ann bot-1996-cline-255-66

Annals of Botany 78 : 255266, 1996

Exogenous Auxin Effects on Lateral Bud Outgrowth in Decapitated Shoots

MORRIS G. CLINE

The Ohio State Uniersity, Department of Plant Biology, Columbus, Ohio 43210, USA

Received: 5 July 1995 Accepted: 11 March 1996

In 1933 Thimann and Skoog demonstrated exogenous auxin repression of lateral bud outgrowth in decapitated shootsof Vicia faba. This evidence has given strong support for a role of auxin in apical dominance. Most, but not all,investigators have confirmed Thimann and Skoogs results. In the present study, auxin treatments were carried outon ten different species or plant types, many of which were treated with auxin in different forms, media and underdifferent light conditions. The ThimannSkoog experiment did work for most species (i.e. exogenous auxin did repressbud outgrowth) including the dgt tomato mutant which is known to be insensitive to auxin in certain responses. Toxicauxin symptoms were observed in some but not all species. The ThimannSkoog experiment did not work forgreenhouse-grown Coleus or for Arabidopsis. Light was shown to reduce apical dominance in Coleus and Ipomoea nil.

# 1996 Annals of Botany Company

Key words : Apical dominance, lateral bud outgrowth, axillary bud, auxin, IAA, decapitation, Vicia faba, Ipomoea nil,Pisum satium, Phaseolus ulgaris, Lycopersion esculentum, dgt, Coleus blumei, Arabidopsis thaliana, Helianthusannuus, ThimannSkoog.

INTRODUCTION

Apically derived auxin in shoots is generally thought tocontrol apical dominance either directly via entry intolateral buds with subsequent repression of outgrowth orindirectly via some other mechanism, i.e. activation of asecond inhibitor messenger, auxin-cytokinin ratio, sec-ondary growth substances, nutrient diversion, etc. (Martin,1987; Cline, 1991; Bangerth, 1994; Stafstrom, 1995; Tamas,1995). As studies of control mechanisms of developmentalresponses in plants have progressed, particularly with theuse of molecular and genetic approaches (Cline, 1994), therehas been increased interest directed towards the role ofauxin in apical dominance, and with the degree of branchingserving as a possible indicator of auxin activity (Tepfer,1984; Lincoln, Britton and Estelle, 1990; Shen et al., 1990;Estruch et al., 1991; Klee and Romano, 1994).

Because of this increased attention concerning auxin inapical dominance and inasmuch as the precise mechanismof auxin action in apical dominance has yet to be elucidated,a closer scrutiny of the evidence supporting auxin in-volvement in the control of lateral bud outgrowth is needed.

The strongest evidence supporting either a direct or anindirect role for apically-derived auxin in controlling apicaldominance comes from the classical ThimannSkoog ex-periment with Vicia faba (Thimann and Skoog, 1933). Theydemonstrated that auxin applied in agar blocks every 6 h tothe stump of a decapitated Vicia faba stem inhibited theoutgrowth of lateral buds situated lower on the stem.Thimann (1937) initially proposed the direct inhibitionhypothesis which presumes that auxin enters the lateral budand directly inhibits its outgrowth. Subsequently, hemodified his views to include indirect auxin action viainvolvement with cytokinins (Sachs and Thimann, 1967) or

ethylene (Russell and Thimann, 1988) as well as effects oflight (Thimann, 1977).

While many investigators have substantiated and ex-tended the results of the ThimannSkoog experiment (i.e.exogenous auxin repression of lateral bud outgrowth indecapitated shoots), others have reported anomalous effectsand there have been questions about the direct role of auxinin apical dominance (Jacobs et al., 1959; Brown, McAlpineand Kormanik, 1967; Ali and Fletcher, 1970; Shein andJackson, 1972; Hillman, 1984). Wareing and Phillips (1981)stated it is now generally considered that auxin does notexert its inhibitory effect on lateral buds in such a directmanner as that originally proposed by Thimann. Someworkers (Gregory and Veale, 1957; McIntyre, 1977) haveproposed nutrition to be the most important controllingfactor in apical dominance. To date, no one has reportedevidence of auxin restoration of apical dominance inArabidopsis via the ThimannSkoog experiment.

The question has also been raised as to how the terminalbud can continue to grow while simultaneously generatinginhibitory concentrations of auxin to the outgrowth oflateral buds below. Klee et al. (1987) have found thatoverproduction of auxin in petunia via transformation withIAA biosynthetic genes of Agrobacterium eliminates branch-ing. With a similar transformed system, Romano, Cooperand Klee (1993) have reported an increase in apicaldominance in Arabidopsis as measured by a reduction infresh weight of secondary inflorescences. However, thisauxin overproduction occurs in every cell of the organismrather than being localized in buds of adjacent nodes asmight occur in a natural system. In addition, Lincoln et al.(1990) reported that total number of inflorescences arisingfrom the rosette does not differ greatly between axr1 [thebushy auxin-resistant mutant] and wildtype even though

0305-7364}96}08025512 $18.00}0 # 1996 Annals of Botany Company

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256 ClineExogenous Auxin Effects in Decapitated Shoots

Fig. 1. Lateral bud outgrowth approximately 1 week following decapitation. A, Vicia faba L. (Broad Windsor Bean). B, Helianthus annuus L.(Mammoth Grey-striped Sunflower). C, Ipomoea nil L. Roth (Japanese Morning Glory). D, Pisum satium L. [Little Marvel Pea (dwarf )], andE, Thomas Laxton Pea (normal). F, Phaseolus ulgaris L. (Blue Lake Bean). The stem stumps of all decapitated plants shown here were treated

with lanolin except for Japanese Morning Glory (C) which has a taped cotton swab for treatment with aqueous solution.

the number of lateral branches did greatly increase in theformer. Beveridge, Ross and Murfet (1994) reported thatgrafting of non-branching wildtype stocks to the branchingrms-2 pea mutant scions did not normalize endogenous IAAlevels even though it did inhibit branching.

White (1976) stated that discrepancies in the results ofdifferent workers using apparently similar methods ofapplication of IAA to decapitated plants of a single varietyof Phaseolus obviously require some explanation. Hecarried out experiments, focusing on this single species with

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ClineExogenous Auxin Effects in Decapitated Shoots 257

variations in plant age, concentrations and quantity of IAA,type of lanolin, region of application and time of year. Hisresults showed that the effectiveness of IAA applied didvary significantly with those conditions. He also found thatin many instances, IAA did completely replace the mainshoot with respect to correlative inhibition of lateral budgrowth.

The objective of the present study was similar to that ofWhites except that instead of analysing a single species, thefocus here was to compare responses to exogenous IAA inrepressing lateral bud outgrowth in ten different species orplant types, including the mutant dgt tomato which isknown to be insensitive to auxin promotion of certainresponses, and Arabidopsis thaliana. In addition, the effectsof auxin in different concentrations (both aqueous and inlanolin), forms (IAA, a-NAA and b-NAA) and underdifferent light conditions were studied in a number of thesespecies.

MATERIALS AND METHODS

Seeds of Vicia faba L. (Broad Windsor Bean), Helianthusannuus L. (Mammoth Grey-striped Sunflower), Ipomoea nilL. Roth, strain violet (syn. Pharbitis nil ) (Japanese MorningGlory),Pisum satiumL. [Little Marvel (dwarf ) and ThomasLaxton (normal) Pea], Phaseolus ulgaris L. (Blue LakeBean) and Lycopersicon esculentum, Mill., strain VNF8 andthe mutant diageotropica (dgt) tomato were germinated inPro-mix, a general purpose peat-vermiculite growing me-dium. The seeds of Ipomoea were scarified in concentratedsulphuric acid for 40 min and soaked overnight in runningwater and germinated in Petri dishes before planting. Coleusblumei Benth. was propagated from stem cuttings. All theplants except the normal pea were grown in a greenhouse(1632 C) during the winter}spring of 199394 and thespring}summer of 1995 with supplementaryGeneral Electric400 watt mercury vapour lamps (total irradiance: up to3300 lmol m# s"). All the above plants, except Coleus,were also grown in growth rooms (2729 C) under con-tinuous light (General Electric, Power Groove cool whitefluorescent and incandescent sources 25450 lmol m# s").

The ages of the plants at the times of decapitation and thebeginning of the auxin treatments varied with the growingconditions (i.e. in greenhouse and growth rooms) and thedevelopmental status of the lateral buds. The ages (in d)ranged as follows: Vicia faba, 1213; Helianthus annuus,1927; Ipomoea nil, 1232; Pisum satium, dwarf, 1021,normal, 1516; Phaseolus, 1018; Lycopersicon esculentum,VNF8, 2754; dgt, 3968; Coleus (from time of stemcutting), 2137; Arabidopsis thaliana, 3552. Intact plantswith elongating lateral buds were excluded. Shoots weredecapitated with a razor blade about 1 cm above the base ofthe lateral bud at the node indicated for each species in theResults section (Fig. 1). Excluding the cotyledons, nodeswere counted upward from the base of the plant. There wassome variation in this distance depending upon the speciesand the relevant internode lengths. It was necessary thatthere be sufficient distance between the lateral bud and thedecapitated stem stump above so that auxin could be

applied to the stump without contact with the bud. Auxinwas applied to the stem stump twice daily beginningimmediately after decapitation in approximately 150 lldoses as IAA, a- or b-NAA at concentrations ranging from10' to 10$ m in 005% Tween 20 aqueous solution in ataped cotton swab (Fig. 1C) or as IAA in lanolin, 01, 05,1%. Treatments normally extended from 4 to 10 d withdaily measurements of the lateral bud situated at the highestnode below the point of decapitation. Two exceptions wereVicia faba and Pisum satium (normal) where the largestrather than highest lateral bud was used to determine budoutgrowth. Each treatment was usually carried out withfour to nine plants. At least two experiments with each ofthe plant types were carried out in both the greenhouse andthe growth room with the exceptions indicated above.

For certain light experiments (as indicated in the Resultssection), Ipomoea seedlings were moved from growth roomsto out-of-doors (irradiance under shade screens, up to210 lmol m# s" and in the open, up to 6900 lmol m# s")during the summers of 1992 and 1994 and Coleus plantspropagated from stem cuttings were grown in growth roomswith greatly reduced irradiance from General Electric coolwhite fluorescent lamps (2530 lmol m# s"). There wereusually four to ten Coleus and Ipomoea plants in each of theauxin treatments. In the outside shade screen experiments,there were eight to 11 Ipomoea plants per treatment. Seedsof Arabidopsis thaliana (strain CS 1072 Chi-O from the OhioState University Arabidopsis Biological Resource Center)were germinated in water-saturated Magik-moss pottingsoil containing perlite and vermiculite in greenhousefollowing a 2-d treatment in the refrigerator (03 C). Afterseveral weeks, some seedlings were moved to the growthroom (2729 C) under continuous light while the remainingwere kept in the greenhouse.

RESULTS

Auxin significantly inhibited lateral bud outgrowth fol-lowing decapitation when applied to the stem stump of mostof the species tested [Ipomoea nil, Helianthus annuus,Lycopersicon esculentum (VNF8), Pisum satium (LittleMarvel and Thomas Laxton), and Vicia faba] at concen-trations of 10& and}or 10% m in aqueous solution or 01and}or 1% in lanolin (Figs 2, 4; Table 1). Inhibition wasalso found in the tomato mutant dgt (Fig. 4) which isinsensitive to auxin with regard hypocotyl elongation andethylene production (Kelly and Bradford, 1986). In Ipomoea,Helianthus and dgt tomato there were no obvious toxiceffects of exogenous auxin applications observed in anyexperiments. In Vicia faba and Pisum, occasional auxin-induced aberrations in stem and leaf growth were observedwhereas in the VNF8 tomato such aberrations were morecommon.

Auxin had no effect on restoring apical dominance indecapitated greenhouse-grown Coleus (Fig. 5) or Arabidopsis(Fig. 3, Tables 2 and 3) and only a partial effect on bean(Fig. 2F). a-NAA was generally more potent than IAA ininhibiting lateral bud outgrowth. b-NAA, the inactive auxin

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Fig. 2. Auxin repression of lateral bud outgrowth over 3 or 4 d following decapitation. A, Vicia faba (growth room, n 4). B, Helianthus annuus(growth room, n 5). C, Ipomoea nil (growth room, n 34). D, Pisum satium (dwarf ) (growth room, n 56). E, Pisum satium (normal)

(growth room, n 7). F, Phaseolus ulgaris (greenhouse, n 4). Decap, Decapitation. Vertical lines represents.d.

analogue, which was tested on seven of the ten plant types,usually had little or no effect on apical dominance in mostexperiments. With some species [Vicia faba ; Pisum (LittleMarvel), Helianthus annuus], the lack of effect of auxin inaqueous solution may have been due to a penetration ormetabolism problem. Auxin in lanolin was always effective.

In the following section are descriptions of apicaldominance in the ten plant types, and their responses todecapitation and to auxin treatments under growth roomand greenhouse conditions. The term strong apical domi-nance as used here signifies little or no lateral bud outgrowthin intact plants. Medium signifies some bud growth andweak indicates substantial and continuing lateral budgrowth in intact plants.

Vicia faba (Broad Windsor Bean)

The intact plant had weak to medium apical dominance.Lateral buds on nearly half of the greenhouse plants and ona quarter of the growth room plants had grown out beforethe start of the auxin treatment. These plants had to beexcluded. Decapitation above the third node resulted, mostoften, in the outgrowth of the lateral buds at the basal nodebut sometimes at the upper nodes (Fig. 1A). All three lateralbuds were repressed by the auxin treatment with the top(third) bud (closest to the auxin application) inhibited themost. Consistent repressive effects on bud growth werefound with 1% IAA in lanolin and partially at 01% (Fig.2A). There was some swelling and abnormal curving of

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Table 1. Outgrowth of lateral buds (mms.d.) oer 3 or 4 dfollowing decapitation and treatment of shoot stump withIAA, a-NAA, b-NAA in aqueous solution or IAA in lanolin.

Decap., decapitation

Days after decapitation

0 1 2 3 4

Vicia faba, growth room, n 4Intact 0 0 0 0 0Decap. control 0 12 83 214 354IAA 10% m 0 55 1410 2713 4314a-NAA 10% m 0 12 56 1611 2314

Helianthus annuus, growth room, n 45Intact 0 0 0 0 0Decap. control 0 11 32 94 207IAA 01% lanolin 0 0 0 0 12IAA 05% lanolin 0 0 0 0 0

Ipomoea nil, growth room, n 10Decap. control 10 31 81 255IAA 01% lanolin 20 21 32 86IAA 1% lanolin 10 20 21 42

Pisum satium (dwarf ), greenhouse, n 4Intact 31 31 31 31 41Decap. Control 21 31 51 83 136IAA 10% m 31 41 52 82 113a-NAA 10% m 21 21 21 31 31b-NAA 10% m 21 31 42 52 72

Phaseolus ulgaris, growth room, n 4Intact 30 41 40 41Decap. control 40 72 113 4511IAA 10% m 31 52 82 238a-NAA 10% m 30 41 62 115b-NAA 10% m 31 52 92 259

stems which accompanied the auxin treatment. Neither IAAnor a-NAA in aqueous solution were very effective inrepressing axillary bud outgrowth (Table 1).

Helianthus annuus (Mammoth Grey-striped Sunflower)

The intact plant had very strong apical dominance withabsolutely no sprouting of lateral buds. Outgrowth of budsin the greenhouse was not observed until nearly a weekfollowing decapitation above the first node, whereas it wasobserved in the growth room within 1 or 2 d (Fig. 1B).Release from apical dominance could be inhibited by 10& ma-NAA (Fig. 2B) or by 01% IAA in lanolin (Table 1). IAAin aqueous solution was ineffective at 10% m (Fig. 2B). Notoxic auxin effects were observed.

Ipomoea nil (Japanese Morning Glory)

The intact plant had medium to strong apical dominance.There was no axillary bud outgrowth in the growth room orin winter-grown greenhouse plants. There was slightoutgrowth of cotyledonary buds of greenhouse plants in thespring (data not shown). Decapitation above the secondnode nearly always resulted in the rapid outgrowth of thebud at the node (Fig. 1C). The bud at the first node alsooften exhibited a small amount of temporary growth.

Both IAA and a-NAA (data not shown) in aqueous

solution (10& to 10$ m, Fig. 2C) and IAA in lanolin (01and 1%, Table 1) significantly inhibited lateral budoutgrowth following decapitation with no toxic auxinsymptoms observed except at 10$ m IAA. As Fig. 2Cindicates, although there was no effect at 10' m IAA, therewas increasing repression of axillary bud outgrowth from10& to 10$ m.

Pisum sativum [Little Marel (dwarf ) and ThomasLaxton (normal) Pea]

The intact plants had moderate apical dominance.Following decapitation above one of the higher nodes(usually the fifth), many of the buds at the lower nodeswould simultaneously grow out to considerable lengths(Fig. 1D, E). In the dwarf pea, the bud at the highest nodeusually grew out most rapidly followed by the buds at thesecond and third highest nodes (Fig. 1D). In the normalpea, it was the bud at the second or third node from the basewhich grew out more rapidly followed by those of the fourthand fifth nodes, respectively (Fig. 1E).

Auxin did significantly restore apical dominance in boththe dwarf and the normal pea plants following decapitation.Significant inhibition of bud outgrowth was obtained indwarf pea with 10% m a-NAA but not with 10% m IAA inaqueous solution (Table 1). IAA in lanolin at 01% waseffective in both dwarf (Fig. 2D) and normal (Fig. 2E) typesalthough some swelling in the stem stump and abnormalstem curvature were observed in the normal peas and in oneexperiment with the dwarf peas.

Phaseolus vulgaris (Blue Lake Bean)

The intact plant had weak to medium apical dominance.Its apical dominance has been described as incomplete (Tamas, 1987). The axillary buds subtended by the primaryleaves (the first node, above which we decapitated) weremore inhibited than the buds at the second node subtendedby the first trifoliate leaf. The bean plant grows rapidly, theinternodes are large and the plant is easy to work with.Decapitation definitely accelerated lateral bud outgrowth(Fig. 1F) but the inhibitory effect of IAA (1% in lanolin,Fig. 2F and 10% m in solution, Table 1) applied to the stemstump was only partial. a-NAA at 10% m had a strongereffect (Table 1). No toxic effects of auxin were observedexcept for a little swelling and bleaching of the stem stumpsupon which the auxin was directly applied.

Coleus

The intact Coleus plant had weak apical dominance. Ithad short internodes, grew slowly and was bushy. The basalbranches were of greater length than the higher branchesbecause of their greater age. These plants were propagatedfrom cuttings. Many intact plants had to be excluded fromthe study because of extensive axillary bud developmentbeginning at the basal nodes. Only plants with very smalllateral buds (! 34 mm) were selected. At the time ofdecapitation, the lateral buds of the lowest of the top three

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Fig. 3. Upper left and right, VNF8 (wildtype) and dgt (mutant) tomatoes about a week following decapitation showing lateral bud outgrowth.The stem stumps have been treated with lanolin. Lower left, Arabidopsis, 1 week following decapitation; A, decapitation above the first node; B,intact control ; C, decapitation above first node with 1% IAA on stump of main stem; Lower right, Coleus showing the branch-promoting effect

of greenhouse high light (B) as compared with indoor low light (A).

nodes were removed. Decapitation above the third node ofthese greenhouse plants had only a small effect onaccelerating bud outgrowth. Auxin (01% and 1% inlanolin) application to the stem stumps had no repressingeffect on lateral bud outgrowth of greenhouse plants (Fig.5).

Lycopersicon esculentum [VNF8 (normal ) and dgt(mutant) tomato]

The intact plants of both dgt and VNF8 had mediumapical dominance with little or no lateral branching. Neitherthe dgt nor the VNF8 in our growth room or greenhouseconditions were categorized as having reduced apicaldominance or as being bushy. In a few experiments, some

lateral bud development was noticed in intact plants. TheVNF8 plants were larger than the dgt plants. The shoot ofthe young dgt seedling initially grows more or less vertically.After several months, the shoot gradually assumes ahorizontal orientation. The seedling shoots of dgt, althoughnot yet at horizontal growth stage, were floppy and hencewere staked in an upright position for convenience in auxintreatment. Since horizontal positioning tends to weakenapical dominance (Prasad and Cline, 1985b), such staking,if anything, would have a countering effect to anomalousbud outgrowth.

Following decapitation, which was usually carried outabove the fifth node, several of the lateral buds on any givendgt or VNF8 plant vigorously sprouted more or lesssimultaneously (Fig. 3). This most often occurred at the

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Fig. 4. Auxin repression of tomato [VNF8 (A, C) and dgt (B, D)] lateral bud outgrowth in growth room following decapitation. Auxin in aqueoussolution, A, B (n 45, 4). Auxin in lanolin, C, D (n 34, 4). Decap, decapitation. Vertical lines represents.d.

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Fig. 5. Lateral bud growth in Coleus blumei over 6 d followingdecapitation and stem stump treatment with 1% IAA (greenhouse,

n 910). Vertical lines represent s.d.

upper-most nodes but sometimes at one of the lower nodes.The points on the curves in the graphs represent the meanlength of the highest lateral bud on each plant. The youngaxillary buds of tomato were difficult to observe and tomeasure because of their small size and narrow shape.

The treatment with exogenous auxin [10& m a-NAAand}or 10% m IAA in aqueous solution (Fig. 4A, B) and01% IAA in lanolin, Fig. 4C, D] did, for the most part,significantly inhibit axillary bud outgrowth in both planttypes. IAA in aqueous solution had variable effects onVNF8 but a-NAA, down to 10& m, had consistentrepressive effects on bud outgrowth (Fig. 4A). b-NAA had

no inhibitory effect. In several instances it appeared even topromote bud growth. IAA at 10% m and 10& m a-NAAgenerally restored apical dominance in dgt (Fig. 4B anddata not shown). IAA in lanolin (both at 01 and 1%) wasquite effective in repressing bud outgrowth in both VNF8and dgt (Fig. 4C, D). Hence, no essential difference insensitivity could be positively detected between dgt andVNF8 in the response to auxin in repressing lateral budoutgrowth except for the toxic auxin effect mentionedpreviously for VNF8.

Arabidopsis thaliana

Arabidopsis is a facultative long-day plant with weakapical dominance consisting of a rosette from which arisesa single branching floral shoot or inflorescence followed bythe generation of several surrounding axillary or secondaryinflorescences also arising from the rosette. The measure-ment of changes in apical dominance status is complexbecause of the two types of branching, lateral shootsdeveloping from the main shoot and axillary inflorescencesdeveloping from the rosette and the rapidity with whichthey occur. The lateral buds (shoots) of the main shootbegin emerging almost as soon as the main shoot begins tobolt. In the many mutants and strains of Arabidopsis nowavailable, there exists a wide range of branching habits.

The CS 1072 Chi-O strain used in the present study hasstronger apical dominance than most Arabidopsis typesinasmuch as it generally lacks axillary inflorescences.Decapitation of the main floral shoot resulted in increased

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Table 2. Effect of 1% IAA on lanolin on Arabidopsis. Outgrowth of first lateral bud (mms.d.) and axillary inflorescence1 week following decapitation of the main shoot and treatment of the shoot stump with 1% IAA in lanolin. Decapitation waseither 35 mm aboe the first node or 35 mm aboe the base of the main shoot (below the first node) as indicated. Axillaryinflorescence deelopment is gien as a percentage of the plants sprouting one or more axillary inflorescences. *Main shoot

length

Decap above first node Decap below first node Decap above first node Decap below first nodeIntact Intactplant Control 1% IAA Control 1% IAA plant Control 1% IAA Control 1% IAA

Growth room Greenhouse

n 7 7 6 7 7 5 5 5 5 5

First Lateral Bud 23521* 14524 13229 12457* 8729 7852 Axial Inflorescences 0 71% 33% 100% 100% 0 60% 60% 100% 100%

Table 3. Effect of 10% M IAA spray on Arabidopsis. Outgrowth of axillary inflorescences 1 week following decapitation ofthe main shoot 35 mm aboe the base of the plant which was sprayed eery other day beginning 15 d before decapitation with10% M IAA. Axillary inflorescence deelopment is gien as a percentage of plants sprouting one or more axillary inflorescences

Axillary AxillaryMain inflorescence inflorescence

n shoot length (mms.d.) percentage

Growth roomIntact control 7 23521 0Intact IAA 10% m 7 21420 3823 38Decap. control 7 7143 100Decap. IAA 10% m 8 8141 100

GreenhouseIntact control 5 12457 0Intact IAA 10% m 9 10946 0 0Decap. control 5 2531 100Decap. IAA 10% m 10 2124 60

outgrowth of lateral (buds) shoots from the main shoot aswell as the subsequent generation of axillary inflorescencesfrom the base of the plant (Fig. 3). If the point ofdecapitation of the main shoot was above the lowest lateralemerging shoot (bud), then that shoot grew out to a muchgreater extent than in the intact plant and took over as themain shoot. If the decapitation of the main shoot was belowthe lowest lateral emerging shoot (bud), then several axillaryinflorescences grew out from the base of the plant. Thegreenhouse plants exhibited somewhat stronger apicaldominance with respect to axillary inflorescence outgrowththan did the growth room plants.

In essentially no plant did auxin, either as 1% IAA inlanolin applied to the decapitated stump of main shoot or as10% m IAA spray applied every other day to the wholeplant, have any effect in restoring apical dominance, i.e., ininhibiting lateral branching of the main shoot or theoutgrowth of the axillary inflorescences (Fig. 3, Tables 2and 3). In the case of decapitation above the first node, thereappeared to be some auxin repression in the percentage ofaxillary inflorescence development in the growth room(Table 2) but this was not confirmed in the greenhouse norin the more direct test when IAA was applied followingdecapitation below the first node.

Attempts to restore apical dominance with auxin sprayapplied to the small and extremely bushy trp 1-1 tryptophan-requiring auxotrophic mutant (suggestive of an auxin defect,Last et al., 1992) were also unsuccessful (data not shown).

Influence of light

Past observation (Hosokawa et al., 1990) indicated thatwhen Ipomoea nil was propagated in growth rooms under amixture of cool white fluorescent and incandescent sources(irradiance: 25450 lmol m# s"), a 30-d-old plant wascharacteristically tall (4070 cm) without branching. How-ever, when the plant was grown outdoors (irradiance: up to6900 lmol m# s"), the plant was short, often with such aproliferation of branching that the study of auxin effects onbud outgrowth was impossible to carry out since most budshad substantially elongated at a very early developmentalstage. Therefore, Ipomoea plants were grown indoors untilthe age of 14 d, at which time they were moved outdoors(still in pots), decapitated and stem stumps treated withauxin in lanolin for 4 d. There appeared to be little or nodifference in sensitivity in auxin repression of axillary budoutgrowth between indoor plants at low irradiance andindoor plants moved outdoors at high irradiance at the time

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Table 4. Outgrowth of lateral buds (mms.d.) of Ipomoeanil plants oer 3 or 4 d following decapitation and treatmentof shoot stump with IAA in lanolin either in growth room(25450 lmol m# s") or outdoors (up to 6900 lmol m# s")

Days following decapitation

0 1 2 3 4

Growth room, n 10Decap. control 10 31 81 255 IAA 01% 20 21 32 86 IAA 1% 10 20 21 42

Outdoors, n 48Decap. control 21 31 61 123 236IAA 01% 30 31 31 61 113IAA 1% 31 81 31 31 52

of decapitation and auxin treatment (Table 4). Hence, therewas no immediate effect of high irradiance on exogenousauxin restoration of apical dominance in decapitated plants.

When 16}17-d-old Ipomoea plants in pots were trans-planted to outdoor plots under heavy shading (irradiance:up to 210 lmol m# s"), they appeared, after 1934 d, verysimilar in overall formandheight (i.e. tallwithout branching)to indoor plants of approximately the same age (no datagiven). Whereas those plants transplanted to outdoor plotsunder full sunlight had, in time, such an extensiveproliferation of lower branches together with reduced height

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2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

0.1

27.

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7

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2.6

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24.

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25.

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79

.6

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.9

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.9

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.4

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00.

2

Fig. 6. Bar graphs comparing lateral bud length (cms.d.) of Ipomoea nil plants grown outside in heavy shade (+) and in the open (*) at differentnodes numbering up from the base of the shoot. n 811.

100

0Weight (g)

Wei

ght

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75

25

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)

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Fig. 7. Bar graphs comparing total fresh weights and heights ofIpomoea nil plants grown outside in heavy shade (+) and in the open

(*). n 811.

that they were hardly recognizable as Ipomoea nil plants.Both the lateral bud length and the total fresh weights of theshaded plants were very much reduced as compared to thoseof unshaded plants (Figs 6 and 7). At the fifth, sixth and

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10

12

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m)

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Fig. 8.Partial auxin repression of lateral bud outgrowth in Coleus atreduced irradiance (2530 lmol m# s") following decapitation over

10 d. Vertical lines represents.d. n 10.

seventh nodes of the shade plants (Fig. 6), there was someanomalous branching due to the fact that the shoot tips hadimpinged against the overhanging shade screen, had bentover and had released the buds at those nodes. Floral budswere found to be much more prevalent in the sun plantsthan in the shade plants. Since the spectral quality ofsunlight in both groups of plants presumably was the same(i.e., the plastic shade screens would not be expected tocause any such changes), the pronounced weakening ofapical dominance as exhibited by the proliferation ofbranching in the sun plants could be attributed to increasesin irradiance (fluence) and not to changes in light quality.

WhenColeus plantswere grown indoors at greatly reducedlight levels (irradiance: 2530 lmol m# s"), branching inintact plants was much decreased compared to that ofgreenhouse plants (Fig. 3) but increased lateral budoutgrowth did occur at the highest node following de-capitation. Definite but partial inhibitory effects of 01 and1% IAA in lanolin were detected in one out of fiveexperiments carried out at the reduced light level (Fig. 8).

DISCUSSION

The results of this study corroborated the Thimann-Skoog(1933) experiment, that exogenously applied auxin to thestem stump of a decapitated plant does restore apicaldominance for many plants under most conditions. This isa classic example wherein the results one obtains dependupon the particular plant system used and upon theconditions employed. There were exceptions where theThimann-Skoog experiment did not work. In those plantswith weak apical dominance such as Coleus or Arabidopsis,auxin had little or no effect on repressing bud outgrowth indecapitated plants. Hence, it is understandable why Jacobset al. (1959) were not able to detect repressive auxin effectson the release apical dominance with greenhouse-grownColeus plants. In plants with incomplete apical dominancesuch as Phaseolus, auxin had only a partially inhibitingeffect on axillary bud growth. Hence, it is understandablewhy Hillman (1984) questioned the role of auxin in apicaldominance in this species. Certain environmental conditionscan proliferate branching to an extent which makes itimpossible to carry out the Thimann-Skoog experiment.

Increased irradiance levels can greatly weaken apicaldominance (Gregory and Veal, 1957; Anderson, 1976).High wind velocity (via thigmomorphic effects) or altera-tions in the direction of gravity can also release apicaldominance (Prasad and Cline, 1985a, b).

Although Thimann, Sachs and Mathur (1971) reportedauxin in aqueous solution to be more effective than inlanolin with Coleus and Pisum, in this study it was foundotherwise. This might have been due to different methodsused for applying the aqueous solution. Thimann employeda fine piece of tubing over the end of the stem for continuousflow whereas two doses of approximately 150 ll wereapplied daily by pipette to a cotton swab taped to the endof the stem in the present study (Fig. 1C). The auxinconcentration (14001700 units) which Thimann and Skoog(1933) used to completely inhibit bud outgrowth in Viciafaba is presumed to be equivalent to approximately 1% inlanolin or to 610# m in aqueous solution (Stafstrom,1993).

In retrospect, Vicia faba, the plant system which Thimannand Skoog (1933) used in their classic study, probably wasnot the best for studying apical dominance. First, it hasweak apical dominance and many plants had to be excludedin the present study because they were already branchingbefore the time designated for decapitation, particularly inthe greenhouse plants. Second, following decapitation, itwas usually the bud at the basal node instead of the highestnode which sprouted first. This complicated the situationbecause of the greater distance between the site of auxinapplication (the decapitated stem stump) and the potentiallyactive axillary buds than when the highest bud sproutedfirst.

It was observed in some plants where several Vicia budssprouted simultaneously that the higher buds located closerto the site of auxin application were repressed the most. Thissuggested that the auxin concentration probably decreasedgradually during transport away from its original source.However, V. faba was sensitive to auxin and its apicaldominance was restored by auxin application. Thoseconcentrations of exogenous auxin (011% IAA) whichinhibited apical dominance release also exhibited toxicauxin effects.

The Thimann-Skoog experiment is carried out with thegreatest facility in a fast growing plant with large internodes,moderate to strong apical dominance, easily observedaxillary buds, and where only the highest lateral bud growsout following decapitation. Of the ten plants tested, Ipomoeanil conformed most closely to the expectations of theThimann-Skoog experiment, followed by Helianthus annuuswhich, however, was somewhat hindered by its slow growthin our greenhouse conditions. Pea (Thomas Laxton) workedwell in many ways except for the uncertainty in which lateralbud would sprout following decapitation. It was usual forseveral buds to sprout simultaneously, which complicatedboth the execution of the experiment and the interpretationof the data.

In Arabidopsis, the complete lack of effect of auxin onrestoring apical dominance following decapitation may bedue in part to the fact that the upright shoot of Arabidopsisis a floral shoot. All experiments done with other species in

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this study were carried out with vegetative shoots, or at leastshoots without the presence of visible floral buds. Forreasons which are not entirely clear, aging and reproductionoften have a weakening effect on apical dominance (Tamas,1987).

dgt mutant tomato

If one accepts the auxin inhibition hypothesis as theexplanation for the mechanism of action of apical domi-nance then the lack of outgrowth of the axillary buds ispresumed to be due to repression imposed by apically-produced}basipetally transported auxin in the shoot. Amutant such as the dgt tomato which has been demonstratedto be insensitive to auxin-induced hypocotyl elongation andethylene production (Kelly and Bradford, 1986) might alsobe expected to be insensitive to auxin repression of lateralbud outgrowth and, hence, would exhibit heavy branching.This was not observed in the present study in growth roomor greenhouse plants up to an age of 45 weeks. It wasnoticed in older plants (810 weeks or more) that high soilfertilization and exposure of plants to open sunlight usuallydid promote axillary bud growth in both dgt and VNF8.

Interpretation of data (not shown) which indicatedinsensitivity of dgt to the auxin transport inhibitors, TIBA(2,3,5-triiodobenzoic acid) and NPA (N-1 naphthyl-phthalamic acid), with respect to releasing apical dominancewas unclear due to significant non-specific effects (abnormalstem and leaf growth) commonly observed in VNF8 in casesof lateral bud outgrowth.

Auxin did inhibit lateral bud outgrowth in decapitateddgt plants following application to the stem stump. Hence,dgt is sensitive to auxin with respect to apical dominance. Ithas been suggested for apical dominance as well as for otherresponses to auxin that theremaybe separate auxin receptors(M. O. Kelly and M. G. Cline, unpub. res. ; Palm et al.,1991).

Light

When irradiance is high, as outdoors, the lateral buds ofmany species (except for those with very strong apicaldominance such as sunflower) will begin to grow out and toproliferate, thus excluding the execution of the Thimann-Skoog experiment which requires lateral buds to be in arepressed state before decapitation and auxin treatment.This proliferation of branching in outdoor-grown plantswas observed in the present study of Ipomoea nil, a plantwith moderate to strong apical dominance. From previousindoor tests, it was clear that once vigorous axillary budgrowth had commenced, it was very difficult to repress, evenwith high concentrations of auxin (data not shown). It isalso possible that other factors in the outdoor environmentbesides light may have a weakening effect on apicaldominance.

When the irradiance is low, the lateral buds of manyspecies (except for those with very weak apical dominancesuch as Arabidopsis) will be in a repressed state, thusallowing for the execution of the ThimannSkoog ex-periment with subsequent inhibition of lateral bud out-growth. In Coleus, a plant with weak apical dominance

where exogenous auxin is known to have no repressive effecton lateral bud outgrowth following decapitation at highirradiance in greenhouse environment (Jacobs et al., 1959),marginal evidence was found in the present study to supportThimanns contention (Thimann et al., 1971; Thimann,1977) that auxin did inhibit bud growth when the irradiancelevel was reduced (Fig. 8).

The fact that outdoor light control of apical dominance inIpomoea could be manipulated wholly by the use of shadescreens suggested that the response under the presentconditions was dependent upon changes in irradiance andnot in spectral differences. This result, while not excludingthe interaction with indirect light quality effects or withauxin, is also consistent with the nutritional hypothesis ofapical dominance via increased photosynthate availabilityfor bud growth at high irradiance. The possibility that theplastic shade screens could affect light quality and, hence,other physiological processes cannot be ruled out.

Light is one of many stimuli (e.g. gravity, CO#, nutrients,

etc.) which often will promote axillary bud outgrowth(Hillman, 1984). The mechanism by which light inducessuch outgrowth is unknown. Field and Jackson (1975) pointout some of the complexities involved in interpreting lighteffects on apical dominance. Gregory and Veale (1957)reported that auxin repression of axillary bud outgrowthincreased with decreasing light in Linum particularly at lownitrogen levels. Thimann et al. (1971) suggested that highlight may promote the synthesis of cytokinins which in turnmay reverse the auxin effect. Accumulating evidence in theliterature for an indirect role of auxin in apical dominancemight also indicate the involvement of certain phytochrome-mediated processes, interaction with other hormones andsecond messengers or by decreasing sensitivity to auxin.

CONCLUSION

The results of this study confirm that the ThimannSkoogexperiment does work for most species and suggests acontrolling role for auxin in apical dominance. Most likelythe role is indirect, perhaps involving cytokinins (Bangerth,1994; Sandberg et al., 1995). It is hoped that the presentaccelerating research involving both traditional physio-logical and molecular approaches with mutants will soon beable to resolve these problems and more fully elucidate thisclassic developmental phenomenon.

ACKNOWLEDGEMENTS

Appreciation is expressed to Daniel Repicz for his diligentefforts with the statistical data and graphs, Annabelle Chernand Liang Shi for their competent assistance with thepreliminary dgt tomato experiments, to the Livingston SeedCo. of Columbus, Ohio for their gracious donation of Viciafaba seeds and to the Ohio State University ArabidopsisBiological Resource Center for their generous help in thedonation of seeds and propagation of seedlings.

LITERATURE CITED

Ali A, Fletcher RA. 1970. Hormonal regulation of apical dominance insoybeans. Canadian Journal of Botany 48 : 19891994.

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Dow

nloaded from


Anderson AS. 1976. Regulation of apical dominance by ethephon,irradiance and CO

#. Physiologia Plantarum 37 : 303308.

Bangerth F. 1994. Response of cytokinin concentration in the xylemexudate of bean plants (Phaseolus ulgaris L.) to decapitation andauxin treatment and relationship of apical dominance. Planta 194 :439442.

Beveridge CA, Ross JJ, Murfet IC. 1994. Branching mutant rms-2 inPisum satium. Grafting studies and endogenous indole-3-aceticacid levels. Plant Physiology 104 : 953959.

Brown CL, McAlpine RG, Kormanik PP. 1967. Apical dominance andform in woody plants : a reappraisal. American Journal of Botany54 : 153162.

Cline MG. 1991. Apical dominance. The Botanical Reiew 57 : 318358.Cline MG. 1994. The role of hormones and apical dominance. New

approaches to an old problem in plant development. PhysiologiaPlantarum 90 : 230237.

Estruch JJ, Chriqui D, Grossmann K, Spena A. 1991. The plantoncogene rolC is responsible for the release of cytokinins fromglucoside conjugates EMBO Journal 10 : 28892895.

Field RJ, Jackson DI. 1975. Light effects on apical dominance. Annalsof Botany 39 : 369374.

Gregory FC, Veale JA. 1957. A reassessment of the problem of apicaldominance. Symposia of Society of Experimental Biology 11 : 120.

Hillman JR. 1984. Apical dominance. In: Wilkins M, ed. Adancedplant physiology. London: Pitman Publishing, 127148.

Hosokawa Z, Shi, L, Prasad TK, Cline MG. 1990. Apical dominancecontrol in Ipomoea nil : the influence of the shoot apex, leaves andstem. Annals of Botany 65 : 547556.

Jacobs WP, Danielson J, Hurst V, Adams P. 1959. What substancenormally controls a given biological process? II. The relation ofauxin to apical dominance. Deelopmental Biology 1 : 534554.

Kelly MO, Bradford KJ. 1986. Insensitivity of the diageotropica tomatomutant to auxin. Plant Physiology 82 : 713717.

Klee HJ, Horsch RB, Hinchee MA, Hein MB, Hoffman NL. 1987. Theeffects of over production of two Agrobacterium tumefaciens T-DNA auxin biosynthetic gene products in transgenic petuniaplants. Genes and Deelopment 1 : 8696.

Klee HJ, Romano C-P. 1994. The roles of phytohormones indevelopment as studied in transgenic plants. Critical Reiews inPlant Sciences 13 : 311324.

Last RL, Borczak JL, Pruitt KD, Radwanski ER, Rose AB. 1992. Thegenetics of tryptophan biosynthesis in Arabidopsis. Journal ofExperimental Botany Supplement 43 : 18.

Lincoln C, Britton J, Estelle M. 1990. Growth and development of theaxr1 mutants of Arabidopsis. The Plant Cell 2 : 10711080.

McIntyre GI. 1977. The role of nutrition in apical dominance. In:Jennings DH, ed. Integration of actiity in the higher plants.Symposium of Society for Experimental Biology, Cambridge:Cambridge University Press, 251273.

Martin GC. 1987. Apical dominance. HortScience 22 : 824833.Palm K, Hesse T, Moore I, Campos N, Feldwisch J, Garbers C, Hesse

F, Snell J. 1991. Hormonal modulation of plant growth: the roleof auxin perception. Mechanisms of Deelopment 33 : 97106.

Prasad TK, Cline MG. 1985a. Mechanical perturbation-inducedethylene releases apical dominance in Pharbitis nil by restrictingshoot growth. Plant Science 41 : 217222.

Prasad TK, Cline MG. 1985b. Gravistimulus direction, ethylene

production and shoot elongation in the release of apical dominance

in Pharbitis nil. Journal of Experimental Botany 36 : 19691975.

Romano C, Cooper ML, Klee HJ. 1993. Uncoupling auxin and ethylene

effects in transgenic tobacco and Arabidopsis plants. Plant Cell 5 :

181189.

Russell W, Thimann KV. 1988. The second messenger in apical

dominance controlled by auxin. In: Phares RP, Hood SB, eds.

Plant growth substances, 1988. New York: Springer-Verlag,

419427.

Sachs T, Thimann KV. 1967. The role of auxins and cytokinins in the

release of buds from dominance. American Journal of Botany 54 :

136144.

Sandberg G, Sitbon F, Eklof S, Edlund A, Astot C, Blackwell J, Moritz

T, Sundberg B, Olsson O. 1995. Regulation of auxin and cytokinin

turnover in plants. 15th International Conference on Plant Growth

Substances. Minneapolis. Program-Abstracts 015.

Shein T, Jackson DI. 1972. Interaction between hormones, light and

nutrition on extension of lateral buds in Phaseolus ulgaris L.

Annals of Botany 36 : 791800.

Shen WH, Davioud E, David C, Barbier-Brygoo H, Tempe! J, Guern J.

1990. High sensitivity to auxin is a common feature of hairy root.

Plant Physiology 94 : 554560.

Stafstrom J. 1993. Axillary bud development in pea: apical dominance,

growth cycles, hormonal regulation and plant architecture. In:

Amasino RM, ed. Cellular communication in plants. New York:

Plenum Publishing Corp., 7586.

Stafstrom J. 1995. Developmental potential of shoot buds.In: Gartner

BL, ed. Plant stems: Physiology and functional morphology. San

Diego: Academic Press, 257279.

Tamas IA. 1987. Hormonal regulation of apical dominance. In: Davies

PJ, ed. Plant hormones and their role in plant growth and

deelopment. Dordrecht : Martinus Nijhoff Publishers, 393410.

Tamas IA. 1995. Hormonal regulation of apical dominance. In: Davies

PJ, ed. Plant hormones, physiology, biochemistry and molecular

biology 2nd edn. Boston: Kluwer Academic Publishers, 572597.

Tepfer D. 1984. Transformation of several species of higher plants by

Agrobacterium rhizogenes : sexual transmission of the transformed

genotype and phenotype. Cell 37 : 959967.

Thimann KV. 1937. On the nature of inhibitions caused by auxin.

American Journal of Botany 24 : 407412

Thimann KV. 1977. Hormone action is the whole life of plants. Amherst :

University of Massachusetts Press.

Thimann KV, Sachs T, Mathur KN. 1971. The mechanism of apical

dominance in Coleus. Physiologia Plantarum 24 : 6872.

Thimann KV, Skoog F. 1933. Studies on the growth hormones of

plants. III. The inhibition action of growth substance on bud

development. Proceedings of the National Academy of Science,

USA 19 : 714716.

Wareing PF, Phillips IDJ. 1981. The control of growth and differentiation

in plants, 3rd edn. New York: Pergamon Press.

White JC. 1976. Correlative inhibition of lateral bud growth in

Phaseolus ulgaris L. Effect of application of indol-3yl-acetic acid

to decapitated plants. Annals of Botany 40 : 521529.

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