feruloylputrescine caffeoylputrescine are not involved ...plant physiol. vol. 92, 1990 1.5 0. 0-0...

Plant Physiol. (1990) 92, 924-9300032-0889/90/92/0924/07/$01 .00/0

Received for publication June 30, 1989and in revised form November 7, 1989

Feruloylputrescine and Caffeoylputrescine Are Not Involvedin Growth and Floral Bud Formation of Stem Explants from

Nicotiana tabacum L. var Xanthi nc.

Markus Wyss-Benz, Luc Streit, and Edith Ebert*Agro Division, Ciba-Geigy Ltd., Postfach, CH-4002 Basel, Switzerland

ABSTRACT

The role of feruloylputrescine (FP) and of caffeoylputrescine(CP) was investigated in an explant system of stem explants fromday-neutral Nicotiana tabacum L. var Xanthi nc. Previously, acorrelation between cortical callus formation and increase in FPcontent, as well as between in vitro flower formation and increasein CP content had been shown. During the explant growth in vitro,the increase of both FP and CP was inhibited by 4-fluor-(1-amino-2-phenylethyl)phosphonic acid and 2-amino-indene-2-phos-phonic acid, both inhibitors of phenylalanine ammonia-lyase (EC4.3.1.5). DL-a-difluoromethylarginine, an inhibitor of arginine de-carboxylase (ADC, EC 4.1.1.19), prevented only the increase inFP, while DL-a-difluoromethylornithine, an inhibitor of ornithinedecarboxylase (EC 4.1.1.17), reduced only that of CP. Increasein dry weight and the formation of cortical callus and of floralbuds of explants were not affected by any of the inhibitors. Weconclude, in contrast to earlier hypotheses, that FP and CP donot trigger growth and differentiation in the explants. It seemsmore likely that FP and CP increase in response to auxin andcytokinin in the media.

HCA' are widely distributed in the plant kingdom (12) andare particularly abundant in plant cell and callus cultures (6,15, 25). Suggestions pertaining to their role in plants includevirus resistance, stress response, and reproduction (15, 25).The latter role was forwarded mainly on the grounds thatHCA accumulate in floral parts of plants and are absent fromcytoplasmic sterile anthers in Zea mays (5, 12-16, 18, 21).HCA were therefore proposed to play a role in induction anddifferentiation of flowers in plants.The majority of investigators examined the role ofHCA as

a part of the action of PA. They hydrolyzed plant extractswith HCI and derivatized the conjugated PA with dansylchlo-ride. This method has been used for thin cell layer explants(29) of tobacco to compare in vitro growth responses such asgenerative or vegetative shoot formation with patterns of freeand conjugated PA, the latter including HCA. Torrigiani et

'Abbreviations: HCA, hydroxycinnamic acid amides; PA, polya-mines; Put, putrescine; FP, feruloylputrescine; CP, caffeoylputrescine;PAL, phenylalanine ammonia-lyase; F-APEP, 4-fluor (1-amino-2-phenylethyl)phosphonic acid; AIP, 2-aminoindene-2-phosphonicacid; DFMA, DL-a-difluoromethylarginine; DFMO, DL-a-difluoro-methylornithine; DW, dry weight; Spd, spermidine; Spm, spermine;NAA, 1 -naphthaleneacetic acid.

al. (28) found an increase in both free and conjugated PA,but saw no difference in the PA pattern between vegetativelyand generatively growing explants, which originated fromdifferent sites of the plant. Tiburcio et al. (27) reporteddifferent patterns of conjugated PA in explants taken fromthe same site of a tobacco plant and subjugated to differentkinetin concentrations for triggering vegetative or generativeshoots. Kaur-Sawhney et al. (8) reported a role of spermidinein flower formation.Other investigators detected each HCA separately by HPLC

or TLC. In leaf explants of tobacco, changes in putrescineand hydroxycinnamoyl putrescine levels were correlated withcell division and bud formation (16). Martin-Tanguy (15, 16)proposed a role for Put and for hydroxycinnamoyl putrescinesas plant growth regulators or as synergists of other plantgrowth regulators.By contrast to the above mentioned reports, previous results

from our laboratory let us question the role of HCA in theprocess of flowering. FP accumulated during callus prolifera-tion while CP accumulated in the second part of cultureduring flower differentiation (31). However, both stem ex-plants from day-neutral tobacco forming floral buds (31) andstem explants from photoperiodical tobacco forming vegeta-tive shoots (32) exhibited a similar pattern of FP and CP.PA and cinnamic acid are precursors of HCA (19, 32). F-

APEP and AIP are specific inhibitors ofPAL (EC 4.3.1.5) (9;N Amrhein, ETH Zurich, personal communication). DFMA,a specific inhibitor of ADC (EC 4.1.1.19) (3), and DFMO, aspecific inhibitor ofODC (EC 4.1.1.17) (17), are widely usedto block PA biosynthesis and to investigate the role of PA (4,10, 23). Using these inhibitors should be a possibility tosuppress HCA formation due to precursor depletion. Thisshould be a means to examine whether endogenous HCAaffect cortical callus and floral bud formation on the explantsor not. A recent report (4) from experiments using leaf ex-plants indicated that the formation ofPut via ADC may affectfree PA pools while that via ODC may affect the conjugatedPut pool.

In this paper we report on growth responses and HCAcontents of tobacco stem explants exposed to inhibitors ofPAL (F-APEP, AIP), ADC (DFMA), and ODC (DFMO), inorder to examine a possible correlation between HCA accu-mulation and tissue differentiation.

MATERIALS AND METHODSPlants

Day-neutral Nicotiana tabacum L. var Xanthi nc. wasgrown with 18 h of light (206 ,umol m-2 s-', 400-700 nm)

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FERULOYL- AND CAFFEOYLPUTRESCINE IN TOBACCO EXPLANTS

and 6 h dark (250C, 65% RH). Stem explants out of theinflorescence of 70 d old tobacco plants were used for in vitroculture. Surface-sterilized internodes were cut into pieces of 1cm length, cross-sectioned, and placed on solidified nutritionmedium as described in Wyss-Benz et al. (31).

Chemicals

Putrescine dihydrochloride, spermidine trihydrochloride,spermine tetrahydrochloride, and dansylchloride were pur-chased from Fluka (Buchs, CH). Acetonitrile (HPLC grade)was purchased from J. T. Baker (Deventer, NL), acetone andtoluene from Merck (Darmstadt, FRG). DFMA, DFMO, andAIP were a gift from N. Amrhein (ETH Zurich, CH). F-APEPwas a gift from L. Maier (Ciba-Geigy Ltd., Basel, CH). Inhib-itors were dissolved in water (HPLC grade) and sterilized byfiltration through ACRODISC 0.2 ;m (Gelman Sciences, AnnArbor, MI). They were added to the in vitro culture mediumafter autoclaving.

Extraction and Detection of HCA

Explants were lyophilized and extracted with water-meth-anol (10 mg tissue/mL solvent). The stem explants had a dryweight of approximately 20 mg at the beginning of in vitroculture and about 40 mg at d 36. The extracts were repurifiedwith an acidic cation-exchanger column (31). The resultingsamples were analyzed by reverse-phase HPLC using a gra-dient of 25 mm sodium acetate pH 4.5 to acetonitrile. HCAwere detected by fluorescence (excitation 320 nm, emission> 389 nm) and absorption at 306 nm. This method waspreviously described (31) and used for the calculation of allHCA-contents.

Extraction and Dansylation of PA

The PA were extracted from lyophilized explants with 0.2N perchloric acid (10 mg tissue/mL acid). Derivatization wasdone according to Smith and Davies (24), except that 300 ,Lof plant extract, 300 IiL saturated Na2CO3, and 600 ,uLdansylchloride (5 mg. mL-' in acetone) were used.

HPLC Analysis of Dansylated PA

HPLC was performed on the same system and with thesame solvents as used previously (31) for the detection of FPand CP. The gradient was changed, starting with 50% aceto-nitrile to 100% in 12 min. Elution was completed after 25min. Derivatives ofPA and ofFP and CP with dansylchloridewere measured by fluorescence (excitation 365 nm, emission> 470 nm). The identification of PA, FP, and CP was done(a) by comparison of spectra from standard chromatogramswith spectra from chromatograms of explants using a diodearray detector and (b) by spiking plant extracts with referencecompounds. This method was used for the calculation of allPA-contents and for verification of the appearance ofFP andCP.

Amino Acid AnalysisThe amino acids were extracted with water-methanol and

derivatized with o-phthaldialdehyde according to Jones andGilligan (7).

RESULTS

PAL Inhibitors

Explants grown on medium containing F-APEP (1 AtM-1mM) or AIP (1-100 ,M) showed neither a difference in theircortical callus and floral bud formation (Fig. 1) nor in theirincrease in DW (results not shown) throughout the cultureperiod of 55 d when compared with explants cultured in theabsence of the inhibitors. Cortical callus and floral buds wereformed in all cultures, with any inhibitor concentration tried.The experiments with both F-APEP (Figs. 2 and 4) and

AIP (Figs. 3 and 4) were done with stem explants of theinflorescence of the same tobacco plant. The inhibition wasconfirmed in two additional experiments using other tobaccoplants. F-APEP (1 mM) blocked the formation of FP duringthe 7 d culture completely, while lower F-APEP concentra-tions were less effective (Fig. 2). The same pattern was seenat 14 d. Between d 14 and d 28, FP decreased in controlexplants grown on medium without inhibitors (Fig. 2) (31)and the differences between the treatments were therefore lesspronounced. The same type ofexperiment was done with AIP(Fig. 3), but with inhibitor concentrations (1-100 gM) 10times lower than those used with F-APEP. At, 7, 14, and 28d, FP content was decreased as a function of inhibitor con-centration (Fig. 3).CP was detectable in stem explants only after d 14 (3 1). At

d 28, floral bud formation could clearly by recognized. Ap-plication of F-APEP (1 uM- 1 mM, Fig. 4a) and AIP (1-100ELM, Fig. 4b), added at the onset ofexplant cultivation, resultedin a concentration dependent decrease in CP content.Even after 49 d of growth in the presence of F-APEP and

? -Control

PEP 100 I

4_AIP 1op'

I.DFMA 100pMI

FX~FMO 100p

Figure 1. Explants from N. tabacum Xanthi after 28 d of growth onmedium without (control) or with inhibitors. APEP = F-APEP.

925

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Plant Physiol. Vol. 92, 1990

1.5

0.

0-

0

Figure 2. Content of FP in explants from N. tabacum Xanthi after 7,14, and 28 d of growth on medium without (C) or with F-APEP.Explants derived from the same plant were grown and analyzed asdescribed in "Materials and Methods."

I_1,

1 0.75

0..; 0.5cl0

00.m. 0.25P.%0

0%"

o

Figure 3. Content of FP in explants from N. tabacum Xanthi after 7,14, and 28 d of growth on medium without (Ctrl) or with AIP. Explantsderived from the same plant were grown and analyzed as describedin "Materials and Methods."

AIP, CP content was still reduced in correlation to increasinginhibitor concentrations (results not shown). At that stage, FPcontent was very low in both treated and untreated explants.F-APEP (50 and 500 Mm) or, respectively, 10 Mm AIP were

added to the medium at time 0 to investigate the pattern ofFP and CP (Fig. 5). The same increase in DW and floral budformation was obtained in all cases, regardless ofthe treatment(Fig. 1). In control explants, FP showed the known pattern(31, 32), peaking at d 15 and decreasing slowly afterward.Addition of 50 Mm F-APEP or 10 Mm AIP resulted in an

approximately 50% inhibition of FP increase, while additionof 500 gM F-APEP lowered FP increase by more than 90%over the entire culture period. In controls, CP increasedbetween d 15 to 22. F-APEP (50 Mm) and 10 Mm AIP inhibited

I_1,

= 0.75C0-s:L

a; 0.5

._a

.fi 0.25C

c0

Go

a6

-

.5

0

._

0

'5

aa

Go4)

cJ,

4

2

0

b

Figure 4. Content of CP after 28 d of growth on medium withdifferent concentrations of F-APEP (a) and of AIP (b). The values arefrom the same experiments as in Figures 2 and 3. nd, Below detectionlimit of 0.1 Amol (g DW)-1.

CP increase by more than 75%, and resulted in concentrationsbelow detection limit (0.1 Mm [g DW]-') throughout culturetime. At d 7 and d 28, I50 values of FP and CP formation(Table I) were 20 times lower for AIP than for F-APEP.The ratio of Put to Spd to Spm in controls changed from d

0 (1:1.5:0.58) to d 29 (1:0.64:0.20). This was due to the factthat the Put content changed during culture (Fig. 5, Ctrl). TheSpm pool remained more or less the same over 36 d ofexplantgrowth and showed the same pattern with F-APEP and AIPin the medium. In controls, Put remained on the same levelup to d 15, increased about three times until d 29, anddecreased afterward. Put pattern for explants grown on 50 MmF-APEP and 10 Mm AIP were very similar with the exceptionthat at d 8 and d 15, Put content was higher than in therespective control. This effect was enhanced in explants grownon 500 AM F-APEP. In controls, Spd content remained almostconstant. The same patterns were seen for explants grown on50 Mm F-APEP and 10 Mm AIP. However, Spd content washigher at d 8 when explants were grown on 500 Mm F-APEP.

ADC and ODC Inhibitors

DFMA and DFMO in 1, 10, and 100 Mm concentrationswere added to the cultures at d 0. Growth rate and growthresponse between explants grown on medium with or withoutinhibitors were equal (Fig. 1). Floral buds formed even incultures with 100 gM inhibitor. However, addition of 100 MmDFMA and DFMO sometimes caused a cessation of growthduring the culture which was accompanied by a drop of PApools. Thus, 100 gM of DFMA or DFMO was the highestconcentration which did not cause growth arrest of all ex-plants. Therefore, only the highest values of putrescine ob-tained from six stem explants were plotted in Figure 7. Thesetendencies were reproducible upon repeating the experiment.

-

926 WYSS-BENZ ET AL.




Figure 5. Content of FP, CP, Put, Spd, andSpm in explants from N. tabacum Xanthi after 0to 36 d of in vitro growth. The time course ofexplant extraction is indicated in the figure be-tween FP and CP. Explants grown on mediumwithout inhibitors were used as controls (Ctrl).Inhibitors were added at the beginning of culture.Values are means of three independently meas-ured explants. Vertical lines over the bars show+SE. nd, Below detection limit of 0.1 AmoI (gDW)-1. *, ±SE was 0.55 jsmol (g DW-'.

Ctrl F-APEP ATP Ctrl F-APEP AlP Ctrl F-APEP AIP=L = = =-sfL mL

=

cm~~~~~~~~~~~~~~~~~~~f

Table I. 150 Values Calculated from Experiments Shown in Figures 2to 4 and in Figure 6

FP CInhibitors at 2dAfter 7 d After 28d after28d

JIMF-APEP ~24 a =26AlP =0.1 11DFMA 9-DFMO <1

a = Not effective.

FP and PA contents were examined at d 7 when FP was

just before its peak level in control explants, while CP was

absent. Increasing the concentration ofDFMA in the mediumlowered the FP content in explants after 7 d in a concentrationdependent manner (Fig. 6a). DFMO in the medium did notblock the formation of FP as a function of its concentration.Nevertheless, FP content was slightly lower compared withcontrols. A combination of both 10 ,uM DFMA and 10 uM

DFMO decreased the FP content to the same extent as 10 uM

DFMA alone (Fig. 6a; 2). Addition of 10 uM AIP resulted ina further reduction of the FP content, corresponding roughlyto the effect of 100 AiM DFMA (Fig. 6a; 3).At d 28, the inhibitory effect of DFMA on FP formation

was smaller than the variation ofFP content. CP content waslower in explants grown 28 d with DFMA than without (Fig.6b). No correlation with increasing inhibitor concentrationswas observed. On the other hand, explants grown on mediumwith DFMO showed decreasing CP contents with increasinginhibitor concentrations. Both inhibitors together reduced thecontent of CP to the same extent as DFMO alone (Fig. 6b;2), and supplemental addition of AIP totally blocked theformation of CP (Fig. 6b; 3).

Figure 7 shows that the Put pool was smaller after 7 and

I

= 0.75-d

0.5

0

.fi 0.25

=L0.

0

-

0

6

-4NC

._

aC.)

To41

cJ

4

2

0

b

Figure 6. Content of FP (a) after 7 d and of CP (b) after 28 d ofgrowth on different media in explants from N. tabacum Xanthi. At thebeginning of culture no inhibitors (C) or various concentrations ofthem were added to the medium. Values are means of three inde-pendently measured explants. Vertical lines over the bars show +SE.2, 10 gM DFMA and 10 AiM DFMO in the medium; 3, 10 AM DFMA,10 lM DFMO, and 10 gM AIP in the medium. nd, Below detectionlimit of 0.1 gmol (g DW)-1.

28 d of explant growth on both DFMA and DFMO whencompared with control explants. The Put pool increased incontrol explants between d 7 and d 28 (Fig. 5). No reductionof Spd and Spm contents was observed with DFMA andDFMO (results not shown).

Evidence for Inhibitor Potency

Amino acid contents were analyzed in order to assess thepotency of the inhibitors employed by a different approach.

1

0.1

3

-4

1,

=L

0

2

1

0

927




2.5

2

- 1.5W-

1.50.

0V 0.5Pe

0

a2.5

- 2

1-

1.5

210

._o.0

0go

b

Figure 7. Content of Put after 7 d (a) and after 28 d (b) of growth ondifferent media in explants from N. tabacum Xanthi. Values are chosenfrom the best growing explant treated and compared with untreatedcontrol explants. Symbols are the same as in Figure 6.

After 28 d of growth of the explants on 10 AM AIP, Phecontent was 415% of that of the control and 500% whenexplants were grown for the same time on 100 fM F-APEP.Contents of other amino acids, i.e. Arg, Asn, Gln, and Tyrwere not changed. At the same time, addition of 100 4MDFMA resulted in an Arg content of 141 % as compared withcontrols. In these cultures, free Orn content was below detec-tion limit and concentrations of Asn, Gln, Phe, and Tyr werethe same. After 28 d of growth on 100 uM DFMO, Orncontent was again below detection limit while Asn, Gln, Phe,and Tyr contents were as in control explants.

HCA Detection with Dansylation

The method of dansylation was used for the calculation ofthe PA contents (Figs. 5 and 7). Dansylation was also an

effective method for the detection of FP and CP in explants(Fig. 8). Therefore, the dansylation was used to verify theaction of the inhibitors, but not for the calculation of FP andCP.

DISCUSSION

It has been postulated that HCA are causally involved infloral development and act as a new class of plant growthregulators (15). According to this postulate, inhibition ofHCAformation should inhibit floral bud formation.Two PAL-inhibitors (F-APEP and AIP) blocked the in-

crease of FP and CP contents as a function of their concen-tration (Figs. 2-5). However, the absence of an increase in FPand in CP content caused neither a change in the growth ratenor in the formation of floral buds on the explants, even withinhibitor concentrations (1 mm F-APEP, 100 tLM AIP) atwhich FP and CP contents remained at the initial level.Therefore, inhibition of PAL seems to be an useful approachfor investigating the functions of HCA in plants. Previousinvestigations (31) have shown a possible correlation between

C-)

Xb o o.O L U

ciu

10.00

retention time, min

Figure 8. HPLC-chromatograms (0-20 min) of dansylated PA andHCA. a, Standard chromatogram with 30 pmol Put, Spd and 5pm,each; b, standard chromatogram with 0.2 nmol FP and 1 nmol CP;c, chromatogram of an extract from an in vitro growth explant of N.tabacum Xanthi.

an increase in FP and cortical callus formation, and betweenan increase in CP and floral bud formation in the explants.Later experiments with photoperiodic Nicotiana questionedthese correlations (32). A function ofEP and CP in the processof cortical callus and of floral bud formation can be ruledout, since neither cortical callus nor floral bud formation wereinfluenced by PAL-inhibitors, albeit the content of FP and ofCP remained at the initial level.

These conclusions were corroborated by experiments withDFMA and DFMO as inhibitors of the biosynthesis of Put(Fig. 7), which was shown to act as a precursor for FP andCP (19, 32). DFMA inhibited FP formation, while DFMOblocked that of CP (Fig. 6). Growth rate and floral budformation were not affected. Surprisingly, FP accumulationappeared to depend on the formation of Put through ADC,while CP accumulation exploited Put formed through ODC(Fig. 6; Table I). Malmberg et al. (11) have demonstrated thatin stems of flowering tobacco, ADC activity was about 10times higher than ODC activity while in callus cultures andin flowers, ODC activity was approximately 3 times higher.Smith et al. (25) discussed a correlation of ADC with stressresponse in plants. ODC was found to be more closely relatedto growth and cell cycle. Slocum et al. (23) indicated thathydrolysis ofDFMA to DFMO occurs in tissues ofN. tabacumcv Wisconsin 38 with high arginase activity. In our explantsthis effect could be excluded, since at d 28 Arg content washigher in explants grown on DFMA than in those grown oncontrol medium, indicating an inhibition ofADC rather thanODC by DFMA. We have found (32) that in stem explantsfrom N. tabacum Xanthi, the FP increase occurred mainly inthe cortex of the stem during the first 10 d of in vivo culture.CP increased, starting after d 14, sequentially from pith tocortical callus to floral buds. High activities ofADC and ODCin different tissues could explain the differences in inhibitionof FP and CP formation by DFMA and DFMO, respectively.FP appears to be formed via Put from Arg in cortex. Thepattern of FP contents suggests a response of the tissue to thestress of stem cutting. CP is mainly produced from Put as the





product of Orn decarboxylation during the second part ofculture, starting in pith tissue and responding to bud forma-tion and growth. The increase in CP content was not due tostress caused by a deficiency in the medium, since explantson medium changed weekly showed the same pattern of FPand CP as explants left for 3 weeks on the same medium(results not shown). All these experiments taken together allowthe conclusion that cortical callus formation, growth, floralbud initiation and differentiation in this stem explant systemwere not triggered by either FP or by CP. This conclusion isalso supported by the observation that FP and CP could bedetected in explants which formed roots or callus from pithtissue (results not shown) or in explants of photoperiodictobacco, which formed vegetative shoots only (32).High concentrations of PAL-inhibitors increased the Put

pool and, to a lesser extent, the Spd pool (Fig. 5). Thisobservation led to the conclusion that conjugation of Put withhydroxycinnamic acids may act as a buffer for free Put. Asexpected, DFMA and DFMO lowered the Put pool (Fig. 7).Explants showed no change in cortical callus and floral budformation when grown on the highest concentrations ofDFMA and DFMO (100 Mm), which reduced the Put pool toabout 25% of the content of controls. In control explants, thePut pool stayed constant from d 0 to 15 and increased twotimes to d 28 (Fig. 7). Spd was less affected and Spm stayedthe same over the whole culture period. Therefore, the Spd toPut ratio changed from the beginning to the later growthstages of the culture (1.5-0.64).

Torrigiani et al. (28) measured a marked increase in solubleconjugated polyamines during in vitro culture of thin celllayer epidermis explants from N. tabacum Samsun whichformed floral buds. In the same cultures, free Put and Spdpools were dramatically enhanced. They demonstrated a cor-relation between changes in free PA and also in conjugatedPA levels on one hand and growth and generative versusvegetative response on the other hand. In a similar systemwith thin cell layer explants of N. tabacum Wisconsin 38,Tiburcio et al. (27) showed a difference in the increase ofconjugated PA between their 'vegetative' and 'floral' system,respectively, induced by different concentrations of kinetin inthe medium. The difference between their and our systemwas, that we used a different cultivar of tobacco, and that wehad, in addition, pith and vascular tissue in our explants.More importantly, we added no plant hormones to the culturemedium. Martin-Tanguy et al. (16) and Burtin et al. (4)observed in in vitro cultured leaf disc explants from N. taba-cum Xanthi, that addition of auxins (2,4-D) and cytokinins(BA) caused enormous increases in Put, Spd, FP, and CPlevels, while in explants grown without hormones these in-creases were minimal but at similar levels as in our system.Addition of high concentrations ofDFMA and DFMO (0.5-3 mM) caused differences in growth response (4), which couldnot be seen in our explants using 0.1 mm or lower concentra-tions of both inhibitors. Leubner and Amrhein (10) used anin vitro system for tuber formation in Solanum tuberosumexplants. The in vitro tubers were formed with or withoutaddition of BA to the medium. The growth response was thesame, but after 30 d of growth with BA in the medium, thecontent ofFP and ofCP was 10 times higher in these explants

than in those growing without the hormone. Therefore, rec-ognizing the discrepancies between the discussed systems, wenevertheless postulate that the ratio of plant hormones in themedium caused the growth responses directly, without theintermediary ofHCA (FP and CP) as plant growth regulators,as had been postulated by Martin-Tanguy (15, 16).

Addition of plant hormones, and changes in their endoge-nous levels may also explain the pattern of FP and CPfluctuations without a direct link of these HCA to the growthresponses seen in recent studies (4, 16, 27, 28). This interpre-tation is supported by the observation that in various systemsthe Put pool was enhanced by the addition of auxin andcytokinin (16, 20, 30). The increase in the Put pool couldresult from higher ADC activity caused by auxins, as wasshown by Roustan et al. (22) in cell suspension cultures ofVitis vinifera.The smaller increases in FP and CP in our cultures as

compared with other systems (4, 16, 27, 28) could be ex-plained as follows: FP increased at the beginning of culture incortex tissue (32), which contained also vascular tissue, at atime when auxins are transported in the stem explants to theformerly basal side of the stem. This auxin accumulationsupposedly causes the formation of cortical callus (1). Smuld-ers et al. (26) investigated this effect in N. tabacum Samsunby addition of NAA. In their explants, low concentrations ofNAA caused floral bud formation on the cortical callus justas in our explants grown in the absence of hormones in themedium, while high levels of NAA caused floral bud forma-tion all over the remaining surface as reported for thin layercultures (29). In our system, the CP increase occurred togetherwith the formation of floral buds and was localized in corticalcallus and floral buds (32) at a time when meristematicactivity was high. Therefore, auxin and cytokinin levels couldbe assumed to be elevated as well.

In N. tabacum Xanthi, N-caffeoyl-4-aminobutyric acid hasbeen reported to occur in floral parts with the same patternof distribution as CP and it was detected in cultured cells ofN. tabacum Xanthi grown on Put as the sole source ofnitrogen (2). Thus, CP and perhaps other Put conjugates maybe intermediates in Put catabolism, since 4-aminobutyric acidis an intermediate in the catabolism of free Put.The results discussed here show that FP and CP do not

trigger growth and floral bud formation in stem explants ashas been postulated previously. AIP seems to be the bestinhibitor to study the role of total HCA, while application ofDFMA or DFMO should be a means to study the physiolog-ical roles of either FP or CP, respectively. The switch fromthe Arg to Put pathway to the Orn to Put pathway may be arewarding focus for further investigations to unravel a possibleconnection between PA-biosynthesis and tissue differentiationand determination.

ACKNOWLEDGMENTS

We thank N. Amrhein (ETH Zurich, CH) and L. Maier (Ciba-Geigy Ltd., Basel, CH) for the generous gift of the inhibitors, forsuggestions and discussions, G. Leubner (ETH Zurich, CH) fordiscussion, and J. Gaudin (Ciba-Geigy Ltd. Basel, CH) for technicalassistance.

929




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2. Balint R, Cooper G, Staebell M, Filner P (1987) N-Caffeoyl-4-amino-n-butyric acid, a new flower-specific metabolite in cul-tured tobacco cells and tobacco plants. J Biol Chem 262:11026-11031

3. Bitonti AJ, McCann PP (1987) Inhibition of polyamine biosyn-thesis in microorganisms. In PP McCann, AE Pegg, ASjoerdsma, eds, Inhibition of Polyamine Metabolism. Aca-demic Press, London, pp 259-275

4. Burtin D, Martin-Tanguy J, Paynot M, Rossin N (1989) Effectsof suicide inhibitors of arginine and ornithine decarboxylaseactivities on organogenesis, growth, free polyamine and hy-droxycinnamoyl putrescine levels in leaf explants of NicotianaXanthi n.c. cultivated in vitro in a medium producing callusformation. Plant Physiol 89: 104-110

5. Cabanne F, Dalebroux MA, Martin-Tanguy J, Martin C (1981)Hydroxycinnamic acid amides and ripening to flower of Ni-cotiana tabacum var Xanthi nc. Physiol Plant 53: 399-404

6. Evans PT, Malmberg RL (1989) Do polyamines have roles inplant development? Annu Rev Plant Physiol Mol Biol 40:235-269

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Plant Physiol. (1990) 94, 3920032-0889/90/94/0392/01 /$01 .00/0

Correction

Vol. 92: 924-930, 1990

Bronwyn J. Barkla, Jeffrey H. M. Charuk, Edward J. Cragoe,Jr., and Eduardo Blumwald. Photolabeling of Tonoplastfrom Sugar Beet Cell Suspensions by [3H]5-(N-Methyl-N-Isobutyl)-Amiloride, an Inhibitor of the Vacuolar Na+/H+Antiport.

Reference numbers cited in the right-most column of TableI, page 929, are incorrect. The correct reference numbersare shown in the table reprinted below.

Table I. Na+/H+ Antiport-Related Suibuinits in Plants, Bacteria, and Animal SystemsSource Method Estimated Mass Reference

kDRat-renal brush border ["4C]-Br-EIPAa photolabeling 65.2 (9)Rabbit-renal brush border Affinity chromatography; analog A35 25 (13)

coupled to Sepharose CL-4BPig-renal brush border [3H]MIA photolabeling 81-107 (27)Dog-renal brush border [3H]MIA photolabeling 40-60 (6)Escherichia coli 35 (15)Beta vulgaris tonoplast [3H]MIA photolabeling 174-38-35 This report

a 5-(N-Ethyl-N-isopropyl-bromoamiloride).

392

feruloylputrescine caffeoylputrescine are not involved ...plant physiol. vol. 92, 1990 1.5 0. 0-0...

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