investigations on nature of auxin-wave cambial region ... · corrections vol.82: 518-522, 1986...

Plant Physiol. (1987) 84, 135-1430032-0889/87/84/01 35/09/$01.00/0

Investigations on the Nature of the Auxin-Wave in the CambialRegion of Pine Stems'VALIDATION OF IAA AS THE AUXIN COMPONENT BY THE AVENA COLEOPTILE CURVATUREASSAY AND BY GAS CHROMATOGRAPHY-MASS $PECrROMETRY-SELECTED ION MONITORING

Received for publication December 30, 1985 and in revised form December 18, 1986

TOMASZ J. WODZICKI2, HIROSHI ABE3, ALINA B. WODZICKI2, RICHARD P. PHARIS*, ANDJERRY D. COHENPlant Physiology Research Group, Biology Department University ofCalgary, Calgary, Alberta, CanadaT2N 1N4 (T.J.W., H.A., A.B.W., R.P.P.,), and United States Department ofAgriculture, AgriculturalResearch Service, Plant Hormone Laboratory, Beltsville Agricultural Research Center- West,Beltsville, Maryland (J.D.C.)

ABSTRACr

The major auxin ofScots pine (Pinus silvestris L.) which is transportedbasipetally into agar strips from the cambial region of the stem wasquantified by the Went Avena coleoptile curvature assay before and afterreversed phase Cis high performance liquid chromatography (HPIL),and then identified by full spectrum gas chromatography-mass spectrom-etry (GC-MS) as indole-3-acetic acid (IAA). The IAA was subsequentlyquantified by GC-MS-selected ion monitoring (SIM) using an internalstadard of J13Cj(C4)-LAA. The amount of IAA collected into 22-milli-meter long agar strips during 10 minutes of contact with the stem cambialregion was estimated by GC-MS-SIM and the Went bioassay to be 23and 2.1 nanogrs- per strip, respectively. The GC-MS technique thusconfirmed the results obtained by the Went curvature assay. The Aremcurature assay revealed the presence of at least one other, more polar(based on HPLC retention time) auxin that diffused into the agar stripswith the IAA. Its bioactivity was only 5% of the UIA fraction. Its HPLCretention time was earlier than IAA-glucoside, IAA-aspartate, or IAA-glycine, but the same as IAA-inositol. No sigifnt amounts of inhibitorsor synergists of IAA activity on the Arena assay were found in extractscorresponding to one or five strips of agar. Thus, the direct bioassay ofthe agr strips immediately after their removal from the cambial regionof P. silhestris stem sections reflects the concentration of the native IAA.For both P. silhestris and lodgepole pine (Pings contorta) a wavelikepattern of auxin stimulation of Arena curvature was found in apr stripsexposed for oaly 10 minutes to the basal ends of an axial ser*s of 6-millimeter long sections from the cambisl region of the stem. This wave-like pattern was subsequently confirmed for P. contota both by A4enacurvature assay and by GC-MS-SIM of HPLC fractions at the retentiontime ofVHIIAA. The wavelike pattern ofauxin diffuing from the cambialregion of Pinus has thus been determined to consist primarily ofIAA andthis pattern has now been quantitated using both the Went APenacurvature assay and GC-MS-SIM with i'3Cj-C6-IAA as an internalstandard.

Oscillations in the basipetal efflux of natural auxin-like growthsubstances from the cambial region of tree stems have beenmeasured using the Went Avena coleoptile curvature test (17,18, 20). When successive axial series of small stem sections were

tested, oscillations of auxin activity were observed. This hasprovided a basis for the formulation ofa new concept concerningthe mechanism ofauxin regulation in the morphogenesis ofstemgrowth in conifers (21-23). Most ofthe past work has been donewith Pinus silvestris. Thus, this species and also one species ofNorth American two-needled pine, Pinus contorta, have beenchosen for the present investigation ofthe postulated auxin-wavein the cambial region of the stem of Pinus trees.The discussion concerning the nature of the auxin in Pinus

began with Fransson's (4, 5) studies of the diffusion rate of thisgrowth stimulator through agar. Fransson concluded that theidentity ofthe P. silvestris auxin was not IAA. Later investigationby paper chromatography ofauxin extracted with methanol fromthe cambial region ofP. silvestris, however, tentatively identifiedthis auxin as IAA (14), and this was confirmed (although by lessthan definitive methods) in extracts of proleptic buds and shootsusing gel filtration after a Sephadex LH-205 column (1). Al-though there were discrepancies in the Avena curvature stimu-lation curves obtained using authentic IAA and the auxin ex-tracted from P. silvestris (16), IAA was conclusively identified asa component of the native auxin extracted from seedlings of P.silvestris by GC-MS (10).An attempt (19) to confirm the auxin wave observed by

bioassay of P. silvestris diffusates was made using a fluorescencemethod after conversion of IAA into indole-a-pyrone (7). How-ever, these workers (19) were not able to confirm the wave inauxin content that had been detected in a number of studiesusing the Avena curvature assay. Thus, the present study wasundertaken in an attempt to confirm by the more definitivemethod of GC-MS the identity of the auxin that diffuses intoagar from the cambial region of Pinus, and to further study the

'Supported in part by a Canadian Forestry Service P.R.U.F. Contract,a University of Calgary Visiting Scholar Award, a Natural Sciences andEngineering Research Council of Canada International Scientific Ex-change Award and, Research Grant A-2585, to R. P. P.

2 Permanent address: Agricultural University of Warsaw, (S.G.G.W.-A.R.), Department of Forest Botany, 26/30 Rakowiecka Str., 02-528,Warsaw, Poland.

3 Permanent address: University of Agriculture and Technology, Fac-ulty of Agriculture, Department of Plant Protection, Fuchu, Tokyo 183,Japan.

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Plant Physiol. Vol. 84, 1987

auxin-wave phenomenon by using GC-MS-SIM4 of IAA afterisocratically eluted reversed phase C,8 HPLC.

MATERIALS AND METHODS

Collection of Auxin-Like Growth Substances. Segments 20 x20 cm, sawn from the stem of mature (about age of 60-80 years)trees were brought to the laboratory and recut into smaller platesof tissue (22 X 100 x 4 mm, or 44 x 72 x 4 mm with the longerdimension paralleling the stem axis) (Fig. 1). This tissue iscomprised of the phloem, cambium, and 1 to 2 recent annualrings of wood. The tissue plates were further sectioned perpen-

dicular to the stem axis into a series of sections (each 6 mm highand 22 or 44 mm wide as measured in direction of stem circum-ference) and one or two 2 x 1.5 x 22 mm strips of 1% agar(Difco)5 were immediately applied opposite to the stem cambialregion at both apical and basal cut surfaces (Fig. 1). Strips fromthe basal ends were collected after 10 min of contact with thetissue, and were subdivided into four pieces. Alternate pieceswere either immediately frozen and freeze-dried, or immediatelyassayed using the Went Avena coleoptile curvature test (usingone-half of the strip) (Fig. 1). The choice of a diffusion time wasbased on empirical testing where efflux at 10 min was shown tobe on the exponential part of the efflux versus time curve, andwas sufficiently long enough to allow for statistically significantcomparisons between successive stem sections. The other portionof the same strips were extracted with 80% aqueous methanolfor C,8 HPLC followed by bioassay and/or GC-MS-SIM. Thefreeze-dried strips had been collected from P. silvestris stems ofapproximately 80-year-old trees in the forest stand in RogowExperimental Forests of the Warsaw University of Agriculture(S.G.G.W.) Poland. They were transported sealed under N2 tothe University of Calgary and stored at -20OC until extracted.There were two series of these strips. The first were collected inlarge numbers during July and August 1984, and the second, a

series of 12 strips each corresponding to a successive axial seriesof stem sections, were collected in May 1985 in Poland fromseveral trees. The latter series allowed the analysis of consecutivestem sections. Agar strips for fresh analysis on the Went curvatureassay, and also for extraction, were collected in an analogoussequence from stems of P. contorta in May 1985 in the RockyMountain foothills about 60 km west of Calgary, Alberta.

Extraction. Dry strips ofagar were extracted with 80% aqueous

methanol three times(1 h plus 2x 0.5 h), each time in aliquotsequivalent to10 ml of methanol per100 strips (or1 ml per strip,if single strips were extracted). The three extracts were filteredusing a Millipore FH 0.5 Mm filter, combined and immediatelyevaporated to dryness with excess methanol under reduced pres-

sure. Extractions were usually performed with 30 to100x 103dpm of[3H]AA (16.7 Ci/mmol) added as an internal standard.Extraction of test agar strips prepared with [3H]IAA showed theextraction method to be >97% efficient.High Performance Liquid Chromatography. The dry residue

of the 80% methanol extract was dissolved in small volumes(1-2 ml) of28% methanol, filtered and subjected to reversed phaseC,8 HPLC fractionation (analytical IA-Bondapak columns) usinga Waters HPLC with solvent delivery system model 6000A andautomated gradient controller model 680. The fractionationprograms were chosen after a series of preliminary runs (seebelow) designed for:

(a) Effective separation of IAA,IAA-sugar conjugates,IAA-amino acid conjugates, IAA-methyl ester, and ABA.

Abbreviations: SIM, selected ion monitoring;lAInos, indole-3-acetyl-myo-inositol; Rt, retention time.

'Mention of a trademark, proprietary product, or vendor does notconstitute a guarantee or warranty of the product by the United StatesDepartment of Agriculture, and does not imply its approval to theexclusion of other products or vendors that may also be suitable.

22or

CPh

A 6 M x

B E B E

FIG. 1. The method of collecting into agar strips the natural sub-stances diffusing from the cambial region of a series of 12 successive 6mm sections of stem. Agar strips, A; cambium,C; phloem, Ph; xylem,x. Segments of the basal agar strips used for bioassay (B) or extraction(E).

(b) The possibility of detection of nanogram amounts ofIAAby in-line spectrophotofluorimetry, and

(c) To determine whether ['H, '2C]IAA, [2H]-(d5)-IAA, and['3C]-(C6)-IAA eluted in the same HPLC-fractions.

Results of the above HPLC runs are shown in TableI, as arethe HPLC programs selected for further use in the investigationof extracts of the natural auxins from Pinus.

Spectrophotofluorimetry. A Schoeffel Spectroflow monitorsystem with L. C. Fluorimeter model SF-970 and Monochro-mator model GM970 were used to determine Rt peaks forIAAand the variousIAA conjugates as well as noting the Rt offluorescent impurities from the extracts of agar. The emission offluorescence was recorded using a 370 nm monochromatic filterand an excitation wavelength of 280 nm, at which the highestefficiency of measurements in the methanol:H20:1% acetic acidsolvents was obtained. By setting the fluorimeter sensitivity at0.05 A, the time constant at 6 s, the recorder input at 10 mV,and the recording rate 30 cm/h, extremely sharp peaks werereproducibly recorded. The lowest detectable amount of IAAwas 500 pg when chromatographed with the 28% methanolHPLC solvent. At lower concentrations of methanol, peaks ofIAA were progressively wider and thus the detection limit washigher (in the range of 1-2 ng).Gas Chromatography-Mass Spectrometry-Selected Ion Mon-

itoring. All data were obtainedusing a Hewlett Packard 5972Agas chromatograph and a 5970A computerized mass selectivedetector, fitted with a direct capillary interface for on-columninjections, and connected to a cross-linked 5% phenylmethylsilicone capillary column (15 mx 0.25 mm DB-5-lSN, J&WScientific). The GC-MS operating conditions were: head pressure3 p.s.i., electron multiplier at 2000 V, initial temperature60°C,final temperature250°C, He carrier gas at 1.1 ml min-' programrate25°C/min, and interface temperature280°C.

Calibration Procedure. Calibration plots were derived fromstandard solutions in cyclohexane containing a fixed amount of['3C]-(C6)-IAA (50 pmol L-') together with known amounts of['2C]IAA (0, 10, 25, and 50 pmol l-') (3). The solutions weremixed in molar ratios of['2C]AA:['3C]IAA varying from 0.05to1.0. The mixtures were methylated by diazomethane andinjected (1,l) directly onto the column. The SIM program wasaccomplished by monitoring m/z 189(M+), 130 (base peak), and

136 WODZICKI ET AL.

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ON THE NATURE OF THE AUXIN-WAVE IN PINE CAMBIUM

Table I. Retention Times (Rt) on a Reversed Phase C18 Analytical pt-Bondapak HPLC Column, Determinedby Fluorescence, and/or Radioassay ofIAA, pH]JIAA, fHJ-(d5J-IAA, f3CJ-(CJ-IAA, Several IAA Conjugates,

andABAFive HPLC programs are shown. The flow rate was 2 ml/min.

Rt,.HPLCProgram 3I21CA 1AA- IAA- 1AA- 1AA- 1A

No. IAA [3H]IAA (2H]IAA ['3C]IAA .nt. IAydne-AA methyl ABA0inostol glucose aspartate glycine estermin after start

la 11.47 11.45 11.07 11.45 4.40 5.92 5.85 5.90 24.092b Same as above 26.803c 14.72 13.80 6.24 7.60 37.004d 29.90 28.79 29.905' 42.20 40.81 11.5-18.1 22.10

aIsocratic 28% aqueous methanol:0.8% acetic acid-(0-16 min), gradient 28% aqueous methanol:0.8%acetic acid > absolute methanol (16-22 min), isocratic absolute methanol (22-34 min, or longer). b Isocratic28% aqueous methanol:0.8% acetic acid. c Isocratic 24.4% aqueous methanol:0.86% acetic acid.d Isocratic 14.5% aqueous methanol:0.95% acetic acid. ' Isocratic 10% aqueous methanol: 1.0% acetic acid.

103 for ['2C]IAA methyl ester, and m/z 195 (M+), 136 (basepeak), and 109 for ['3C]-4C6)-IAA methyl ester. A linear relation-ship was shown between the molar ratio and the peak area ratioderived from the three characteristic ions (Fig. 6).

Quantification of the endogenous ["2C]IAA in the extracts wasmade by reference to the calibration plots.

Hydrolysis. The HPLC fractions that contained the bioactiveauxin-like substances were suspended in 200 ,ul of 7 N NaOH inan ignition tube, frozen on liquid N2, sealed in vacuo with flame,then heated for 4 h at 100C (3). The hydrolyzed sample wasdiluted and adjusted to pH 3.0 with H2SO4, then extracted threetimes with an equal volume of ethyl acetate. The acidic, ethylacetate-soluble fraction was rechromatographed and purified byHPLC, and 1 min fractions were bioassayed with the Avenacoleoptile curvature test.

Bioassay. The Avena (cv Seger I) coleoptile curvature test hasbeen applied throughout this study following a somewhat mod-ified technique of Went's bioassay as recommended by Funke(6). The modifications concerned the time of separating thecoleoptile from the seed, the details of seeding, coleoptile decap-itation, and the curvature measurement. The procedure wasexactly the same as in all previous works on the natural auxin ofpine (15, 17-19). The method is highly reproducible and is verysensitive (8-10 pg of IAA per coleoptile can be assayed). Theextracted substances were taken to a dry residue in the bottomof a 10 ml conical Pyrex centrifuge tube, then dissolved in 100Al of 1% hot agar to prepare the 6 x 8 mm agar plates fromwhich twelve 8 mm3 agar cubes were cut for placing on thedecapitated coleoptiles. Either 10 coleoptiles or 2 replicates of 8coleoptiles were used in testing each fraction.

Standard Compounds. The IAA used throughout the study wasSigma lot 70F-0045. 3-[5n3H]IAA (16.7 Ci/mmol) code TRKbatch 3, and [3H]ABA (24 Ci/mmol) were from Amersham Co.The [2H]-(d5)-IAA was custom synthesized by Merck/Frosst,Canada. Indole-3-acetyl-L-aspartate [2-acetyl-'4C] (42.2 mCi/mol) (2) and indole-3-acetyl-glycine [2-acetyl-'4C] (42.2 mCi/mol) (2) as well as ['3C1-(C6)-IAA (3) and unlabeled IAA glucoside(prepared by D Keglevic) were used as noted. IAInos was a giftfrom R. S. Bandurski. The 5-[3H]IAInos (27 Ci/mmol), preparedenzymically from [3H]-L-tryptophan (8), was a gift from WPengelly.

RESULTS

Preliminary Fluorimetric Screening of Substances Extractedfrom Pinus Agar Diffusates in a Range Allowing Detection ofIAA. HPLC separation using program No. 1 (Table I) for the

methanol extract of agar strips containing diffusates from thecambial region ofthe stem ofPinus revealed a variety of fluores-cent substances when monitored at excitation X 280 nm with aX 370 nm filter (Fig. 2). An extract equivalent to five strips ofagar (2 x 1.5 x 22 mm each) produced a very large fluorescentpeak which cochromatographed with [3H]IAA. The height of thepeak corresponded to about 8 ng of IAA except it was consider-ably wider than 8 ng of authentic IAA. The peak amount (8 ng)was thus comparable to the quantity of natural auxin measuredby Went's bioassay (e.g. 9.1 ng). Some ofthe fluorescence presentwas produced by substances extracted from pure agar (Fig. 3),but most originated from the diffusates of the cambial region ofPinus. It thus may be feasible to use in-line fluorescence duringHPLC to quantitate the IAA-like substance from cambial diffus-ates if contaminating substances did not vary from those presentin the extract shown in Figure 2.Growth Stimulators in the Pinus Agar Diffusates-Avena

Coleoptile Curvature Test. Agar strips used for 10 min to collectdiffusable substances from the cambial region at the basal end ofthe Pinus stem sections were extracted, subjected to HPLC, andbioassayed (Table II). The bioactivity could be separated intotwo regions by HPLC program No. 1 (Table I). The first HPLCregion included fractions collected between 2 and 5 min. Thesecond major HPLC region included fractions collected between11 and 15 min which corresponded to the [3H]IAA internalstandard (Fig. 4A). Slight stimulations of bioactivity were oftennoticed in fractions eluting at .9 to 10 min and 27 to 33 min.However, these could not be consistently confirmed by consec-utive HPLC runs or by rechromatography.The major peak fractions (Rt 1 1-15 min) were combined and

rechromatographed on isocratic HPLC program No. 4 (Table I),but no other regions of stimulation were revealed and the peakappeared again at the Rt of the [3H]IAA internal standard (Fig.4B). Fractions of the lesser peak (Rt = 2-5 min) were combined,hydrolyzed, and the acidic, ethylacetate-soluble fraction of thehydrolysate chromatographed on HPLC program No. 1. Theonly bioactive substance detected in the hydrolysate eluted at thesame Rt as [3HJIAA (Fig. 4C).

Earlier work (17-19) had detected the auxin wave in crudeagar diffusates. Theoretically, 'valleys' in the wave phenomenoncould be due to increased amounts ofbioactive 'inhibitor,' ratherthan reduced levels of auxin. To investigate the possibility ofbioactive inhibitors in the extract, the consecutive fractions ofthe HPLC program No. 1 were extended to 60 min in its isocratic100% methanol stage. These fractions of an extract equivalentto five agar strips were tested on the Went Avena bioassay inagar to which authentic IAA was added at a concentration of 0.5

137




0 10 20 30

H PLC FRACTIONS ( Rt minutesi

FIG. 2. Spectrophotofluorimetric investigation of HPLC fractions of

an 80%-methanolic extract of agar strips containing diffusate from the

cambial region of P. silvestris stems. The equivalent of 5 agar strips

(diffused for 10 min) were tested. HPLC fractions collected at minintervals using program No. 1 (Table I). An internal standard [3H]IAA

was added to the extract before chromatography (dotted peak). Results

of the Avena coleoptile curvature test of the fractions at the Rt of

[3H]IAA are also shown (dashed peak). The time lapse between fluori-

metric records and collection of the corresponding fraction is approxi-

mately 0.5 to 0.7 min.

,uM. No interaction of inhibitors, and no other stimulators except

for the substances at the Rt of [3H]IAA were detected (Fig. 4D).

The first peak (Rt = 2-5 min) of stimulation was no longer

detectable in the presence of 0.5 MM IAA, while the interaction

of IAA with substances at Rt = 11-15 mmn (where [3H]IAAelutes) may be synergistic, but cannot be proved so since extrap-

olation of the bioassay standard curve would be necessary to

draw such a conclusion. An apparent synergism, as well as

inhibition, is apparent in Figure lOA, for bioassay before purifi-

cation, relative to bioassay after purification.

Identification by GC-MS of the bioactive Auxin Substance

Present in the Pinus Agar Diffusates. The HPLC fraction con-

taining auxin-like growth activity (e.g. from a 80% methanol

extract of 40 agar strips previously applied for 10 min to P.

silvestris stem sections at their basal ends) was methylated with

CH2N2, then analyzed by GC-MS and GC-MS-SIM.

Figure 5 shows the mass spectrum of the active component,

together with mass spectra of methyl esters of authentic

['2C]IAA and ['3CJ-(C6)-IAA. The bioactive component from

0

11 38

STOP

10HPLC RETENTION TIME (min

22

FIG. 3. Spectrophotofluorimetric spectrum of the HPLC fractionsfrom an 80%-methanolic extract obtained from a single (about 100 Al)pure Difco agar strip, to which 4 ng of authentic IAA was added. HPLCprogram No. 1 (Table I). The peak at Rt 11.38 is due solely to the 4 ngof authentic IAA. The other fluorescent peaks (2.51 min and 4.92 min)were extracted from the pure agar.

Table II. Results ofAvena Coleoptile Curvature Bioassay ofthe 80%Methanolic Extract ofAgar Strips Containing Diffusatesfrom the

Cambial Region ofP. silvestris StemsAn extract equivalent to one agar strip was tested. The means are from

two replicates of eight coleoptiles each.

IAA as ng/Strip ofAgar, or ng

Substance Tested Curvature Present in a 100Ml Cube of Agar(authentic IAA)

(degrees)±SE

Pinus extract before HPLC 20.1 ± 0.4 2.4HPLC fraction 11-15 min

([3H]IAA Rt, the samefractions were used forGC-MS 19.0± 0.2 2.1

IAA controls-AM0.01 8.0 ± 0.6 0.1750.05 11.1 ± 0.2 0.8750.1 18.1 ± 0.3 1.750.5 37.0 ± 0.6 8.751.0 50.3 ±0.6 17.55.0 62.7 ± 1.9 87.5

l-,:

138 WODZICKI ET AL.

w

z

w

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w

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100 -

sc>

Pinus was identical in mass spectrum, and also in HPLC andcapillary GLC Rt with authentic IAA.For the purpose of quantifying the endogenous Pinus auxin

we used ['3C]-(C6)-IAA as an internal standard. For SIM threecharacteristic ions: the molecular ion, the quinolinium ion, andthe styrenyl ion were used (13). As shown in Figure 5, ['3C]-(C6)-IAA has no significant '2C isotopic contributions and no correc-tion was therefore necessary in the mixture of ['2C]IAA and['3C]-(C6)-IAA. Furthermore, a linear relationship was observedbetween the area ratio and the molar ratio (Fig. 6).

['3C]-(C6)-IAA (90.5 ng [0.5 nmol]) was added to the bioactive/radioactive fractions after HPLC. The fractions were then meth-ylated and subjected to GC-SIM. Figure 7 shows a typical SIMchromatogram. The measured molar ratio derived from the arearatio at m/z 189/195, 130/136, and 103/109, was 0.495, 0.504,and 0.567, respectively. From this we calculated a mean valueof 2.3 ng IAA/strip.

Results from the A vena coleoptile curvature bioassay of a one-third portion of the same HPLC fraction (IAA Rt) from themethanol extract of agar diffusates gave an amount equivalentto 2.1 ng IAA per agar strip (Table II). The final one-thirdportion of the IAA Rt HPLC fraction, (equivalent to 20 strips ofagar in total) was tested simultaneously for presence of gibberel-lin-like substances on the microdrop dwarf rice cv Tan-ginbozuassay (9), with negative results. However, significant gibberellin-like activity of a broad range of polarities was present in otherHPLC fractions (B Stiebeling, T Wodzicki, R Pharis, unpub-

z

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FIG. 4. Avena coleoptile curvature tests of HPLC frain legend of Fig. 1). For A, B, and C dotted bars denotof the total radioactivity recovered from the intern[3H]IAA cochromatographed with the samples, and opbioassay results. A, C, D, fractions obtained with HPLCand B-fractions obtained with HPLC program No. 4 (Talof direct HPLC of the crude extract; B, a second HPLCHPLC fractions at Rt = 1 I to 15 min from A; C, a seco

acidic, ethyl acetate-soluble fraction of the 7 N NaOl-HPLC fractions at Rt = 2 to 5 min from A; D, HPLCcrude extract (HPLC program No. 1 extended to 60 miuMeOH section) tested with (open bars) or without (daddition of 0.5 gm of IAA; x, y, extracts of only the rtotal and the combined fractions at Rt = 11 to 15 n

program No. 1.

FIG. 5. Mass spectra of the methyl esters of: A, an auxin-like sub-stance(s) from Pinus agar diffusates which promotes the curvature ofAvena coleoptiles (e.g. from HPLC fraction at Rt = 11-15 min; program1, Table I). The agar strips were applied for 10 min to the cambial regionof the stem of P. silvestris; B, authentic ['2C]IAA; C, authentic ['3C]-(C6)-IAA which was used as an internal standard.

16-02428-32 lished data), thus indicating that gibberellin-like substances arepresent in the diffusates.

D Interaction of the Pinus IAA, with its Putative IAA Conju-gate(s) and with a Less Polar HPLC Fraction in the Avena

,trcct only' Coleoptile Curvature Assay. The possibility of an interaction inthe bioassay of the various HPLC fractions (program No. 1) ofthe extract from Pinus agar diffusates was tested at two concen-trations (e.g. equivalent to 1 and 5 agar strips). Stimulation ofAvena coleoptile curvature produced by the crude agar extractand that produced only by the fraction eluting at Rt = to 15

min ([3H]IAA Rt) was similar at both concentrations (Fig. 8).The stimulations corresponded to 0.1 and 0.5 lM of authentic

)N (m,n) AA (iMt IAA in extracts equivalent to I and 5 strips of agar, respectively(or about 2.1 ng per 1 agar strip as corrected for 15% workup

Lctions (as noted losses monitored with the internal standard of [3H]IAA). The:ethe percentage fraction at Rt = 2 to 5 min (putative IAA conjugates) did producenal standard of a bioactive stimulation of coleoptile curvature when bioassayed)en bars refer to separately. However, the Rt = 2 to 5 min substance was less thanprogram No. 1, additive to the stimulation produced by the Pinus IAA (Rt =

ble I). A, Results 1 1-15 min) when they were assayed together.of the combined The fraction at Rt = 29 to 34 min would be expected tond HPLC of the contain less polar substances (including some pine resins). TheI hydrolysate of Rt = 29 to 34 min fractions did not affect the coleoptile curvature'fractions of the at the concentrations tested, and only very slightly reducednin the absolute stimulation produced by the Pinus IAA (Rt = 11-15 min)lashed bars) the fraction.pure Difco agar, Investigations on the Wave-Like Pattern of Auxin-Like Sub-nin after HPLC stances Collected in Agar from a Series of Stem Sections of P.

silvestris. A series of 12 agar strips corresponding to an axial

139

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WODZICKI ET AL. Plant Physiol. Vol. 84, 1987

<: 0.5 /

0.010.0 02 0.L 0,6 08 10

MOLAR RATIO I12C- IAA/ '3C- IAA 50pmole)

FIG. 6. Calibration plot of IAA against the peak area ratios derivedfrom M+, quinolinium and styryl ions, using 50 pmol ['3C]4Q)-IAA as

an internal standard.

103

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IIpi, I I. .r r o1 ,..

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7 8 9 10RETENTION TIME (min)

FIG. 7. Selected ion chromatograms from the methylated extracts ofauxin from the cambial region of P. silvestris (eluted from agar) using50 pmol ['3C]4C6)-IAA as an internal standard.

series of 12 consecutive stem sections from two P. silvestris treeswere collected on May 20, 1985. Each agar strip was extractedseparately. The extracts were filtered, divided into two equalvolumes and evaporated in vacuo. Half of each agar extract was

subsequently chromatographed with HPLC program No. 1 (Ta-ble I). Fractions at Rt = 2 to 5 min corresponding to putativeIAA conjugates and fractions at Rt = 11 to 15 min (co-chromat-ographing with [3H]IAA) were bioassayed. As well, the crudeextracts of the agar strips (the other half) were also bioassayed.The results (Fig. 9) confirmed on two separate trees a closeagreement between the amounts of stimulation (Avena curva-ture) produced by crude extracts of the agar strips and thatproduced by HPLC fractions at Rt = 11 to 15 min ([3H]IAARt). Stimulation by the putative IAA conjugates (fraction at Rt= 2-5 min) was low and did not reveal any correlation with thewavelike pattern of stimulation by the crude extract. Thus, thebioactive auxin wave could be detected not only in crude agardiffusates but also in the HPLC fractions that contain IAA. TheseHPLC fractions (IAA Rt) should not contain known IAA con-jugates, or ABA, and do not appear to contain significantamounts of inhibitors ofAvena curvature.

Investigations on the Wavelike Pattern of Auxin-Like Sub-stances Collected in Agar from a Consecutive Series of StemSections of P. contorta. Two parallel stem segments (44 x 72 x4 mm) were cut from a single tree of P. contorta on May 31,1985. They were subsequently cut perpendicular to the grain ina succession of twelve 6-mm high and 44 mm wide sections.Two agar strips 22 mm long were attached for 10 min to thebasal end of each stem section. These strips were divided intotwo equal (11 mm long) parts immediately after collection. Onepart of each strip was then taken into a glass vial (two per vial)for extraction while the remaining two parts were bioassayedimmediately with the Avena coleoptile curvature test (Fig. 1).

Methanolic extracts ofthe remaining parts ofthe agar strips werechromatographed with HPLC program No. 1 (Table I) and theRT = to 15 min HPLC fractions (corresponding to the[3H]IAA internal standards) were collected for either further

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13% during HPLC); c, C, fractions at Rt = 2 to 5 min (where IAA-inositol will elute); d, D, fractions of Rt = 29 to 34 min (nonpolarregion); e, E, combined fractions at Rt = 11 to 15 (IAA Rt) and 2 to 5min; f, F, combined fractions at Rt = I I to 15 and 29 to 34 min; g, G,authentic IAA at 0.1 and 0.5 ,M, respectively.

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SUCCESSIVE STEM SECTIONS FROMTHE APICAL END

FIG. 9. The wavelike pattern ofauxin (as measured by Avena coleop-tile curvature assay) collected in agar over 10 min (e.g. the basipetalefflux from an axial series of 6-mm high sections of the stem cambialregion ofP. silvestris). From crude methanolic extracts (solid lines); fromHPLC fractions corresponding to the Rt of ['H]IAA (broken lines).Dotted lines denote bioassay activity of the polar HPLC fractions where[3H]IAA inositol will elute. This activity has been tentatively determinedto be an IAA conjugate based on hydrolysis and rechromatography on

C18 HPLC followed by Avena curvature assay. A and B refer to twodifferent trees sampled on May 20, 1985. The data for the LAA fraction(Rt = 11-15 min) in (B) were corrected for losses as determined with an

internal standard of [3H]IAA. The standard errors of bioassay determi-nations of auxin efflux from successive stem sections 1 to 12 are shownin Table III. The x mark in (B) fraction of the putative IAA conjugateat section No. 6 means that it was rejected due to an anomalously highvalue on the Avena bioassay.

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bioassay or GC-MS-SIM. For this latter determination knownamounts of ['3C]4C6)-IAA were added as an internal standardafter HPLC, but prior to methylation.The results (Fig. 10) show a close similarity of the wave-like

patterns between: (a) two neighboring stem segments as detectedby bioassay, (b) bioassays of fresh agar strips and bioassays of theHPLC fractions (['H]IAA Rt) from the methanolic extracts ofthe agar strips, and (c) bioassays of the crude agar strips andquantities of ['2C]IAA detected by GC-SIM. Interestingly, thepeaks of the wave, when quantified by GC-SIM, gave higheramounts of IAA than measured by bioassay in the fresh agarstrips.Stem segments of two other trees of P. contorta were analo-

gously investigated but only with serial bioassays of fresh agarstrips. Both revealed wavelike patterns of stimulation (data notshown).

DISCUSSION

Fractionation by HPLC of the 80% methanolic extract fromagar strips that had previously been applied for 10 min to thebasal end of cambial region stem sections of P. silvestris allowedseparation oftwo groups ofauxinlike substance(s) that stimulatedcurvature of Avena coleoptiles. The main auxin co-chromato-graphed in various HPLC solvents with authentic [3H]IAA and['H]IAA (as monitored by fluorescence emission [X 370 nm],after excitation at A 280 nm, or by bioassay). The full massspectrum (Fig. 5) of the bioactive component indicated that it

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FIG. 10. The wave-like pattern of auxin (as measured by Avena co-

leoptile curvature assay) collected in agar over 10 min (e.g. the basipetal

efflux from an axial series of 6-mm high sections of the stem cambial

region of P. contorta). By bioassay from the fresh agar, measured directly

(broken lines A and B) or from HPLC fractions corresponding to the Rtof[3H]IAA (solid line, A); The standard errors ofbioassay determinationsof auxin efflux from successive stem sections 1 to 12 are shown in TableIII. By GC-MS-SIM using a ['3C]4C6)-IAA intemal standard added to

HPLC fractions corresponding to the Rt of [3H]IAA (solid line, B). The

two segments of sectioned tissue (A) and (B) were situated side-by-sideon the stem of the same tree.

was IAA. The amounts measured in extracts equivalent to 1 stripof agar (corresponding linearly to a 22 mm sector of the stemcambial region cylinder) were 2.3 and 2.1 ng as measured byGC-MS-SIM and by the Avena coleoptile curvature bioassay,respectively. These results thus confirm previous findings whichidentify the major auxin of P. silvestris as IAA.The second, more polar auxin yielded, after hydrolysis, a

bioactive substance co-chromatographing with IAA. It is tenta-tively identified as an IAA conjugate. It is more polar than IAA-glucoside, IAA-glycine, or IAA-aspartate, and its HPLC Rt co-

incides with IAlnos. This more polar putative IAA conjugateappears to differ from IAA conjugates found in the extracts fromP. silvestris needles (11, 12), and appears to be only one of theputative IAA conjugates that can be extracted from scrapings ofthe cambial region of P. silvestris (G Stiebeling, R Pharis, AWodzicki, CHA Little, unpublished data).No significant inhibitors of the Avena coleoptile curvature

assay were found in crude extracts of the agar strips containingPinus diffusates (Fig. 4D), although their presence is impliedfrom Figure 10, A and B. Spectrofluorimetric investigation ofthe methanolic extract from the agar strips previously in contactwith the stem cambial region, performed at the same conditions

which allowed tracing of IAA, revealed many highly fluorescentsubstances, a few being originally present in the pure Difco agar.Synergistic substances to IAA action are implied from Figures9A and 10, A and B (in certain stem segments), but examinationfor these (Fig. 4D) gave no, or equivocal evidence of theirpresence.IAA and the putative IAA conjugate from Pinus diffusates do

not interact synergistically, and are less than additive when testedtogether. This apparent inhibition of bioactivity of the putativeIAA conjugate when tested in the presence of authentic IAA isan interesting interaction, and deserves a separate investigation.An analogous elimination of theIAInos stimulation of the Avenacoleoptile curvature response by simultaneously added IAA hasbeen found (TJ Wodzicki, RP Pharis, AB Wodzicki, unpublisheddata).The definitive identification of IAA by GC-MS, and the ab-

sence of highly significant synergistic, or inhibitory interactions(results from present study) allow us to conclude that the auxinwave pattern as detected by bioassay directly from agar strips ofPinus diffusate is a valid reflection of the concentration of nativeIAA diffusing from the cambial region into agar over the 10 minperiod.The wavelike patterns of diffused IAA were found for both P.

silvestris and P. contorta. The auxin-wave phenomenon wasconfirmed not only by bioassay of the crude agar strips, but alsoby bioassay of HPLC fractions corresponding to the Rt of[3H]IAA, and by using an internal standard of ['3C]-(C6)-IAA.The auxin producing the wave-like pattern is thus natural IAA.

In most of our purified fractions the amounts of IAA deter-mined by GC-MS-SIM agreed reasonably well with our resultsof bioassays of the crude extracts. However, at two maxima wavepeaks (P. contorta) the amounts of IAA measured by GC-MS-SIM were greater than by bioassay, in one case as much as 20times greater (Fig. lOB). There are several possible explanationsfor this difference: (a) that there was some unknown growthinhibitor (but not ABA) which reduced the expression of IAA inthe Avena coleoptile curvature when this particular fraction wasbioassayed; (b) that the concentration of IAA was supraoptimaland therefore, the bioassay underestimated the amount present;(c) that the curve of stimulation for the pure IAA bioassaystandards, and of the P. contorta IAA tested in the presence ofother extracted (and somewhat inhibitory) compounds differ atvery high concentrations, thereby causing an underestimation ofendogenous P. contorta IAA when calculated from the standardcurve (which shows a proportional response of curvature to thelog of IAA dosage). Nevertheless, the present results (Fig. lOB)confirm that stimulation-waves of diffusable auxin from the stemcambial region of Pinus (e.g. auxin-waves) are indeed primarilydue to a wavelike basipetal effiux of natural IAA.

Acknowledgments-The authors gratefully acknowledge gifts of authentic com-pounds from R. S. Bandurski and W. Pengelly, and thank Miss Yan-Yan-Huangfor her very skillful technical assistance.

LITERATURE CITED

1. ALDEN T, L ELIASSON 1970 Occurrence of indole-3-acetic acid in buds of Pinussilvestris. Physiol Plant 23: 145-153

2. COHEN JD 1981 Synthesis of '4C-labeled indole-3-acetylaspartate. J LabelledCompd Radiopharm 18: 1393-1396

3. COHEN JD, BG BALDI, JP SLOVIN 1986 '3C6-[Benzene ring]-indole-3-aceticacid: a new internal standard for quantitative mass spectral analysis of indole-3-acetic acid in plants. Plant Physiol 80: 14-19

4. FRANSSON P 1953 Studies on auxin in young stem of Pinus silvestris. PhysiolPlant 6: 544-550

5. FRANSSON P 1959 Studies on a shoot and root cell elongation stimulator inPinus silvestris. Physiol Plant 12: 188-198

6. FUNKE H 1939 Uber den Nachweis kleines Wuchsstoffmengen. Jahrb WissBot 88: 375-388

7. KNEGT E, E VERMEER, J BRUINSMA 1981 The combined determination ofindolyl-3-acetic acid and abscissic acid in plant materials. Anal Biochem114: 362-366

8. MICHALCZUK L. JR CHISNELL 1982 Enzymatic synthesis of 5-3H-indole-3-

142 WODZICKI ET AL.




acetic and 5-3H-L-tryptophan. J Labelled Compd Radiopharm 19:121-1289. MURAKAMI Y 1968 A new rice seedling bioassay for gibberellins, "microdrop

method," and its use for testing extracts of rice and morning glory. Bot MagTokyo 81: 33-43

10. SANDBERG G, B ANDERSON, A DUNBERG 1981 Identification of 3-indole-aceticacid in Pinus silvestris L. by gas chromatography-mass spectrometry, andquantitative analysis by ion-pair reversed-phase liquid chromatography withspectrofluorimetric detection. J Chromatogr 205: 125-137

11. SANDBERG G 1984 Biosynthesis and metabolism of indole-3-ethanol andindole-3-acetic acid by Pinus silvestris L. needles. Planta 161: 398-403

12. SUNDBERG B, G SANDBERG, E JENSEN 1985 Identification and quantificationof indole-3-methanol in etiolated seedlings of Scots pine (Pinus silvestris L.)Plant Physiol 77: 952-955

13. WILLIAMS CM, AH PORTER, M GREEN 1969 Mass spectrometry of biologicallyimportant aromatic acids. University of Florida, Medical School and Veter-ans Administration Publication, Gainesville

14. WODZICKI TJ 1968 On the question of occurrence of indole-3-acetic acid inPinus silvestris. Am J Bot 55: 564-571

15. WODZICKI TJ 1978 Seasonal variation of auxin in stem cambial region ofPinus silvestris L. Acta Soc Bot Pol 47: 225-231

16. WODZICKI TJ, AB WODZICKI 1973 Auxin stimulation of cambial activity in

Pinus silvestris L. II. Dependence upon basipetal transport. Physiol Plant29: 288-292

17. WODZICKI TJ, AB WODZICKI 1981 Modulation of the oscillatory systeminvolved in polar transport of auxin by other phytohormones. Physiol Plant53: 176-180

18. WODZICKI TJ, AB WODZICKI, S ZAJACZKOWSKI 1979 Hormonal modulationof the oscillatory system involved in polar transport of auxin. Physiol Plant46: 97-100

19. WODZICKI TJ, E KNEGT, AB WODZICKI, J BRUINSMA 1984 Is indolyl-3-aceticacid involved in the wave-like pattern of auxin efflux from Pinus silvestrisstem segments? Physiol Plant 44: 122-126

20. ZAJACZKOWSKI S, TJ WODZICKI 1978 On the question of stem polarity withrespect to auxin transport. Physiol Plant 44: i 22-126

21. ZAJACZKOWSKI S, TJ WODZICKI 1978 Auxin and plant morphogenesis-amodel of regulation. Acta Soc Bot Pol 47: 233-243

22. ZAJACZKOWSKI S, TJ WODZICKI, J BRUINSMA 1983 A possible mechanism forwhole-plant morphogenesis. Physiol Plant 57: 306-310

23. ZAJACZKOWSKI S, TJ WODZICKI, JA ROMBERGER 1984 Auxin waves and plantmorphogenesis. In TK Scott, ed, Encyclopedia of Plant Physiology (NewSeries). Hormonal Regulation of Development. II, Vol 10. Springer-Verlag,Berlin, pp 244-262

143



Plant Physiol. (1987) 84, 5600032-0889/87/84/0560/01/$0 1.00/0

CorrectionsVol. 82: 518-522, 1986

Eleni Selinioti, Yiannis Manetas, and Nikos A. Gavalas. Coop-erative effects of light and temperature on the activity ofphosphoenolpyruvate carboxylase from Amaranthus panicu-latus L.

Page 519, in the legends of Tables I and II, the correct units foractivities are ,umol CO2- min' g-' fresh wt.

Vol. 83: 262-264, 1987

Hilary V. Martin and Paul-Emile Pilet. Effect of pH on IAAUptake by Maize Root Segments.

Page 264, column 2, the last line ofthe article should read: Based

on these results, the distribution of the carrier along the root,and its apparent kinetic properties (10), the saturable carrierfor IAA uptake could play an important part, along with thecarrier for auxin efflux, in the regulation of IAA movementand levels in the enlongation zone of the maize root.

Vol. 84: 135-143, 1987

Tomasz J. Wodzicki, Hiroshi Abe, Alina B. Wodzicki, RichardP. Pharis, and Jerry D. Cohen. Investigations on the Natureof the Auxin-Wave in the Cambial Region of Pine Stems.Validation of IAA as the Auxin Component by the AvenaColeoptile Curvature Assay and by Gas Chromatography-MassSpectrometry-Selected Ion Monitoring.

Page 140, in Figure 6, the equation for ions m/z 103/109 shouldread y = 1.223x + 0.046.

560

investigations on nature of auxin-wave cambial region ... · corrections vol.82: 518-522, 1986...

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