowth resides in the shoot tips, not the leaves (1 3). Therefore,tissue specificities for the regulation of GA biosynthesis andmetabolism may be required because the two developmentalprocesses are temporally and spatially separated.On a relative basis there was 47 times more endogenous

KA in NI than TI shoot tips (Table II). This is an indicationthat the metabolism of KA to GAs is blocked in NI shoottips, resulting in the accumulation of KA.The C- 13 desoxy GAs, GA9, GA25, and GA51 were detected

only in TI shoot tips. Moreover, GA9 was the only GA forwhich there was mass spectral evidence for the biosynthesisfrom [2H]-KA in the shoot tips and this was observed only inTI material. Together, this indicates that only TI plants (shoottips) synthesize C- 13 desoxy GAs and is consistent with thehypothesis that GA9 controls the initiation of thermoinducedstem growth.

ACKNOWLEDGMENTS

We wish to thank Amy Hassebrock and Marcia Mardaus for theirassistance in the isolation of KA and the synthesis of ['4C]-GA12-aldehyde. We also wish to thank Tom Gianfagna for his experttechnical advice on the Wittig reaction.

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