Hormone Production Plants Research Morphich

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Biol. Ret:. rg7S), 48, 0.509-559

BRCPAFI a8-a509

THE PRODUCTION OF HORMONES IN HIGHER PLANTS

Bv A. R. SHELDRAKEDepartmentof Biochernistry, Jniztersity f Cambridge

(Receizted April rgn)

CONTENTS

I. Introduction Sro

IL The biochemistry of auxin production . 5ror. Animals, fungi and bacteria 5roz. Higher plants . Srz

3. The biochemical control of auxin production 5r5

4. Other indole compounds 5r8

5. Bound auxin 5rg6. The production of auxin by autolysing tissues S2z

III. Sites of auxin production in higher plantsr. Coleoptile tips

z. Young leaves, shoot tips and buds

3. Senescent leaves

4. Dicotyledonous seedlings

5. Stems

6. Roots

7. Flowers, fruits and seeds8. Cellular sites of auxin production

IV. Auxin production under pathological conditions

r. Fungal and bacterial infections

z. Animals

3. Viruses

4. Crown gall

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V.

VI .

VII.

Environmental auxin

Auxin and lower plants

The production of other plant hormones

r. Abscisic acid

z. Gibberellins

3. Cytokinins

4. Ethylene

VIII. The wound response

IX. The control of hormoneproduction and distribution

X. Conclusion

XI. Summary

XII. References

n n r 4 8



5ro A. R. Snnlonern

In order o understandro*thlr;)::ff""oilt:,1, is necessaryo analyse hatarein fact coherent,continuousprocessesnto a seriesof causes nd effects.A greatdealhas been found out about the effects of hormones on the control of growth anddifferentiation in plants; this has provided some understandingof the causesofdevelopmentalchanges.But the production of hormones s itself an effect whichrequires a causalexplanation.Something s known about the regionsof hormoneproduction n the plant and he way n which hormonesmove rom these egions.Butvery little is known about the cellular sitesof hormone productionor about he wayin which hormone production s controlled.Physiologicalnvestigations f hormoneproduction n whole organsor partsof organsdo not in themselves hedmuch lighton theseproblems. Biochemical nvestigations f hormoneproduction by tissuesorhomogenates f tissuescontaining a mixture of cells provide information about thebiochemistryof hormoneproduction under experimental onditions,but they do notrevealwhich cellsproduce he hormonesn,uizto.Unless he cellular sitesof hormoneproduction

areknown,

t is almost mpossibleo understand ow hormoneproductionwithin the plant is controlled at eithera physiologicalor biochemicalevel.The majority of this review s concernedwith the productionof auxin,aboutwhich

a vastand confusing iterature hasgrownup over the last forty-five years.The produc-tion of the more recentlydiscovered ormones, he gibberellins,cytokinins,abscisicacid and ethylene,will be consideredonly briefly. The results of biochemicalandphysiologicalnvestigations f hormone production will be discussedn an attemptto obtain a clearerunderstanding f the cellularsitesof hormoneproductionand of theway in which hormoneproduction s controlled.The major conclusion o which thisdiscussioneads s that much of the hormoneproduction in plants takesplace as a

consequence f cell death.

II. THE BIOCHEMISTRYF AUXIN PRODUCTIONThere is a greatdeal of evidencehat the naturalauxin of plants s indol-3yl-acetic

acid(IAA) (Thimann, 1969).Members of almosteverygroup of living organisms re

known to be capable f producing AA; it is formed by numerousspecies f bacteria

(Roberts& Roberts, 939;Stowe,1955;Wichner& Libbert, 1968) nd ungi (Gruen,

1959); t hasbeen ound n a varietyof animals Went & Thimann, rg37; Gordon &

Buess,ry6il and is produced n developingchick embryos Robinson& Woodside,

r%7). Considerablequantitiesare excreted n human urine, which is one of the

sources rom which IAA wasfirst isolated.The IAA in human urine is not simply

derived rom plant material n the diet, nor can more than a third of it be attributed

to auxin production by the microflora of the gut: the majority is actually ormed n

the human body (Weissbach,King, Sjoersdma& Udenfriend, 1959).The rate of

human auxin production, expressedn terms that permit comparisonwith auxinproduction in plants, is about 5-5ox ro-12g/*g/h. Coleoptile ips of Avenayield

5ox ro-1' gl^glh (Went & Thimann, rg37).This comparisonemphasizeshat IAA

production n plantsshould not be regardedas an isolatedbiochemical henomenon.



Theproduction of hormonesn higherplants 5 I I

t. Animals, ungi and bactuia

IAA is produced n animalsas a consequencef tryptophancatabolism. t is by nomeans he major breakdownproduct: for example, he increasedexcretionof IAA

which follows he oral administrationof tryptophan o humansaccounts or less han

o.ro/oof the tryptophan

metabolizedWeissbacht al., ry59).

The productionof

IAA

can occur as a resultof the transamination r the decarboxylation f tryptophan, but

the former is the predominant oute (Weissbach t al., ry59; Gordon& Buess,1967).

These reactions ield indolepyruvic acid and ryptaminerespectively Fig. r). Indole

pyruvic acidcanundergodecarboxylationo indoleacetaldehyde, hich is also ormed

by the action of amineoxidases n tryptamine.IAA is producedby the oxidationof

indole acetaldehyde.

The production of IAA by bacteriaand fungi which occurs when tryptophan is

added o the culturemedium ollowssimilarpathways.Although tryptophandegrada-

tion via tryptamine has been conclusivelydemonstrated,most bacteria and fungi

resembleanimals n the greater mportanceof the transamination oute (Libbert,

Erdmann & Schiewer, gTo).Again, it is important to bear n mind that IAA is only

one of several ossible roductsof tryptophan catabolism;or examplendole pyruvicacid s not only decarboxylated ut canalsobe reduced o indole acticacid;and ndole

acetaldehydes not only oxidized to IAA but can also be reducedto tryptophol

(Fig. r). These substances ave often been detectedn microbial cultureswhich are

degrading ryptophan e.9.Kaper & Veldstra,1958;Rigaud, g7oa,b). Only a small

proportion of the tryptophan supplied is converted o IAA: in cultures of Agro-

bacterium urnefaciens,or example, he maximum efficiency s less han zo/o(Kaper &

Veldstra,1958).

The way in which indole pyruvic acid is converted o IAA in animalsand micro-

organismss not fully understood. ndole pyruvic acid s a rather unstablecompound;

in aqueoussolutions, especiallyunder alkaline conditions, it breaks down spon-

taneously o give a numberof differentproducts, ncluding IAA, indole acetaldehyde

and ryptophol Bentley t a1.,1956;Kaper& Veldstra,1958;Moore & Shaner, y67).Againstthis backgroundof spontaneous egradationt is difficult to obtain evidence

for the participationof enzymes;and indeed here seemso be no reason o believe

that indole pyruvic acid does not decarboxylate pontaneouslyin ohso,or that indole

acetaldehydes not spontaneously xidized.Enzymicoxidationor reductionof indole

acetaldehydemay also akeplace,and there s someevidence hat both occur (Kaper

& Veldstra,1958;Rigaud,rg7oa, D).The relativecontributionsof enzymicand non-

enzymicprocesses re, however,difficult to assess. ryptophan can be converted o

IAA inoitro by incubating he aminoacidwith apurified ransaminasefromEscherichia

coli (Gunsalus& Stamer,1955) n the presence f a-keto glutarate an amino group

acceptor) ndpyridoxal phosphate the co-factornecessaryor transamination) ut in

the absencef any other enzymesA. R. Sheldrake, npublished esults).This ishardly

surprising n view of the instability of indole pyruvic acid, but it emphasizeshat, inoioa, IAA could be producedsimply asa consequence f tryptophan transamination,

although he yields might be increased y enzymes apableof decarboxylatingndole

32-2



A. R. Snrlonernyt,

<\-fcu,CHco,n\,4"/

512

<'\--11-cH2cH2NH2\,,\"/

H

T ransaminay/Tryptophan

\carboxvlase

o H o. I - ^ I I

a\yn-cgrCuco,tr t-.--- cH,dcortt

V\"1--'--7

\An/i i H

Indole lactic acid Indole pyruvic acid Tryptamine

\ /

4--t-T-.cH,cH2oH L <-\---1cH,cHo<-\"/ =:==--;\-\-/H H

Tryptophol Indole acetaldehYde

tCl-'-r-cH2co2H

\A*/H

IAA

Fig. r. Pathways of tryptophan degradation'

pyruvicacidandoxidizing ndoleacetaldehyde.onversely,he yieldsmight be dimin-

ishedby enzymeswhich ieduce hesecompoundso indole acticacidandtryptophol'

IAA is not known to play a hormonalor indeedany other role in animals, ungi or

bacteria. t is perhuptb.tt tegardedasa minor by-productof tryptophancatabolism,

although or organisms atholenic o plants t maybe of importancen influencing he

response f the plant to the pathogen.

z. HigherPlants

Numerous investigationshave shown that IAA is producedfrom tryptophan in

plant tissues for reviewsseeGordon, ry6r; Mahadevan,1964;Libbert et al', rgTo;

Wightman, 1973).Doubt was caston the validity of someof the earlier resultsby

Libbert et al. tigOOlwho showed hat under somecircumstances piphytic bacteria

could account or much of the auxin production by nonsterileplant tissues.The

finding that sterilecoleoptilesectionsof Atsena athtawhich grew in the presence f

IAA did not elongatewhen tryptophan was supplied (Winter, ry66; Thimann &

Growchowska, 9OAlseemed o provide further evidenceagainst ryptophan as a

precursorof IAA. In order to explain heir results,both Libbert et al. (1966)and

fuir,t", (1966)proposedhypotheticalpathwaysof IAA synthesis rom indole which

did not involvetryptoph"tt. Wittt.r's conclusionswerebasedon the assumptionhat

coleoptileissues"ot*"tty

synthesizeAA in the sameway asotherplant tissues'This



Theproduction of hormonesn higherplants 513

assumptions not valid, for it has ong beenknown that coleoptile ips havean ano-

malous auxin economywhich dependson a supply of auxin and/or 'inactive' auxins

from the seed; here s no evidenceo suggest hat denooo AA production occurs n

coleoptiles Section III, r). Therefore the inability of sterile coleoptile tissues oproduceauxin from tryptophan n sufficientquantities o stimulategrowth doesnot

support a generalargumentagainst he role of tryptophan as a precursorof IAA. In

fact, sterile coleoptile issuescan produce small quantitiesof IAA from tryptophan

(Libbert et al., 1968; Libbert & Silhengst, y7o; Black & Hamilton, r97r). The

ineffectiveness f exogenousryptophan as an auxin precursor n sectionsof.Aoena

coleoptilesappearso be due to its rapid incorporation nto proteins,preventing any

significant ncreasen the intracellular evelsof free tryptophan (Black& Hamilton,

ry7r). There is good evidence hat other sterile tissuescan convert tryptophan toIAA (e.g.Kulescha,gSz; Libbert et al. 1968;Sherwin& Purves, 969; Mitchell &Davies, rgTz).Evidenceagainsthe hypothetical athwayof IAA synthesisrom indole

without tryptophan as an intermediatehasbeen obtained by Erdmann & Schiewer

GgZr) and Black& Hamilton (r97r). Libbert et al. (tqZo) havenow concludedhat

IAA is, after all, formed from tryptophan n higher plants.Evidence or this view has

continued o accumulatee.g.Gibson,Schneider Wightman,rg72;Wightman, ry73).The predominantway in which plant tissuescatabolize ryptophan is by trans-

amination Libbert et al., ry7o; Wightman, ry73).As in other organisms,he produc-

tion of IAA occursasa resultof the breakdownof indole pyruvic acid. There is some

evidence halthis reaction can be catalysedenzymically,possibly by an oxidative de-

carboxylationanalogouso the oxidativedecarboxylation f pyruvic acid to acetyl-

coenzymeA. This evidencedependson the stimulatory effectsof thiamine pyro-

phosphate Gordon, 196r; Moore & Shaner,1968)and lipoic acid (Gordon, ry6r),which are co-factorsn other oxidative decarboxylations. here is also evidencehatindole acetaldehyde, hich can be detectedas an intermediate n the production ofIAA from tryptophanby the useof radioactive racersand/or rappingagents uch as

2,4-dinitrophenyl yd razine Phelps& Sequira,1967;Khalifah, ry67; Wightman&

Cohen, 968; Moore & Shaner,1968;Gibson,Schneider Wightman, rg72),maybeoxidizedenzymically.Aldehydedehydrogenasesapable f carrying out this reactionhavebeendetectedn a variety of plant tissues Rajagopal, 967;Wightman & Cohen,1968;Gibson,Schneider& Wightman, r97z),although he presence f theseenzymesdoesnot in itself prove that they are normally involved n IAA production. It is atpresentalmost mpossible o assesshe relativecontributionsof enzymicand spon-taneous eactions n the formation of IAA from indole pyruvic acid n ahto.

In their recent review, Libbert et al. (tgZo) critically examined he evidegce nfavour of the formation of IAA from tryptophan via tryptamine and concluded hatin higher plants the formation of tryptamine from tryptophan was unproven andunlikely. However, there seems o be no doubt that tryptamine can be detected n

some,but not all, plant tissues for references eeSchneider,Gibson & Wightman,

rgTz).Severalhypothetical pathways o account or its formation without the involve-ment of tryptophan havebeenproposed Libbert et al., rgTo\but there is now per-suasive videncehat somehigherplant tissuesdo in fact containenzymes apableof



5r+ A. R. Snnronern

TOMATO

Tryptophan121tg

/ \Tryptophan / \ TryptophantransamnaseT/

\ecarboxylasc

/ \Indole dctic uri63:s:=;Indole pyruvic acid Tryptamine0.005 g

--dARLEY

Tryptophan2opg

/Tryptophan

/transanlrnase /

4 4 /

V

Indole pyruvic acid

\ Tryptophan

\"'o;*ttu'"Tryptamine

2 us./ ' -

/ \ / " 'Tryptophol*=;Indole acetaldehyde

Tryptophol I tndo," acetatdehydedehydrogenase

I dehydrogenaset 7 J 8

IAA0.05pg

Fig. z. Pathways of IAA. formation. The numbers below the names of the indole compoundsrepresent the concentrations in untreated tissue in pglg fresh weightl numbers below the

names of enzymes represent their rates of activity as pg product/g fresh weight/h. (Gibson,Schneider & Wightman, tgTz).

converting ryptophan to tryptamine (Sherwin, rgTo; Gibson, Schneider& Wight-man, ry72; Gibson,Barrett& Wightman, gTz;Wightman, rg73).Some,but not all,plantscontainamineoxidaseswhich can converttryptamineto indole acetaldehyde.The most intensive nvestigationof theseenzymeshas been carried out with pea(Pisamsatioum) issues Mann, 1955;Clark & lVlann,rySil but unfortunately his isaspecies hich doesnot contain ryptamine Schneideret al., ry72).Gibson,Schneider& Wightman (1972)haveshown hat in tomato (Lycopersiconsculentum\nd barley(Hordeurnoulgare) issues,both of which normally contain tryptamine, tryptophancan be converted o tryptamineand this can n turn be converted o IAA. Enzymescapableof forming IAA from tryptophan via indole pyruvic acid are also present.

These authorshaveestimated he relativeactivitiesof the enzymesnvolved in thedegradationof tryptophan and also the naturally occurring amountsof tryptophanand its degradation roducts n these issues.Their results Fig. z) indicate hat thetryptaminepathway s relativelyunimportant.Further results rom the same abora-tory haveconfirmed hat in tomatoshoots he primary pathway or the production ofIAA from tryptophan is via indole pyruvic acid (Wightman, rgn).

A number of plants from a wide range of families have been found to contain

5-hydroxytryptamine, nown to animal physiologists s serotonin(Schneideret al.,r97z).In some, or example n the stinginghairsof nettles Collier & Chesher,1956),it hasa role in defence gainstanimals; n others ts function is unknown. In animals

5-hydroxytryptamine is formed by the decarboxylationof 5-hydroxytryptophan(Udenfriend,Titus, Weissbach& Peterson,1956)but in plants t is probably ormed

by the hydroxylationof tryptamine (Gibson,Schneider& Wightman, rg72).Tomatotissues ontainabout ive imesmore5-hydroxytryptaminehantryptamine Schneideret al., rgTz).

Indole acetaldehyde

II Indole acetaldehyde

I dehydrogenaset 1 0J

IAA0.012pg



Theproduction of hormonesin higherplants 5r5Tryptophan, in common with a number of other amino acids, can be degraded

in oitro by reactionwith phenolsunder alkalineconditions; he mechanismappears

to involvean oxidativedeamination y the quinonesormedby the oxidationof phenols

suchascatechol Gordon, 196r).The incubationof catecholwith tryptophanat high

pHs, and under lessalkaline conditions n the presence f phenolase, eads o theproduction of small amountsof IAA (Gordon & Paleg,196r; Gordon,

ryfi;Whit-

more &Zahser, t964i Wheeler& King, 1968).Gordon & Paleg 196r ) suggestedhat

this route of IAA production s probably of little significancen oiao because f the

compartmentalizationof the substrates n living cells, but they stressed ts possible

importance n macerated lant tissuesand during extractionprocedures.This point

hasalsobeenemphasized y Whitmore & Zahner 1964).

Wightman(rgZl) hasshown hat when abelledphenylalanines supplied o tomato

shoots,a number of breakdownproducts ncluding phenylpyruvicacid and phenyl-

aceticacid are produced.The major pathwayof phenylalanine egradation, ike that

of tryptophan, is by transamination; ndeedthe same ransaminases probably in-

volved.Phenylacetic cidhasweakauxin activityandoccursnaturally n tomatoshoots(Wightman, ry73).

3. Thebiochemical ontrol of auxinproduction

Plant tissues onvert ryptophan o auxin with efficiencies s ow as,or lower than,

thosefound in animals,bacteriaand fungi. In short-term experiments, AA rarely

accountsor even asmuch as o.r o/oof the tryptophan supplied.Tryptophan is con-

verted to auxin by auxin-requiring tissue cultures and can substitute for auxin as a

growth substancef supplied at concentrations bout a hundred timeshigher, mply-

ing an efficiencyof conversionof about r o/o(Kulescha, gsz). None of the enzymes

thought to be nvolved n the production of IAA from tryptophanhavehigh specifici-

ties. For example,a purified ' tryptophan transaminase' rom Phaseolusureus as.a

higher activitywith alanine, eucine,methionine,arginine, ysine,phenylalanineand

tyrosine han with tryptophan(Treulson, rgTz). A purifiedtryptophandecarboxylase

fromtomato shoots

smuch

more specific ortryptophan,but

thisenzyme s of littleimportance n the production of IAA (Wightman, tg73). An amine oxidase rom pea

seedlings apableof oxidizing ryptamine o indole acetaldehyde lsooxidizesa widerangeof monoamines Mann, 1955).And there s no evidenceo suggesthat enzymes

which are able to oxidize ndole acetaldehydeo IAA are specific or this substrate.

The unspecificnature of theseenzymes,he probability of spontaneous reakdownof

intermediates uch as ndole pyruvic acid and the low efficiencies f IAA production

from tryptophan suggest hat in plants, as n other organisms, AA is formed rather

unspecificallyas a by-product of tryptophan catabolism. t would be misleading o

think of the reactions eading to the production of IAA as a biosynthetic pathway

directly comparable o the efficient and specificenzymepathways nvolved in inter-

mediarymetabolismor in most biosyntheses. consequence f this view is that the

control of IAA production from tryptophan is not likely to involve any very specificregulatorymechanisms t the enzymic evel.

Severalattempts o explain he control of IAA production n this way have been



516 A. R. Snnronerp

made,but they arenot persuasive. ajagopal Larsen tgZz) purified an enzyme rom

non-sterileAztena oleoptile issueswhich catalysedhe conversionof indole acetal-dehyde o IAA. They found that the bestpreparations nder optimal conditionshada turn-over number of six moleculesof indole acetaldehyde/min/enzyme oleculeand suggestedhat this extremelysluggishactivity might be of decisive mportance

in the control of IAA biogenesis. his conclusionseemsmprobable f only for thereason hat coleoptile issuesdo not normally synthesizeAA. For similar reasons,

the findings hat gibberellicacid enhanceshe growthof non-sterilecoleoptile ectionsin the presence f tryptophan (Sastry& Muir, 1965)and that gibberellic acid has apromotive effecton the decarboxylation f tryptophan by homogenates f coleoptile

tissues Valdovinos& Sastry,1968) do not support these authors' conclusions hatauxin biosynthesiss regulatedby gibberellic acid. These attempts o demonstrateadirect effectof gibberellicacid on auxin biosynthesis temmed rom observationshatauxin production wasenhancedn organsstimulated o developby the applicationofgibberellins. For example, omatoes stimulated to develop parthenocarpicallybygibberellicacid produceauxin (Sastry& Muir, rg6l). But sodo tomatoesdevelopingparthenocarpically fter other chemical reatments Section III, 7), indicating that

auxin is probably produced as a consequence f fruit development.Similarly, theenhancedauxin productionby dwarf peasand Helianthu.s lants stimulated o grow

by gibberellins Kurashai& Muir, ry62)and n rosetteplantsof Centaurea timulated

to bolt by gibberellins Kurashai& Muir, 1963)seemsikely to be a consequence f

the developmental hanges rought about overa period of days ather han asa direct

effectof gibberellicacidon auxinbiosynthesis.t is thereforedifficult to evaluate he

significance f the finding that enzymepreparations rom pea issuespretreatedwithgibberellicacidproducedmore ether-insolubleauxin'from tryptophan han controls(Muir, ry6+).Valdovinos & Ernest (1966) ound that homogenates f plant tissues

which had beenpretreatedwith gibberellicacid n a detergent olution releasedmoreuCOz from [r-1aC]tryptophanhan controls; but even if this effectwas due to the

gibberellicacid ather han he detergent,ts relevanceo the controlof IAA production

is far from clear.The widespread ccurrence f enzymes apable f catabolizing ryptophan with the

consequent roductionof IAA in animals, ungi, bacteriaand higher plantssuggests

that the major factor controlling the production of IAA is the availability of trypto-

phan; many cells and tissuesdo not produce IAA in significant quantities unless

exogenous ryptophan is supplied. But all cells capableof protein synthesismust

contain a pool of free tryptophan. Therefore it is probable hat theseendogenous

levels of tryptophan are normally too low for tryptophan breakdown o occur. This

suggests hat the affinities of the tryphophan-activating enzymes (responsible for

charging specific ransfer RNAs) arehigher than the affinitiesof tryptophan-degrading

enzymesor tryptophan. The few availabledataon Michaelis constants i.e. the sub-

strateconcentrationat which an enzyme s half saturated) upport this view: amino-

acid-activating nzymesgenerallyhaveK-s between x ro-o and r x ro-4 ivr Novelli,1967),while the tryptophan ransaminase f Phaseolusureas as a K*of 17 x ro-3 M

accordingo Gamborg& Wetter (tg6:) and3'3 x ro-4 wraccordingo Treulson(tgZz).



Theproduction of hormonesin higherplants 5r7Tryptophan transaminase nd tryptophan decarboxylaserom tomato shoots haveK-s of 5 x ro-a vI and 3 x ro-s rvr espectively Gibson,Barrett & Wightman, ry72).

The pathwaysof amino-acidbiosynthesis ave been studied most extensivelynmicro-organisms. n general, hey are controlledby feed-back nhibitions wherebythe amino acidwhich is the product of the pathway nhibits an enzyme, r enzymes,involved in its production, often at the beginning of the pathway; this inhibitionoccurswhen the concentration isesabovea certain evel (Umbarger, ry6g). Similarcontrol mechanisms ave been ound to regulateamino-acidbiosynthesisn higherplants (Miflin & Cave, rg72; Miflin, rgn). In micro-organisms, ryptophan bio-synthesis s regulatedby the inhibition of anthranilatesynthetase, n enzymeat thebeginning of the pathway,by tryptophan (Umbarger, 1969).There is evidence hattryptophan is synthesized y a similar pathway n higher plants (Delmer & Mills,1968)and it seems easonableo assumehat the control mechanismmay be similar.Tryptophan biosynthesismust be regulated n such a way that levelsof tryptophansufficient or protein synthesis re maintained,but theseconcentrationsmust be toolow for tryptophan degradation o occur under most circumstances;otherwise allplant tissueswould produceauxin all the time.

It is conceivablehat auxin productioncould beregulated ya change n this controlmechanism such that higher concentrationsof tryptophan were synthesized.But asimpler way in which the tryptophan evelscouldbe elevated s by the degradation fproteins.The freeamino-acidpools n plant tissues epresent nly a small proportion,often ess han So/o, f the total amino acidswhich canbe released y the hydrolysisof the proteins Allsop, 1948;McKee, 1958). n living cells,where here s a steadyturnover of proteins,proteolysispresumablycontributes o the steady-state ools offree amino acids.But when net protein degradation ccurs, or example n senescentleaves, he levels of free amino acids are elevatedconsiderably Chibnall, r939iMcKee, 1958).

The amountsof free ryptophan n plant issues sually ie in the rangeof ro-5o trglgfresh weight (Schneideret al., rgTz).Higher amountsare found in shoot ips, young

leaves, enescentotyledons Nitsch & Wetmore, tg5z) and senescenteaves Kim &Rohringer,tg6g),allof whicharesitesof auxinproduction Section I). Thesemeasure-ments give no information about the cellulardistribution or intracellular ocalizationof tryptophan. It is therefore mpossible o deduce he intracellularconcentrations.

The elevatedryptophan evels n senescenteaves nd n the cotyledonsof seedlingscanbe explained saresultof proteindegradation. he elevated mountsof tryptophanper unit weight in shoot tips and developing eavescould in part reflect the higherratio of cytoplasm o cell wall material n young issues,and in part be explainedbythe net protein breakdownwhich occursduring vasculardifferentiation:xylem cellsand most ibresundergoacompleteautolysisas hey differentiate, nd partial autolysisoccurs n differentiating ieve ubes.

Many o-amino acidsare convertedby plant tissues o their N-malonyl derivatives

(Rosa& Neish, 1968)by what appearso be a detoxificationmechanism. n analogousconversionof o-amino acids o their N-acetyl derivativesoccurs n yeast (Zenk &Schmitt, 1965).Malonyl-o-amino-acidconjugates ccur naturally n a wide rangeof



- -

5r8 A. R. SHnronern

<t\--T-.cH,co2H\A"/

H

IAA

H

Glucobrassicin

{

I l l l l +Glucose\,A*/ +so12-

ii +scN-

Ascorbic z 3-Hydroxymethyl

acid4/

indole

Ascorbigen I

J<i----"-cH,----At l t i l i l i l tV-*/ \*A/

H H

3,3'-diindolylmethane*formaldehyde

Fig.l.

The degradation of glucobrassicin (after Gmelin, 1964\.

plants(Rosa& Neish,1968)andmalonyl-o-tryptophan asbeendetected n a numberof vegetative issuesand fruits (Good & Andreae, rgST; von Raussendorf-Bargen,

ry62; Zenk& Scherf, rg6l). Apple (IlIaluspumila) ruits containabouto.ztrglg freshweight (Zenk& Scherf, g6:) andtomato shootsabouto.Spglg freshweight(Good &Andreae, rg1il.This compound appears o be quite stable n plant tissues Good& Andreae, ry57). It is possible hat in autolysing issues t could be hydrolysed,releasing ree o-tryptophanwhich can be brokendown by someplant tissues o IAA(e.9. Kim & Rohringer, 1969).However, he amountsof malonyl-o-tryptophanaresosmall comparedwith the amountsof L-tryptophan n plant tissueshat it is unlikely tobe of any significance or auxin production.

4. Other indole compounds

A number of plantshavebeenused or centuriesasa sourceof indigo dye,which isformed by the oxidation and polymerization of indoxyl. Indigofera and Polygonumtinctoriumcontain ndoxyl-p-o-glucoside nd woad (Isatistinctoria)contains ndole-

B-o-5-ketoglucuronic cid (Stowe,Vendrell& Epstein,1968). ndoxyl is releasedromthesecompoundsby hydrolysis.Little is known about the indole metabolismof theplants which produce hesecompounds,but there is no reason o suppose hat theyare precursorsof auxin or play any part in growth control.

A variety of mustard oil glucosides, r glucosinolates, re found in the Cruciferae,Resedaceae, ropaeolaceae nd certain other families. They appear to be stablein oivo, but when tissuesarecrushedor otherwisedisrupted hey rapidly breakdownunder the influenceof the enzymemyrosinase, hich is probably

confined o specialcells (Virtanen, 1965).This reaction s of culinary importance n the preparationofmustard and is alsoresponsibleor the release f the compoundswhich give plants




suchascress Lepidiumsatirrum)and Tropaeolwntheircharacteristic lavours Virtanen,

1965).Brassica nda numberof othergenera ontain he indole-glucosinolatesluco-

brassicin and neoglucobrassicin. hese compoundsare present in both roots and

shoots n quantitieswhich canaccount or up to lo/o of the dry weight (Gmelin, W64;Virtanen, 1965;Elliott & Stowe, ry7r). Either spontaneously nder acid or alkaline

conditions,or as a result of the action of myrosinase, lucobrassicin reaksdown toform a variety of indole compounds Fig. 3), the nature of the productsbeing deter-mined by the conditionsof the reaction(Gmelin, lq,6+;Virtanen, 1965).One of the

breakdown products, 3-hydroxymethyl indole, dimerizes with the elimination offormaldehyde,but if ascorbicacid is present reactswith it spontaneously o form

ascorbigen.The pH optimum of this reaction is 5 (Schraudolf & Weber, ry69).Indole acetonitrile s produced enzymicallyby myrosinase,but only if the pH is

below 5'z (Schraudolf& Weber, 1969).Reportsof the occurrenceof indole acetoni-

trile and ascorbigen n tissuesof Brassicawere madebefore the discovery of gluco-

brassicin,but thesecompoundsare now known to have arisenasextractionartifacts.Little or none can be detected after extraction procedures hat minimize the break-down of glucobrassicin Kutddek& Prochizka, ry64; Schraudolf & Bergmann,

r965).Indole acetonitrile orms indole acetamide pontaneouslynder acidic conditions;

indole acetamide anbe hydrolysed o IAA. The conversionof indole acetonitrile oIAA is alsoeffectedby the enzymenitrilase which is present n some, but not all,plant tissues Thimann, 1953). ndole acetonitrilecan, if supplied o tissueswhichcontain nitrilase,act as an auxin precursor; but there is no evidence hat it has any

such role in oizto.The hypothesis hat indole acetonitrilewas involved as an inter-

mediate n auxinproduction (e.9.Wightman, tg6z) depended n the belief hat it wasof natural occurrencen species ow known to containglucobrassicin.

The functions of glucobrassicin nd neo-glucobrassicin,ike thoseof many othersecondaryplant products, are unknown. There is no evidence hat under normal

conditions hey act as auxin precursorsor are involved n the regulationof growth.

But since hey break down when tissuesare crushedor damaged, t is possible hatauxin could be produced rom glucobrassicin nder pathological onditions.Gmelin(rg6+) has suggested hat a processof this sort might explain the existence n the

cabbageamily of growth abnormalities imilar to those nduced by exogenous uxin.

Some support for this view comes rom the finding that in club-roots of Brassica,

causedby the pathogenPlastnodiopherarassicae,ndole acetonitrile s present,where-as t is not detectablen uninfected oots (Tamura, Nomoto & Nagao, rgTz).

5. Bound a:uxin

It has ong beenknown that animalspossess variety of detoxifying mechanisms

which involve the formation of amino-acid or sugar conjugates.Benzoicacid, for

example, s conjugatedwith glycine o form benzoylglycine(hippuric acid)which is

then excreted n the urine. In humansand chimpanzees lutaminecomplexes re also

formed (Thierfelder& Sherwin,r9r4). A wide rangeof compounds ncluding phenyl-

acetic acid and various substituted benzoic acids are converted to glycosidesof



52o A. R. Supronerp

o-glucuronic acid (Teague, 1954). IAA complexes from which IAA can be released

by acid hydrolysis are present in human urine (Weissbachet al., ry59) and in Hartnup

disease,which is associatedwith a greatly elevated excretion of IAA, up to r5o mg/day

of IAA-glutamine is found in the urine (Jepson, 1956).

Similar detoxification mechanisms occur in plants. The administration of unphysio-

logically large amounts of IAA results in the formation of IAA complexeswhose nature

depends on the speciesof plant. fn some only IAA-r-aspartic acid is formed, in others

only IAA-p-o-glucose; but in most species both these compounds are produced

(Zenk, ry6+). Indole acetamide, which was previously thought to be formed from

IAA as a detoxification product (Andreae & Good, rgST) s now known to have arisen

as an artifactby the breakdown of IAA-glucose during chromatography in ammoniacal

solvents (Zenk, 196r). A number of synthetic auxins are converted by the same

mechanisms to aspartic-acid and/or glucose derivatives (Andreae & Good, rgST;

Zenk, y62;Stidi, y6+).'IAA-glucose can be detected soon after the application of exogenous auxin to leaf

discs of Hypericurn hircinum, but IAA-aspartate begins to appear only after a lag

period of about zh at zS"C (Zenk, ry6+).The levels of IAA-glucose decline as

IAA-aspartate is formed. The lag period preceding the formation of IAA-aspartate

can be eliminated by pretreating tissues with IAA or synthetic auxins such as

naphthalene acetic acid (Andreae & Ysselstein, 196o; Stdi, ry6+). This effect is

prevented by inhibitors of protein synthesis (Venis, r97o), suggestingthat it is due to

enzyme induction.

One day after the administration of IAA to Hypericum tissues, the majority of the

IAA taken up by the tissue is found to have been converted to IAA aspartate (Zenk,

ry6+). A small proportion of the IAA remains in a free form at a steady-state evel of

about 6 pSlS fresh weight (Zenk, ry6+).This is well above the normal levels of IAA

encountered in vegetative tissues, which usually lie in the range of o'oo5-o'o5o pglg

fresh weight (for references see Schneider et al., rg72).The fact that neither IAA-

aspartate nor the enzyme system which forms it are normally detectable in untreated

vegetative tissues indicates that the endogenous auxin levels are too low for this

conjugation of auxin to occur under physiological conditions. There are, however,

times and situations in plants where relatively large quantities of auxin are produced,

for example during seed development, and the conjugation of auxin may be of con-

siderable physiological importance under these circumstances. A number of dicotyl-

edonous seeds and fruits have been shown to contain IAA-aspartate (Klambt, 196o;

von Raussendorf-Bargen, ry62; Zenk, ry6+) and it has been known for many years

that large quantities of bound auxin are produced during the development of cereal

grains.

In developing rye seeds here is at first a large increase n the amount of free auxin

followed by a decline associatedwith the formation of substances from which auxin

can be releasedby mild alkaline hydrolysis (Hatcher & Gregory, r94r ; Hatcher, 1943),

suggesting that an ester linkage is involved. Developing maize kernels contain bothfree and bound auxin; the levels of free auxin decline as the seed matures (Avery,

Berger & Shalucha, rg4z; Hemberg, 1958; Hamilton, Bandurski & Grigsby, 196r).



Theproduction of hormonesn higherplants 52rAbout half the bound auxin of maizeseedsconsistsof low molecular weight esterswhich have been identified as IAA-inositol complexes Ueda & Bandurski, 1969).Both lipid-soluble and high molecular weight esters are also present (Takano,Bandurski& Kivilaan, tg67; Ueda & Bandurski,1969).Hemberg rgSS)suggestedthat the bound auxin formed during seed developmentservesas a source of free

auxin in the germinatingseed. t is on the auxin released y the hydrolysisof theseauxin 'precursors' that the vicarious auxin economy of the coleoptile ip depends(Section II, r).

Auxin is not the only hormonewhich is converted o a bound form. Glucosidesofabscisicacid (Milborrow, rgTo) and gibberellicacidsoccur naturally n plants. Thelatter are formed during seeddevelopment rom free gibberellic acids; these, ikeauxin, are iberatedby hydrolysis rom their bound forms in germinatingseedsandembryos Barendse,Kende & Lang, 1968; Barendse, gTr; Dale, ry6g).

For many years the possible existenceof auxin-protein complexeshas beenshrouded n confusionandcontroversy. he release f auxin from proteinsby alkaline

hydrolysisor by proteolyticenzymes asoften been akenasevidence or the existence

of protein-bound auxin. But resultsof this type can alsobe explainedby the release

of tryptophan, followed by the conversionof tryptophan to IAA. This conversion

takes place spontaneously nder alkaline conditions (Gordon & Wildman, rg$).Auxin is producedby the alkalinedigestionof casein,but not of gelatinewhich con-

tains negligible amounts of tryptophan (Gordon & Wildman, ry$). Wildman &Bonner (rg+il found that auxin was eleased y the hydrolysisof spinach eafprotein

in quantities greater han they estimatedwould be produced by the spontaneous

conversion f tryptophan; they concluded hat an auxin-proteincomplexwaspresent.

However,Schockenrg+g) showed hat Wildman & Bonner'sassumptions bout therate of spontaneous roductionof auxin from tryptophan were wrong. He re-investi-

gated he productionof auxin rom the spinach auxin-protein' and ound that similar

yields of auxin were obtainedby the hydrolysisof a number of animalproteins; the

amount of auxinproducedwas oughlyproportional o the tryptophan content of the

protein.Wildman & Bonner's videncen favourof the'auxin-protein'alsodepended

on enzymicdigestions, ut the incubationconditions +8h at 37oCunder non-sterile

conditions)make t probable hat much of the auxin detectedwas ormed by micro-

organisms.

Winter & Thimann (1966)claimed hat part of the IAA which is immobilized in

coleoptile issuesduring polar auxin transport is in the form of an auxin-protein

complex. The majority of labelled IAA present n the tissues at the end of the

transport period was found in the sedimentwhen ground-up coleoptile issue was

centrifuged.Radioactivitywasreleasedrom this fraction by treatment with proteo-

lytic enzymesor urea. However, the binding of IAA wasvery labile sinceIAA could

be recoveredby extraction with ether. These authors were apparently unawareof the

ability of IAA to partition into lipid membranesn a pH-dependentmanner(Hertel,Thomson & Russo, rgTz) much in the sameway that IAA partitions nto non-polar

solventsat pHs below about 5. Their experimentsnvolved either unbufferedsolu-

tions, or a comparisonof various treatments n solutions buffered at different pHs.



522 A. R. SHnr-onexn

Their resultsmay thereforebe explicablen terms of a pH-dependentassociation f

IAA with membranes.

Siegel& Galston(rgSg) claimed o havedemonstrated he formation of an IAA-

protein complex n aitro by plant homogenates. he binding of IAA to protein was

stimulatedby nucleotides. heseresultsare now known to havebeendue to an arti-

fact causedby the use of tri-chloro-aceticacid as a protein precipitant Zenk, 1964).Evidence or the formation of an IAA-transfer RNA complex Bendana t a1.,1965)

has alsobeen efuted(Davies, rgTr).In a recentpublication rom the sameaboratory

theformationof IAA-polysaccharide omplexes asbeenpostulated Davies& Galston,

ry7r), but the evidence or their existences indirect.

Zenk (tg6+) has provided the only convincing evidence or the existenceof anIAA-protein complex. A small proportion of labelled AA supplied o pea epicotyl

tissueswasound to be bound o a protein raction romwhich it couldnot be removed

by treatmentwith acetone, ialysisor by exchange ith unlabelled AA. These esults

suggest hat the IAA waschemicallybound to the protein. It is not known whether

this IAA-protein complexhas any physiological ignificance, r indeedwhether t is

formed at all under natural conditions.

In extractionprocedures,free'

auxin is taken o be the auxin obtainedafter shortperiods in the cold, whereas he additional auxin obtained after longer periods ofextractionhas beenconsideredo be due to the release f free auxin from a 'bound'

form (Bentley, 196r). This definition of bound auxin is confusing. n some cases,for example n the extraction of cerealseeds e.g. Hemberg, 1955)and coleoptiles(Wildman & Bonner,1948) his bound auxin represents enuine AA complexes uchas the lAA-inositol compounds.The free auxin which can be liberated from the'bound auxin' of Brassica issues e.g. Avery, Berger & White, 1945) s probablyformed as a consequence f the breakdown of glucobrassicin.n other cases he'bound auxin' represents AA produced from tryptophan during extraction. Theadditionof proteolyticenzymeso tissues eads o largeryields of IAA during extrac-

tion (Skoog& Thimann, r94o; Thimann, Skoog & Byer, ry42) probably because

more tryptophan s releasedrom proteins.No auxin production occurswhen boiledtissuesare subjected o prolongedextractionwith ether (Thimann & Skoog,r94o).

6. Theproductionof auxin by autolysing issues

The continuedproductionof auxin by plant tissuesas hey autolyse uring extrac-

tion with ether s probablydue o the continued elease f freetryptophanfromproteinsand its subsequent egradation.But it is not only during etherextractions hat auto-lysis results n auxin production. Yeast,plant and rat liver tissuesproduce auxin as

they autolyse n oitro (Sheldrake& Northcote, ry68a). fncreases n the amount of

auxin of up to a hundredfoldcanoccur within z+h.The production of auxin by autolysing tissues n aitro suggests hat autolysing

tissueswithin the plant might alsoproduceauxin. But it would be almost mpossible

to duplicate in the test tube the complexsequenceof changes hat occur duringthe autolysisof a differentiating xylem cell, to takeonly one example. The pH of the

different cellular compartments may change as differentiation proceedsand as the



Theproduction of hormonesn higherplants 523intracellular membranesbreak down; the concentrationsof tryptophan would be

expected o changeas it is released rom intracellularcompartments e.g. plastids)

and the amount producedby proteolysiswould be affectedby the changingcompart-

mentalization of proteolytic enzymesand he changingpH in which they are operating.

These factors would also affect enzymeswhich break down tryptophan. It is also

probablethat someof the substanceseleased s the cell autolyseswould be meta-

bolized by adjacent iving cells.

The techniquesof biochemistry are at present oo crude to investigate n any detail

changes f this type as hey occur n ztizto. rom a biochemicalpoint of view it seems

probablethat autolysingcells would produceauxin; but biochemical nvestigations

alone cannot reveal the extent and significanceof auxin production by such cells

within the intact plant. The problem must now be consideredat the physiologicaland anatomicalevels.

III. SITES OF AUXIN PRODUCTION IN THE PLANT

With the exceptionof senescenteavesand coleoptile ips, practicallyall the sites

of auxin production n the plant are n regionsof meristematic ctivity. This correla-

tion betweenauxin production and meristematicactivity has led to the widely, ifimplicitly, accepted ypothesishat auxin is producedby meristematiccells. A very

different hypothesisof auxin production has recentlybeen proposed,according owhich auxin is produced as a consequence f cell death (Sheldrake& Northcote,

1968 , b). The production of auxin by autolysing issues n oitro and by senescentleavesn vivo indicates hat dying cellscanproduceauxin; the sitesof auxin produc-

tion in the plant canbe explainedby the association f regionsof meristematic ctivitywith the cell deathswhich occur as nutritive tissues egressand asvascular issuesdifferentiate. n the following sectionshe sitesof auxinproduction will be discussedwith thesehypothesesn mind.

The best reviewson the productionand distribution of auxin in the plant are by

Siiding (tg;z, 196r) but these, ike many of the publications n this field, are in

German.This importantand nteresting iteratureseemso be ittle known n English-

speakingcountries.Investigationson auxin in plants have dependedeither on the use of extraction

techniques, free' auxin being akenas hat obtainableby short periodsof extractionin the cold, or by the trapping of auxin diffusingfrom plant patrs n agar. The dis-advantage f the diffusionmethod s that it dependsnot only on the amountof auxinin the tissuebut alsoon the auxin transportsystem; he disadvantage f extractionis that it may give a distorted picture of the amount of auxin availableas a hormone,sincesome may be immobilizedor compartmentalized ithin the tissue.

r. Coleoptile ips

Auxin was clearly identifiedas a plant hormone n the classicalnvestigations fWent (1928)on the growth and tropisms of the coleoptiles f grassseedlings.Sincethat time the coleoptilehas continued o occupy a centralposition n auxin research.



52+ A. R. Snnlonern

The classicalwork on coleoptiles, summarized by Went & Thimann (1937), established

that the coleoptile tip is rich in auxin; that the growth of the coleoptile depends on

a supply of auxin from the tip; that the auxin is transported basipetally from the tip in

the polar auxin-transport system; and that tropic movements of the coleoptile could

be explained by the asymmetric supply of auxin from the tip.

The decapitation of coleoptiles results in a cessation of growth; but after a fewhours growth is resumed as a result of the'regeneration of the physiological tip', a

phenomenon whereby the apical region of the coleoptile stump becomes a source of

auxin (Went & Thimann, rg37). This shows that the role of the coleoptile tip as an

auxin source does not depend on any special anatomical features of the tip itself, but

rather on its position at the apex of the coleoptile. The removal of the seed results in

a decline in the amount of auxin in the coleoptile tip and also prevents the regeneration

of the physiological tip (Skoog, r%7).The removal of the roots also results in a

decline in the amount of auxin in the tip; the removal of both the seed and the roots

results in a further decline (van Overbeek, r%il.These results indicate that the auxin

economy of coleoptile tips depends on a supply of a substance or substances rom the

seed and that the roots are involved in this process in some way.

Germinating cereal seeds are rich in auxin and in bound forms of auxin, mostlyIAA esters Section II, 5). Pohl (t935, r936) showed that a depletion of the amount

of auxin in the seedresulted in a decline in coleoptile growth. He suggested hat auxin

from the seed moved acropetally in the vascular tissues and accumulated at the

coleoptile tip. This conclusion was rejected by Skoog (tgll) who was unable to detect

auxin in agar blocks placed on the apical cut surfaces of coleoptiles. He postulated

that a precursor of auxin moved from the seed o the coleoptile tip where it was con-

verted into auxin. Van Overbeek (tg+r) and Wildman & Bonner (tg+8) found that

excised coleoptile tips yielded several times more auxin by exhaustive diffusion into

agar than could be obtained from tips by extraction immediately after excision. Their

conclusions that coleoptile tips actually produce auxin depended on the questionable

assumptions that diffusion and extraction were equally efficient and that no bacterial

auxin production occurred during the prolonged diffusion periods under non-sterileconditions. However, even if appropriate corrections are made to their results, they

still indicate that up to four times more auxin can be obtained by diffusion than by

extraction (Sheldrake, rg73).

This production of auxin in coleoptile tips was interpreted as being due to the

conversion of an auxin precursor supplied by the seed. Skoog (rgSZ) showed that

tryptamine could produce curvatures in coleoptiles similar to those induced by auxin

but after a delay of several hours and concluded that it could act as an auxin pre-

cursor. There is, however, no evidence that it doesso inoizto. Raadts & Soding (tgSZ)

found that both IAA and a labile substance which could undergo spontaneous con-

version to IAA could be detected in coleoptiles. Chromatographic investigations of

diffusates rom coleoptile tips by Ramshorn (r955), Bohling (rgSg) and Shen-Miller

& Gordon (1966) also showed that, in addition to IAA, at least one other compoundwith auxin activity in the coleoptile extension bioassaywas present. This compound,

called P by Shen-Miller & Gordon, is not transported in the polar auxin transport




system and is therefore inactive in the coleoptile curvature bioassay. Shen-Miller &

Gordon (1966) showed that P was apparently converted to IAA by mild heat treat-

ment and also that P and another compound with auxin activity in the coleoptile

extension bioassay were converted to IAA in coleoptile tips. In some experiments the

amounts of IAA increased considerably while the total amount of auxin activity in the

coleoptile tips determined by the extension bioassay remained constant or evendeclined. By this criterion no auxin production could be said to have occurred; but

the curvature bioassay, which detects only IAA, would indicate that auxin had been

produced. Van Overbeek (tg+r) and Wildman & Bonner (tg+8) used the curvature

bioassay; the auxin production they observed can therefore be equated with the

production of IAA.

The substance in coleoptile tip diffusates identified chromatographically as IAA

has recently been conclusively identified as IAA by mass spectrometry (Greenwood

et al., rgTz).

The chromatography of ether extracts of germinating maize seeds Hemberg, 1958)

reveals the presenceof IAA and other zones of auxin activity similar to those found

in ether extracts or diffusates from coleoptile tips (Bohling, rySg; Shen-Miller &

Gordon, ry66). If P and these other compounds represent precursors of IAA inthe coleoptile tip and are also present in the seed, their movement from the seed to

the coleoptile tip could explain the classical results on the auxin economy of

coleoptile tips.

Guttation fluid from coleoptiles contains both IAA and other forms of auxin, in-

cluding P and esters of IAA (Sheldrake, 1973).A similar pattern of auxin activity is

found in the guttation fluid from primary leaves and from decapitated coleoptiles,

showing that auxin is present in the xylem sap and is not merely eluted from coleoptile

tips as guttation takes place. These results indicate that IAA and auxins inactive in

the curvature bioassay (but active in the extension bioassay) move acropetally in the

xylem from the seed. It can be shown by the use of dyes or radioactive IAA that

substancesmoving in the xylem accumulate at the tips of coleoptiles, or indeed at the

tips of the veins in any organ (Sheldrake, rg73).In decapitated coleoptiles substancesaccumulate at the apical part of the stump, although they are not detectable in agar

blocks placed on the apical cut surface. This finding refutes Skoog's (tglil evidence

against the acropetal movement of auxin.

The auxin economy of coleoptile tips can be explained as follows (Sheldrake, 1973):

both free IAA and'inactive'auxins move acropetally n the xylem from the seed o

the coleoptile tip, or to the physiological tip of decapitated coleoptiles, where they

accumulate. This process is affected by transpiration and root pressure, which may

account for the influence of the roots on the amount of auxin in coleoptile tips. Both

the free IAA and IAA released from'inactive'

auxins in the coleoptile tip can then

be transported basipetally in the living cells of the coleoptile where it controls exten-

sion growth. The accumulation of auxin at the apical limits of the xylem could result

in an asymmetry of auxin distribution since the anatomical distribution of the xylemin the extreme tip of the coleoptile is asymmetrical (Thimann & O'Brien, 1965). Such

an asymmetric accumulation of auxin would account for the autonomous curyature

r n n 4 8



526 A. R. Snnronern

of coleoptileswhich is observedwhen Aoena seedlingsare grown on a horizontal

clinostat(Pisek, 19z6; Lange, ry27).The major unresolvedproblem is the identity of the 'inactive' auxins. Obvious

candidateswould be the lAA-inositol estersound in such argequantities n the seed.

Perhaps ompounds uch as P detected fter chromatographyn ammoniacal olvents

representdegradationproducts of IAA esters, ormed either during the extraction

procedures r as a result of ammonolysis.

The production of auxin from 'inactive' auxin in the coleoptile ip was referred oin the earlier iterature as auxin actioation,a term which emphasized he differenceof

this process rom the de nouo synthesisof auxin which takesplace n other parts of

plants. However in more recent,progressively implified, accountsof this classical

work, the production of auxin in coleoptile ips is describedas auxin synthesise.g.Bonner& Galston, gsz; Leopold, y6+).The vicarious atureof the auxineconomy

of coleoptile ips is thus obscured nd his has esultedn considerable onfusion.For

example, he fallacious onclusions f Winter (1966) eferred o on p. 513 depended

on the assumptionhat the coleoptilewasa typicalsiteof auxinbiosynthesis. roblems

of this sort disappearwhen t is rememberedhat there s no evidenceor the de novosynthesisof IAA by coleoptiles n oioo.

z. Young eaaes, hoot ips and buds

Avery (rgSS)wasamong he first to recognize he generalpatternof auxin produc-

tion by developing icot leaves;n hisstudieson Nicotianahe oundthat "auxin is

presentonly in growing leavesand that its concentrations roughly inverselypro-

portional to the age of the leaf". Similar results havebeen obtainedwith leavesofPhaseohn Shoji, Addicott & Swets,r95r; Humphries & Wheeler, ry6+; Wheeler,

1968),Solidago Goodwin, 1937),Aster (Delisle, rg37), Coleus Jacobs& Morrow,

rg1il andfronds of OsmundaSteeves Briggs,196o).Auxin is produced hroughout

the period of leaf development n the basal meristematic egion of the leaf of the

unifoliate dicot, Streptocarpuswendlandii,while very little is present n the mature,

apicalparts of the leaf (Hess, 1958).Developingmonocot eavesalsocontain

moreauxin in the basal meristematic egion than in the rest of the leaf (van Overbeek,

r938).

Leaf development nvolves both meristematicactivity and vasculardifferentiation.

Jacobs& Morrow (tgSZ) found a close correlation betweenauxin production and

xylem differentiation in Coleuseavesand interpreted this as showing hat the differ-

entiation of xylem is controlledby auxin; but the resultscould also ndicate hat auxin

is produced as a consequencef xylem differentiation: hese nterpretationsare not

mutually exclusive.DevelopingNicotianaleaves ontainmoreauxin n the veins hanin the lamina (Avery, 1935)which might indicate hat it is produced n the differ-

entiatingvascular issue.

It is well known that relatively arge amountsof auxin are producedby shoot tips

(Thimann & Skoog, 934i du Buy & Neurnbergk,1935;Delisle,ry37; S<iding, 938;Eliasson, g6g) and developing uds (Czaja, g3+; Zimmerman, 1936;Sdding, rg37;

Avery, Burkholder & Creighton, g37; Gunckel & Thimann, 1949; Diirffiing, 1963).



Species

Phaseolus uulgais

Cucurbita pepo

Phaseolus aulgaris

Aaena satiaa

Age of attached leaves(days)

r 9

3 340

4o (shrivelled

leaves)

Days after detachment

of leaves

o

I I

Theproduction of hormonesn highn plants

Table r. Theproduction f auxinby smescenteaoes

Free auxin content,pg IAA equivalents/kg

fresh weight

e )

6 3 1, q6|

3 3 z l

527

Author

Wheeler t968)

Conrad rq6S)

Sheldrake&Northcote (r968c)

o.sI50 1

,; \

,17

o

6o

+

The tissuesused in all these nvestigations ncluded not only the meristem but also

the submeristematic egion and young leavesand therefore containedboth meri-stematic cells and differentiating vascular issue.

3. Smescenteaoes

The production of auxin by young, developing eavesdeclinesas he leavesmature

(seeabove).Shoji et al. (r95r), on the basisof a singlemeasurement f the auxin evel

in senescenteavesof Phaseolus sulgares,oncluded that a further decline occurred

during leaf senescence. his conclusion appears o have been widely accepted,

especially y workerson leaf abscissione.g. Addicott, rgTo).But there is now con-

siderableevidence hat auxin is produced as leavessenesce. his effect has been

observed n Bryophyllummmaturn Raadts, g6z), Cannabis Conrad, ry62), Cucurbita

PePo Conrad, 1965),AcerpsatdoplatanusDcirffiing,

1963),PhaseolusulgarisShel-

drake & Northcote, 1968c;Wheeler, 1968),Aoena sativa (Sheldrake& Northcote,

1968 ),HeaeabrasiliensisChua,ry7o)andPrunus erasusKaska, 97z).The increases

in auxin levels are arge,often from 30- to a roo-fold (Table r). The treatment of

detachedeaveswith kinetin, which retards heir senescence,esults n a suppression

of auxin production (Conrad, 1965).The production of auxin during senescence

could account or the f indings that Bryophyllurnplantscontainhigh levelsof auxin in

the autumn(Raadts, y62)and hat auxin evelsncrease onsiderablyn plantskept in

darknessor protractedperiods von Guttenberg& Zetsche,1956).

The hydrolysis of proteins which occurs in senescent eaves results in elevated

levels of free amino acids (Chibnall, rg3g) including tryptophan (Commoner &

Nehari, 1953;Pearse Novelli, 1953;Lihdesm?iki, 968;Kim & Rohringer,1969).

The degradationof tryptophan and the consequent roductionof IAA could occurby transaminationand possibly, n somespecies,o someextentby decarboxylation;

the phenol/phenolaseystem ould alsobe of some mportance n cells n an advanced

33-2



528 A. R. Sunrpnern

stateof disintegration.But the relativecontributionsof thesepathwayshavenot been

investigated.

Measurements f the auxin in senescenteavesof Acerpseudoplatanusnd several

otherspeciesn the autumn revealedhat the highest evelswerepresent n leaveshat

were actually falling from the trees (A.R. Sheldrake,unpublished results). This

auxin s thereforeof no importance or the restof the plant. But it seems ossiblehat

someof the auxin produced n senescenteavescould play a part in the control of

abscission.Numerous investigationshave shown that the application of auxin to

petioles etardsabscissionAddicott, rgTo).The auxin of senescenteaves ould there-

fore havean abscission-retarding ffectas ong as he transport systemby which auxin

moved down the petiole o the abscission onecontinued o function. This might be

of considerablephysiologicalsignificance: eavesdo not generally abscind until

senescences well advancedand it is this delay n abscissionwhich enablesnitro-

genousand other compounds o be translocated ack nto the stem. Auxin produced

in the senescenteaf might help to retard abscission ntil the transportsystems hem-

selvessenesced. hus the relativelysmall amountsof auxin which can be collected

by diffusion from the petiolesof senescenteaves Bcittger, y7o) reflecta declineof

the auxin transportsystem atherthan adecline n the amountof auxin n the leaves.

4. Dicotyledonous eedlings

Auxin is produced n the cotyledons f seedlings f Lepidium vanOverbeek, g3z),

Raphanu.r van Overbeek,1933),Lupinus (Navez, 1933) and PhaseolusWheeler,

1968).The productionof auxin in cotyledons or in the endospermof seedlingswith

endospermouseeds) an be explainedas a consequencef the breakdownof reserve

materials.The auxin production of cotyledons ould alsobe regardedas analogous

to the production of auxin by senescenteaves.The shoot ips of seedlings re sites

of auxin production (Soding, rg1z), as are shoot ips in general.

The demonstration hat the growth of grasscoleoptiles s controlled by auxin

moving basipetally rom the tip led to numerousattempts o explain he growth and

tropismsof dicotyledonous eedlings n an analogousmanner. fn some species, .g.

Raphanu.svan Overbeek,1933) t was ound that the removalof the cotyledonsand

the shoot tip inhibited the growth of the hypocotyl. But in other species uch as

Lupinus Dijkman r934i Jahnel, %il andHel:ianthusdu Buy & Neurnbergk, rg32)

decapitationhad little or no effect on the growth of the hypocotyls. The attempt to

explain all growth in terms of auxin meant that theseresultswere interpreted by

postulating hat a diffuseproduction of auxintook place n the growingregions hem-

selves Jahnel, g37; du Buy & Neurnbergk,1935;Sciding,1952,196r). However,

Jost(r94o) concludedrom his studieson beanepicotyls hat auxin alonecouldnot beresponsibleor the control of growth and suggestedhat a secondhormonewas alsoinvolved. It is now known that gibberellins have striking effects on the growth ofstems Cleland,ry69).A widely usedbioassayor gibberellinsdepends n the stimula-

tion of hypocotylgrowth by thesehormones Frankland& Wareing,196o).The recentdemonstration hat gibberellinsbecomeasymmetricallydistributed in shoot tips of

Helianthu.r s a result of geotropic Phillips, tgTza) and phototropic (Phillips, rgTzb)




stimulationand he finding that stemgrowth n this speciess controlledby gibberellin

rather than auxin (Phillips, rgTza) mean hat much of the early work on growth and

tropisms n seedlings on which the auxintheoryof tropisms s based)must be recon-

sidered. n the light of these acts t no longer seems ecessaryo supposehat auxinis produced n the elongating egionsof seedling tems.

5. StemsAuxin is present in the basal meristematic region of growing monocot stems

(Schmitz, 1933) where both meristematicactivity and vasculardifferentiation are

taking place.Mature stemsof monocots in which no secondaryhickening occurs)

contain ittle or no auxin (Schmitz,1933).The growthof maturenodes anbe resumed

as a result of geotropicstimulation; in sugarcane, he intercalaryzonegrows from a

few millimetres to more than a centimetre n a wedge-shaped ay when the stem s

placed horizontally. A marked increase n auxin occurs during this process van

Overbeek,Olivo & de Vasquez,1945). t is interesting o note that these authors

concluded hat rather han beinga cause, uxinwasproducedasa consequencef the

geotropic esponse. uxin production has alsobeen observed n geotropically timu-

lated nodes n other grassspecies Schmitz, 1933).Unfortunately n these eportsno

informationwasprovidedabout he anatomical hangeswhich accompaniedhe geo-tropic reactionand the associated roduction of auxin.

Auxin is produced in secondarily hickening dicot stems (Zimmerman, $36;Sciding, y37, 1938, g+o;Jost, ry+o; Allary, 1958;Hatcher,1959;Diirffiing, 1963i

Sheldrake& Northcote, 1968&).The site of auxin production s the cambialregion

itself (Stiding, 1937,1938, g+o; Ddrffiing, 1963). n young eaves, eveloping uds,etc., a direct investigation of the cellular sites of auxin production has not beenpossible; but the cambial region has the advantage rom an experimentalpoint ofview that the tissuescanbe separated asily, irst by stripping off the bark and thenby u selective crapingof tissues rom the outsideof the wood and the insideof thebark. In this way Sciding(1937, 1938, r94o) showed hat much more auxin waspresent n the cambium and its young derivatives han in the mature phloem andxylem tissues.Because econsideredt intrinsically mprobable hat the differentiatingvascular issuescould produceauxin, he concluded hat the auxin was producedbythe cells of the cambium itself. However, if the undifferentiated cambial cells areseparatedrom the young, differentiatingphloemand xylem tissuesand their auxincontentsare analysedseparately, he highestamounts are found in the differentiatingxylem cells, ess n the cambiumand least n the phloem (Sheldrake, gTra). Unlessthere is a radial movement of auxin againsta concentration gradient (which seemsunlikely), these results suggest hat auxin is produced in the differentiating xylemtissue rather than in the cambium.

The same conclusion has been reachedby"

different experimentalapproach.Segmentsof tobacco nternodesmaintained n sterile culture produce auxin andcontinue o do so for many months.This productionof auxin s associated ith con-tinuing secondary hickening; if cambial activity is eliminated,auxin productionceases. n experiments with separated, egeneratingbark tissue it was found that



530 A. R. Snnlonern

auxinproductiondepended n xylem differentiation, ndicating hat auxin s producedasa consequencef xylem differentiation Sheldrake& Northcote,1968D). he possi-bility that someauxin might alsobe produced n differentiating hloem issuewas noteliminated.

The continuing activity of the cambium n isolatedstem segmentsn the absenceof exogenous ormones Jost, t893, rg+o; Sheldrake& Northcote, 19680) ndicates

that the system s self-catalysing.Auxin is known to be involved in the control ofcambial activity and vasculardifferentiation; t is alsoproducedas a consequence ftheseprocesses. ome of the auxin produced n the cambialregion is transportedbasipetallyby the polar auxin transport system. n isolatedstem segmentshis leadsto considerablyenhanced ambialactivity towards he basalend of the explant andalso o the formation of a basalcallus Jost, rg4o; Sheldrake& Northcote,19680).Bycontrast, obacco stem segments ultured in the presenceof.2,3,5-tri-iodobenzoicacid, a specific nhibitor of polar auxin ransport,exhibit pronounced ambialactivityall along he explants, esulting n the productionof serried anksof tracheidswhichcan exceedby several imes the amount of xylem originally present(Sheldrake&Northcote, 1968 ). This demonstratesery strikingly the 'positivefeedback' nherentin the cambial ystem,which under normalcircumstances ustbe'damped'to someextentby the removalof auxin by polar transport.

6. Roots

Auxin is produced n isolated oots cultured n oitro (van Overbeek,1939),but in

the intact plant it is also ransported rom the shoot nto the roots (McDavid, Sagar& Marshall, ry72)where t may be conjugated r destroyed.In Lens oots ittle auxinis found in the tip itself; most s found about 5 mm behind the tip (Pilet & Meylan,1953)where xylem differentiation s taking place. Similarly, in Zea roots the tipcontains essauxin than the regionbehind the zoneof elongation,and nearly all theauxin is found in the stelerather than the cortex (Greenwood,Hillman, Shaw &Wilkins, ryn). The sourceof this auxin could be the differentiatingvascular issues,

althoughsomeof it could havebeen ransportedacropetallyn the stelar egion.Othersites of cell death where auxin could be produced are the differentiatingvasculartissuesn the cambial egion of secondarilyhickening oots, he regressingoot hairsand the root cap. The latter two may be of particular mportanceunder non-sterileconditions.

Roots are very sensitive o exogenous uxin which is often present n soils in con-centrationssufficient o affect hem considerably SectionV). Thereforeenvironmental

as well as endogenous uxin could be important in the control of root growth undernatural conditions.

7. Flowers,ruits and seeds

Auxin is produced n considerable uantities n developinglowerbuds; its produc-

tiondeclinesas the flower matures and little or none is formed by fully

developed,unfertilized flowers (Sciding,1938;Kaldewey, 1959).The sites of auxin production

in the earlieststagesof flower developmentare not known, but it seems easonableo



532 A. R. Snrllnerr

seen in apples with an asymmetric distribution of pips (Audus, 1953) and close

correlations between the number of developing achenes and the growth of the re-

ceptacle have been demonstrated in the strawberry (Nitsch, r95o). But although

hormones formed in the developing seeds nfluence the development of the fleshy

parts of the fruit to a striking extent in some species, n others the developing fruit

tissues may be more or less autonomous from a hormonal point of view. This must be

the case n naturally parthenocarpic fruits such as seedlessvarieties of orange and

cultivated bananas. In a number of other species whose fruits normally develop

only after fertilization, parthenocarpy can be triggered off by wounding (Haberlandt,

rgzz),treatment with a variety of chemicals including synthetic auxins and gibberellic

acid, or environmental influencessuch as exposure to cold (Nitsch, r95z). Once fruit

development has been nitiated it proceedsmore or lessnormally, although partheno-

carpic fruits are sometimes smaller than normal ones. The development of these

fruits must depend on hormones produced within the fruit tissues themselves. In

tomatoes, the production of auxin in parthenocarpic fruits has been directly demon-

strated by extraction of the tissues of fruits induced to develop by phenylacetic acid

(Gustavson, 1939) and by diffusion of auxin from fruits developing parthenocarpically

after gibberellin treatment (Kurashai & Muir, 196z).

Fruit development takes many forms, but in fleshy fruits involves the expansion of

cells formed before and sometimes after fertilization; in the larger fruits there is also

considerable vascular differentiation. The highest amounts of auxin in partheno-

carpically developing tomatoes are found in the tissues of the central axis and parti-

tions (Gustavson, r939) where vascular differentiation is pronounced. It seemspossible

that the differentiating vascular tissues could be a major site of auxin production in

developing fruits, both parthenocarpic and normal.

8. Cellular sitesof auxin production

It cannot be a general property of dividing cells to produce auxin, since most callus

tissuescultured in aitro require auxin; if dividing cells produced it, these issueswould

become autonomous with respect to auxin once cell division had been initiated. The

hypothesis that auxin is produced by meristematic cells is not supported by the finding

that meristematic embryos and cambial tissue contain less auxin than the dying

tissuesadjacent o them; and it is clearly unable to explain the production of auxin by

senescenteaves.This hypothesiswould require meristematic cells to contain elevated

levels of tryptophan, for which there is no evidence. The only evidence in favour of

the meristematic hypothesis of auxin production is the general correlation between

regions of meristematic activity and auxin production, a correlation which can be

explained equally well by the presence of dying cells. It is not possible to exclude the

possibility that some meristematic cells produce auxin some of the time, but there is

at present no reason to believe that this is the case.

The essential features of the hypothesis that auxin is produced as a consequence of

cell death are that tryptophan is the limiting factor for auxin production; that in living

cells the concentration of tryptophan is regulated and maintained at a level too low

for the degradation of tryptophan, and hence the production of auxin, to occur to a



Theproduction of hormonesn higherplants 533significantextent; and that autolysis esults n increasedevelsof tryptophan.Someof the tryptophan released y autolysingcells may be converted o auxin by adjacentliving cells; it is for this reason hat I have referred to auxin production as takingplace asa consequence f cell death, ather than simply in the dying cells hemselves,although hesemaywell be the majorsiteof tryptophan degradation.With the possibleexceptionof geotropicallystimulatedgrassnodes, all the known sitesof auxin pro-

duction in plants aresitesof cell death.The sitesof cytolysis within plants which, according o this hypothesis,are likely

to be sitesof auxin production are: regressing utritive tissues the tapetum, nucellus,endosperm nd cotyledons),he dying cells of senescenteaves, ossiblydying roothairsandroot-capcells,differentiatingxylem cells,most differentiating ibres,possiblydifferentiating sieve ubes, and differentiating cork cells.Auxin is probably alsopro-duced by damaged nd woundedcells SectionVIID.

IV. AUXIN PRODUCTION UNDER PATHOLOGICAL CONDITIONS

Attacks by fungi, bacteria and animals involve the death and breakdown of cells

in the infected region; tryptophan released by the dying cells could be converted to

IAA by enzymesof the host cells themselves or of the pathogen.

r. Fungal and bacterial infections

Tissues infected by bacteria or fungi usually contain considerablyelevated evels of

auxin (e.g. Wolf, 1956; Gruen, 1959; Sequeira, 1965;Kim & Rohringer, 1969) an d

tryptophan (Kim & Rohringer, 1969). Many fungal and bacterial pathogens are

known to be able to convert exogenous tryptophan to auxin and it seems likely that

they may play an important part in the production of auxin in infected tissues. By the

use of rather dubious biochemical criteria, Sequeira (tg6S) attempted to determine

the contributions of the host and the pathogen to auxin production in tobacco tissue

infected by Pseudomonas;he concluded that both were important, the host being more

so in the early stages of infection. But whatever are the relative contributions of host

and pathogen, the production of auxin can be seen as a consequenceof the releaseof

tryptophan by the lysis and digestion of the infected cells.

z. Animals

Some animals which infect plant tissues feed by secreting digestive enzymes and

then sucking up the digested material (Krusberg, 1963; Miles, 1968). As in fungal

and bacterial diseases,t is possible that tryptophan releasedby proteolysis could be

converted to auxin in the host as well as in the pathogen. Other animals digest the

material internally and hence auxin is likely to be formed within the animal and may

be released from it by excretion. For example, leaf-mining insect larvae which munch

their way through the mesophyll deposit faecal pellets around which intumescences

develop, probably as a response o auxin (La Rue, 1937). The application of mouse

faecal pellets to mesophyll tissues has similar effects (La Rue, 1937).

These observations provide an approach to the understanding of galls produced by



53+ A. R. Snrronexn

more or lessstationaryanimalswhich live entirely enclosedwithin the plant tissues.Animals whose posterior parts remain outside he plant do not produce galls (Mani,1968).There is evidence hat galls nducedby Meliodogyne ematodes ontainelevatedlevelsof amino acidsand auxin (Dropkin, 1969); t seems robable hat auxin mayalso be produced in other galls. However, the wide diversity of galls suggests hat

hormonesother than auxin are also nvolved; the balanceand sequence f hormones

releasedmust be characteristic f the gall-inducing pecies.

3. Viruses

Unlike fungal, bacterial and animal pathogens,virusesdo not sustainthemselvesby digesting he contentsof the host cellsbut rather by perverting the metabolismofthe cells o their own ends.Thus amino acidswithin the cellsare useddirectly in thesynthesisof viral proteins.Tryptophan is found in the protein of many plant viruses(Fraenkel-Conrat, 968); its utilization n infectedcells might be expectedo lowerrather than raise he levelsof free tryptophan. This is indeedknown to be the case nvirus-infected otato ubers Andreae& Thompson,r95o). t is therefore nteresting

to note that tissues nfected with viruses generallycontain less auxin than normal,

uninfected issues S<iding, 96r; Sequeira, 963).

4. Crowngall

In many species,umours develop n the vicinity of wounds infected by virulent

strains of the crown gall bacterium,Agrobacterium urnefaciens. he proliferation ofthe tumour cells continues even f the bacteria are killed, showing that a permanent

transformation,analogouso animal cancer,has akenplace.The actual ransforming

agentmay be a virus, carriedasatemperatebacteriophageby . tumefaciensBeardsley,

r97z). Sterile tumour tissue continues o proliferatewhen grafted nto healthyplants

and canalsobe cultured n ztitroon a simplemedium containingsugarand salts.These

facts indicate either that crown gall tissuesdo not require auxinsand other growth

factors that are necessaryor the growth of normal callus tissues,or that they have

acquired he ability to produce hem. The evidences n favour of the atter explanation

(for reviewsseeBeardsley, gTz; Wood, r97z). Non-sterile Link & Eggers, 94r;Dye, Clark & Wain, 196r)and sterile Kulescha, 95z) crowngall tissues ave been

shown o produceauxin.But they do not containunusually igh evelsof aminoacidsin general Lee, r95z) or tryptophan n particular Henderson& Bonner, rg5z).

All existing heoriesof crown gall seem o assume hat the tissues ontaina more orlesshomogeneousopulationof tumour cells e.g.Braun,196z;Wood, rgTz),Braun

(tqS8) hasproposed hat the systems ynthesizing rowth hormonesare'activated'in the transformed cells. An 'activation' of the auxin-synthesizingsystem wouldpresumably nvolve an increasedability of the cells to convert tryptophan to auxinand/or an increasedsynthesisof tryptophan. But in fact crown gall tissuesneither

show an enhancedability to convert tryptophan to auxin (Kulescha, r95z) nor dothey contain more tryptophan than normal callus issues Henderson& Bonner, rgsz),

It is therefore necessaryo questionBraun's assumptions.Perhapscrowngall tissues ontain a more or lessstablemixture of cells,some rans-



Theproduction of hormones n higher plants 535formed, others normal. Even 'clones' grown from singlecells can contain a mixture

of normaland ransformed ells, ince eversions known o be possible Braun,1959).

The differencesbetweenpartially and fully transformed issues Braun, r96z) could

be explainedon this basis n terms of the former havinga lower propoftion of trans-

formed cells. Furthermore, only some of the transformed cells may be involved in

the production of the hormones to which the other cells respond.This would be

possible, or example, f at any given time some of the transformedcells died, withthe consequentproduction of auxin and also, possibly, cytokinins (Section VII, 3).Crown gall tumours do in fact containconsiderable umbersof dead and dying cells;

it has often been observed hat cell divisions occur in crown galls around necrotic

areas r zones f tracheiddifferentiationRobinson& Walkden, 9z3 Banfield,1935;

Manignault,1953 Therman,1956;Kupila, 1958).No comparable istological tudies

of crown gall tissues cultured in zsitrohave been published, but in all the cultures

which I have examineddeadcellsare quite common. The tumourous transformation

viewed n this light would not involve an 'activation' of the auxin synthesizing ystem

in all the cells but would lead to auxin production by causinga more or lessconstant

percentageof the cells to die. This could be thought of by analogy with lyso-

genic bacterialcultures where at anygiven time a minority of the cellsare killed by

bacteriophageswhich in the other cells are integrated with the genomeand remain

latent.

The autonomy of 'habituated' tissueswhich arise spontaneously rom normal

calluses fter more or lessprolongedperiods n culture might be explicable n a similar

way. But in the absenceof any quantitative data on cell deathwithin these issues,

or indeed within normal callus issues, hehypotheses dvanced bovecan be no more

than speculative.

V. ENVIRONMENTAL AUXIN

Auxin hasbeendetectedn a wide varietyof soils Parker-Rhodes,94o; Stewart&

Anderson, g4z; Hamence, g4+, 1946; Whitehead,1963;Sheldrake, 97rD). It is

produced rom tryptophanby many soil micro-organismsRoberts& Roberts,1939)

and is found in the highest amounts n soils rich in decaying organic matter. The

auxin content of the soil represents n equilibrium betweenproduction and destruc-

tion (Parker-Rhodes,94o; Hamence,1946)and s presumablyalsoaffectedby factors

such as rain and leaching,

The elongationof root hairscan be stimulatedby extraordinarily ow concentrations

of auxin: r x ro-4 pgllis sufficient o bring about a significanteffect(Jackson,196o).

The growh of roots themselves s also sensitive o exogenousauxin, often being

inhibited by concentrationsn excess f about to pgll (Whitehead, 1963).Thus roots

grown in this order of auxin concentrationhave a more densecovering of root hairs

which are also onger (Ekdahl, 1957).The concentrationof auxin in the soil solution

usually ies n the rangeof r-5opg/l (Whitehead, 963; Sheldrake,y7tb). Theseare

averagevalues; it should beremembered hat the soil is made up of many micro-

environmentswhere locally higher or lower concentrationsmay be present.

The effectsof auxin on roots may well be of adaptive significance; he relatively





Theproduction of hormonesn higlterplants J 5 t

VII. THE PRODUCTION OF OTHER PLANT HORMONES

This subjectwill not be discussed t such ength as he production of auxin, partly

becausehe literature s more recent,smallerand ess angled;partly because everal

recent eviewsareavailable: n cytokininsby Fox (r969),Skoog& Armstrong (rgZo)

and Kende OgZr); gibberellinsby Cleland (t969), Lang QgTo)and West (tgli;

abscisicacid by Addicott & Lyon (tg6g) and Dcirffiing tg7t); and on ethyleneby

Burg (1962),Pratt & Goeschl t969), Mapson tq6q) and Abeles tglz).I shall con-

centrateon thoseaspects f the literature that have a bearing on the understandingof

the sitesof productionof thesehormones:aswith auxin, t is only when the cellular

sitesof synthesisare known that a clearer understandingof the control of hormone

production will becomepossible.

In the light of the evidence n favour of auxin production asa consequence f cell

death, it seemsworth considering he possibility that other hormonesmight alsobe

formed as a result of cytolytic processes. here is no a priori reasonwhy they should

be; but conversely here is no a priori reason or assuming hat they are synthesized

in living cells. In the following sections have attempted o weigh up the evidence

for and against hesepossibilities.On balancet seems robable hat abscisic cid s

synthesized y living cells; that the synthesisof gibberellin precursorsoccurs n

living cells, but that the final oxidativereactionsnecessaryor the production of these

hormonesmight takeplaceasa consequencef cell death; that cytokinins hough pro-

duced by living cells, at least n root tips, are also formed by dying cells; and that

ethylene s produced as a consequence f cell damageand cell death.

t. Abscisicacid

Two pathways f abscisic roductionhavebeenproposed: y direct synthesisrom

mevalonate r by the oxidativebreakdownof carotenoidsAddicott & Lyon, 1969).

The photo-oxidation of violaxanthinresults n the productionof xanthoxin, a naturally

occurring compoundclosely elated o abscisic cidandwith similar biologicalactivity

(Taylor & Burdon, rgTo).Abscisic acid has been found in a wide range of plants and in a variety of tissues(Milborrow 1967, 968).t ispresent ndprobablyproducedn the shoots f seedlings

(Teitz & Diirffiing, 969), in young eavesand buds (Milborrow, 1967)and in de-

veloping fruit tissue (Rudniki, Pieniazek& Pieniazek,1968; Rudniki & Pieniazek,

r97o; Ddrffiing, r97r; Davis & Addicott, rgTz).A striking ncreasen abscisicacid

occurs n leavessubjected o water stress Wright & Hiron, ry72) and mature leaves

of treesproduceabscisic cid n responseo short-day onditions Phillips& Wareing,

rg59).Abscisicacid s produced n considerableuantitiesn senescenteavesChin

& Beevers, 97o; Biittger, r97o) and n ripening and senescentruit tissuesRudniki,

Machnik & Pieniazek, 968;Goldschmidt,Eilati & Goren, tgTz;Davis & Addicott,

rg72).

Taylor & Smith (tg6l) suggestedhat abscisic cid s produced n,uiaoby the photo-oxidative breakdownof carotenoids,especially n senescenteaves.This hypothesis

at first sight seems o provide a plausibleexplanation or the increasedabscisicacid



538 A. R. Sunronerc

production which occurswhen etiolated issuesareexposed o light (Wright, r968).

However, ight brings about many changesn etiolated issuesand there is no reason

to suppose hat the influence of light on abscisic cid production is direct. There is n

fact no evidence or the production of abscisicacid from carotenoidsn aioo; and the

availableevidence eemso be against t. In pea seedlingsreatedwith gibberellicacid

no relationship could be found betweenabscisic-acid roduction and changesn the

amounts of carotenoidpigments ('Iietz & Diirffiing, 1969). There is evidence hat

abscisicacid is producedde nozton ripening strawberries, ather than by the break-

downof carotenoidsRudniki& Antoszewski, 968).The direct synthesis f abscisic

acid from mevalonatehas been demonstrated n wilting leaves (Milborrow, r97z)

and in this case again the evidence is against carotenoids acting as precursors.

Abscisicacid is producedby senescenteaves Chin & Beevers, 97o) and ripening

fruits (Rudniki, Machnik & Pieniazek,1968) n the dark. Thus although the photo-

oxidation of carotenoidsmay result in the formation of growth inhibitors under some

circumstances,t doesnot appear o be he major pathwayof abscisic-acidroduction

in the plant, even n senescentissues; the predominant route of abscisic-acidpro-

duction in aioo may well be the direct one. This de noao synthesisof abscisicacid

presumably ccursonly in living cells.

z. Gibberellins

Gibberellinswere irst solated rom fungi; they arealsoproducedby somebacteria

(Vandura,196r) and have been detected n algae Radley,196r). In fungi and in

higher plants they are known to be formed from ( - ) kaurenewhich is in turn derived

from mevalonate Lang, r97o; West & Fall, ry72). A number of inhibitors (e.g.Amo

1618)which block the synthesis f kaureneprevent he productionof gibberellins

(Dennis, Upper & West, 1965)and when applied o higher plants act as growth

retardants.The conversionof kaurene o gibberellicacids nvolves a seriesof oxidative

reactionswhich are ittle understood West& Fall, tgTzi West, 1973).

Gibberellinsare produced n young, developing eavesof.PhaseolusHumphries &

Wheeler, ry64),Helianthus Jones& Phillips, r966)and Taraxacum Fletcher,Oegema& Horton, 1969).The levelsof gibberellins n mature eaves re ow. In Helianthus

shoot tips, gibberellinproduction occurs n the young leaves ather than in the

meristemtself(Jones& Phillips, 1966).Gibberellinshavebeendetectedn the xylem

sap of a numberof species e.g.Phillips& Jones, 964; Skene,1967) ndicating hat

they areproduced n roots; and the productionof gibberellinsby root tips+caps of

Helianthushas beendirectly demonstrated Jones& Phillips, r966). There is indirect

evidence hat they areproduced n the cambial egion.The applicationof auxin to

woody stems nduces cambial development and tracheid differentiation; but for

normal vessel differentiation another hormone is necessary.This secondhormone

was shown by Rehm (tg:6) and Jost (tg+o) to be formed not only in young

leavesbut also as a consequence f cambial activity. In the presenceof auxin the

effects of gibberellic acid on the differentiation of cambial derivatives (Wareing,Hanney & Digby, 1964) are the same as those attributed by Rehm and Jost to

the unknown hormone, suggestinghat it was in fact gibberellin. Gibberellins are



Theproduction of hormonesn higherplants s39formed in considerable uantitiesduring seeddevelopment Cleland,1969),by far

the largest amounts being produced as the nucellus and/or endospermdegenerate

(Corcoran& Phinney, 962 Jackson Coombe 1966;Luckwill, Weaver& MacMillan,

1969; Chacko,Singh & Kachru, r97o). In developingseedsof Echinocystishe great

majority of the gibberellin is found in the nutritive tissues ather than in the embryo

(Corcoran & Phinney, 196z).As seed developmentproceeds owards dormancy,

free gibberellins are convertedto bound forms, as glycosidesof gibberellic acids(Barendse t al., 1968; Barendse, 97r; Sembdneret al., ry72). fn a number of

dicotyledonousspecies, ree gibberellinshave been shown to be releasedrom these

bound forms after germination of the seeds;and at least he early stagesof seedling

development re ndependent f denozto ibberellinsynthesis Barendse t al., 1968;

Dale, r969). Similarly, he 'production' of gibberellinsby newly germinatedbarley

embryosmay be due to their releaserom a bound form (Cohen& Paleg,1967).Therelease f free gibberellins rom abound form hasalsobeenshown o occur n etiolated

wheat leavessoon after an exposure o red light (Loveys & Wareing, ry7r).Taken as a whole, theseobservationswould seem o exclude the possibility that

meristematic issuesaremajor sitesof gibberellin production: the apical meristemof

Helianthus does not produce gibberellins; the meristematic embryonic tissues of

developing seeds contain relatively little gibberellin and the apparent productionof gibberellinsby germinating embryoscan be explained n terms of a release rom a

bound form. Growing cells in general also seemunlikely to be sites of gibberellin

synthesis,otherwise hesehormoneswould not be a limiting factor for the growth of

stems.The only featurewhich all the sitesof gibberellin production have n common s

the presenceof dying cells; in root caps, n regressing utritive tissuesand in differ-

entiatingvascular issue. n this connexion t is interesting o note that gibberellinsare

produced at or near the wounded surfacesof potato and Jerusalemartichoke tuber

tissues hortly after cutting (Rappaport& Sachs, 967; Kamisaka& Masuda,1968;

Bradshaw& Edelman,1969).The simplesthypothesis uggestedy thesedata s that

gibberellins are produced as a consequence f cytolysis.This suggestionmay seem

both surprising and improbable rom a biochemicalpoint of view. But it is not neces-

sary o suppose hat gibberellinsaresynthesizedrom mevalonaten dying cells; more

immediate precursorssuch askaureneor kaurenoicacid could alreadybe presentand

then only the final oxidative eactions ecessaryor the conversionof thesecompounds

to gibberellins eed akeplaceasaconsequencef celldeath.This hypothesis ppears

to be in conflict with the finding that gibberellin levels decline rather than increase

in senescent eaves Fletcher et al., t96g; Chin & Beevers, 97o) and fruits (Gold-

schmidt et al., ry72) but perhaps n thesecaseshe cellsdo not contain the necessary

precursors.

Fungal cultures do not producegibberellins n significantquantitiesuntil the phase

of exponentialgrowth hasceased. here is, however,someevidence hat gibberellins

are produced in the stationary phase metabolically rather than autolytically, since

autolysis within these cultures does not becomeapparent until a rather later stage

(Borrow et al., tg55; Jefferys, r97o). It is difficult to assesshe relevanceof these

findings to gibberellin production in higher plants,but they perhapsweaken he case



540 A. R. Snoronexr

for thinking that gibberellinsareproducedas a consequence f cell death, or at leastof autolyticprocesses.

3. Cytokinins

A diphenyl urea isolated rom coconut milk has cell-division-stimulating activity

(Shantz & Steward, 1955);and cell division factors, hought to be nicotinamidederivatives,were isolated rom several ypes of cell cultured 'in oitro (Wood, Braun,Brandes& Kende, 969). Thesecompounds avenowbeen e-identified spurinones(Wood, r97o). With these exceptions,he naturally occurring cytokinins are adeninederivatives, ound in plants as ree basesand as ribosidesand ribotides. The sameorclosely elated compoundshave been ound in the transfer RNA (IRNA) of animals,bacteria, ungi and higher plants Skoog& Armstrong, gToi Kende, 97r). There isstrong evidence,summarizedby Kende (rg7t), that cytokinins n tRNA are synthe-sizedby the attachmentof an sopentenylgroup, derived rom mevalonate,o adeninein preformed IRNA, that plant tissues equiring an exogenous upply of cytokininas a growth factor are capable of synthesizingcytokinins in IRNA and that freecytokinins are not involved in any direct way in the formation of cytokinin nucleo-

tides in tRNA. But while the cytokinins of IRNA are not derived from freecytokinins, the free cytokinins could be derived from the cytokinins in IRNA byhydrolysis(Sheldrake& Northcote, 19686).Hydrolysates f IRNA from animals,micro-organisms nd plantsare active n cytokinin bioassaysBellamy, 966; Skooget al., 1966;Letham& Ralph,1967).

Cytokininsare found and probablyproduced n developingruits and seeds,par-ticularly n the nutritive tissuesSteward& Shantz,1959;Skoog& Armstrong, ryJo)tin germinatingseeds Barzilai & Mayer, 1964), n young eaves nd buds(Engelbrecht,r97r), developinglowerpetals Mayak,Halevy&Katz, tgTz),root ips+ caps Weiss& Vaadia,1965;Short & Torrey, ry7za) and n the cambial egion Bottomleyet al.,1963;Nitsch & Nitsch, 1965).Although the hydrolysisof IRNA with the release fcytokinin ribotides, ribosidesand free basesmight takeplace n living cells, t would

be almost inevitable in dying, autolysing cells. The tissues n which cytokinins areproduced contain dying cells, either in regressingnutritive tissues,n root capsor indifferentiating vascular tissues; in the latter caseRNA breakdown akes place notonly as xylem cells and fibres differentiate and die, but alsoduring sieve-tubediffer-entiation which involves he loss of the nucleus, ribosomesand most cell organelles(Northcote& Wooding, 968).

The production of cytokinins by autolysing cells might lead to an apparentlyparadoxicalsituation in senescenteavessince these hormonesare known to retardleaf senescencen many species.Nevertheless,here is evidence hat a cytokinin isproduced in senescenteavesof Populusand Acer (Engelbrecht, ry7r). fn senescentleaveswhich contain ow levelsof cytokinins, any cytokinin production which occursmay be maskedby a rapid destruction and metabolismof the hormones Srivastava,

1968).There is evidencehat cell-division-stimulatingsubstances re produced n theyellowing regions of slowly senescing herry laurel leaves;Godwin Qgz6) observedzonesof cell division on the outskirts of the yellowing regions, resembling hose hat



:;:fi:fr,T"l':"0::,#:r::::ofhormonesnhighertants

,lT[',:1;"rl$lll*ll$***ii*:i:H,'"x'.:'t":F*evr",,,,s"1i

r.i'i..iv,"""f;.jl1**'..i'."f.?l,:I"+#,iJ,"'.tmmediatetyadjacent*

"ri.'i"

j#l:1t".r is impliedby tspecies

remain ].,r"."^i.::_"" "'

reavesofcherry laurol' n;;;^r.-""'s

rnat the regions

iiir:;1:*i*l*$'{t+}l}i#H,T1ffilx:1;$,;;;';['iJ:lJ'TJ[fffiTjJlsion1(Motn".,;"r;i:l

areasointo'ir,".*o,o-

cyt knns "- :" :ensl d 'u"' l' ""- ';^i* ";;?#[ili;T d*ffi :ffi

ffi${*f*il*?liffi#i'ii*H*::rii,'*:"*.",*1,f::":F,,,:.::i::'*r,,i",,-,ii"it

:r:*i:r,{:"".,;JilT,'#H,rj;Ei*;;4;ni,:j'ffi lT:,:Tff::#il";T:ly'l[fi'f;'T'I:;#i'.",;:lT:Tfff;numbersorayinnll',,lould

e';;;;.'?2.")'.1 #;;;':is'placeif acriticalevel

5r.':J::".""'iif':: fr",1;i**ifi"*i,TT'lp'*:***#lr,nt"rt'"f;Jt:it,1rJffi"T:":i,i::""#i,('ffiT:l.lilL";1..1"'"''nNa.

p."'3 rm sm r",,,o,1l'oknn;; i;,h: ltli: **"r;r#f "

i',ii,::il:

;nffirx*ltTfitl;:::J"x*"::ai"r""r"u'"frfi'H"'#:".T:'fif;;;ru-;;g;;;ffi "T,#;'1#l,l[tf:i,,g.*,*ril;*;";,ffi"xg**qr:ry;'*::n*iril,tt:t'':J:*'";';:ffit";:ilI:"^,1'+'Fi#td*":IIII#:'#T#"?:,hemeris,em;';;;fif;"li::["jg]::i*iti-,,*l-*'i[iilii:TT,":ff:#:,:T["fl.*],1t:1i*ttri;i#"fr.1,1i';','ffj[ii::'H:?quantitative

investigatKrnrnsnotherur,;rilt'

*itt".'""t il'.=:y-t:T

to1r<Jti";+iJ %TiH;::

t['ilx:"i.ffidfiril:::::*:.llrfill'#il*f"l*ffi;#;34_-_, yrwrurrnsare releasedfrom the IRNA

"::r."rjr.a*



542 A. R. Snnronexr

micro-organisms, ecaying rganicmatter,etc., n the soil.Presumablyhey are alsodegradedn the soil; but perhaps he levelsof environmental ytokinins might be

sufficient to influence parts of plantssensitive o exogenous ytokinins. For example,the inductionof buds on mossprotonemata y low levelsof cytokinins Bopp, 1968)

could represent n adaptive esponseo environmental ytokininsanalogouso the

adaptive esponse f liverworts o environmental uxin (Sheldrake, gTrb).

4. Ethylene

Several ossibleprecursors f ethylene n the plant havebeenproposed Mapson,

1969),but the mostprobable s methionine Yang & Baur, r97z). Methionine at the

C-terminal end of peptidesproducedby proteolysis an alsoact as an ethylenepre-

cursor (Demorest & Stahmann,rgTr).Ethylene can be releasedrom methionine

non-enzymicallyby the action of hydrogen peroxide n the presence f certai.nmetal

ions,and, inaitro,by the actionof peroxidase,ut the enzymicmechanism f ethylene

production in aivo is not yet known (Yang & Baur, ry72; Lbeles, ry72).A burst of ethyleneproductionoccurssoonafterplant tissues re wounded Burg,

ry62)andconsiderableuantities reproduced n diseasedissuesWilliamson, 95o).

Under normal conditions thylenes producedby senescingeaves Morgan,Ketring,Beyer& Lipe, rgTz),senescingetioles Rubinstein& Abeles,1965), enescinglower

petals (Mayak, Halery & Katz, rgTz), ripening fruits (Burg, ry62) and germinating

seeds Spencer& Olson, 1965). t is alsoproducedn young, growing issues Burg,

1968) n which vascular ifferentiations takingplace.The applicationof toxic com-

poundssuch as coppersulphateo plant tissueseads o'stress-induced'ethylene

production (Abeles & Abeles, ry72). Ethylene production is also induced by the

administrationof unphysiologically igh concentrations f auxin (e.g. Chadwick &

Burg, 1967;Burg & Burg, 1966)probablyasa resultof non-specific amageMuir &

Richter, rg72). A rise in ethylene production follows the irradiation of fruits or

vegetativeissues Pratt& Goeschl, 969). n all these ases thylene roductioncould

be explainedasa consequencef cell damage, enescencer death.

Ethyleneproduction s ncreased henColeusAbeles& Gahagan, 968)and omato(Leather,Forrence& Abeles, 97z) plants areplacedhorizontally. n the latter case

theplantswere otatedon a clinostat; he ncreased thylene roductionwas herefore

unlikely to be a consequencef the geotropic esponse.t seems ossiblehat these

observations ouldbe explained s nstances f stress-inducedthyleneproductionas

a result of literal, physicalstress.

Ethylene s produced n the soil by micro-organisms Lynch, ry72) and somesoils

contain sufficientquantities o affect he growth of roots (Smith & Russell, ry6g).

VIII. THE WOUND RESPONSE

It has long been known that cells adjacent to wounded or necrotic areas react to

form a protective layer. The nature of the wound response depends on the tissue and

on factors such as humidity and osmotic pressure (Lange & Rosenstock, 1963). Inmany cases t involves cell division with the plane of division roughly parallel to the

wound (Bloch, ry4t, tg5z). Haberlandt's (e.g. rgr3, rgr+, rgzt, tgzz) classical



The production of hormonesn higherplants543

studies showed that the wound responsewas influenced by substances eleasedby thedamaged cells which he called wound hormones, or more generally necrohormones.The more pronounced wound.response n the neighborrrhoJdof vascular bundles wasinterpreted by Haberlandt (rgra) to be due to;leptohormones,difiusing from th evascular tissues.cell division around dead or dyinj ceils has been observed in manyother situations,

for example n tissuecultures (Jones,Hildebrand, Riker & wu, r96oicrown gall tumours (section IV, 4),'genetic'

tumours of Nicotiana(Hagen, Gunckel &sp1t19*, 196r) and around necrotic areas nfected with viruses (Esau, r93g).

cell division around wounded areas usually ceasesafter a few days; the woundresponse s self-limiting. This shows that the dividing cells do not themseiv.. p.od.r""the necessarystimulus for cell division, but that ceil division dependson the woundstimulus. It is interesting to imagine what would happen if the cells around thewounded area possessed ome heritable instability (e.g.'as a result of a ,lysogenic,type of virus infection) such that some of them died after dividing. In this casea newwound responsewould take place around the dying cells and then, after this newwave of division, further cell deathmight ensue,and hence urther division, and so on.The result would be an autonomous, umourous tissue.This is essentially he mechan-

ism proposed for crown gall in Section IV,4.. .Haberlandt Q9z8) suggested that necrohormones might be of importance in theinitiation of the periderm, which often occurs below necrotic areaswhere hair cellshave died or where epidermal cells are disrupted by the growth in circumference ofthe stem' He was unable to explain the continued activity'of the cork cambium onceit had been initiated. However, by a simpre extension oi the necrohormone conceptthis could be seen as a consequenceof the differentiation of the cork cells,which dieas they differentiate' Similarly, the division of the cells of the vascular cambiumadjacent to differentiating- xyl,em cells provides a striking analogy to the woundresponse. Thus the idea that hormone production o".rrrr1, a consequenceof cell

*:l^:tbe arrived at independently ofa knowledge of the chemical identity of the

normones.

- A-substancecapableof inducing cell division in beanpods was solated by Bonner &English (tqr8) and given the name traumatin. But the bean-podassays very unspecificand traumatin is inactive in other cell-division assays Fox, 1969); it i, prorutty oilittle importance in the wound response in most tissues u.rd

"rr",in bean pods its

physiological significance is far from clear.Haberlandt never identified the necrohormones but it now seems likery that

wounded cells could be a source of auxins, cytokinins, gibberellins and ethyrene.Auxin is known to be prodrrced as a .on..q_,.r".rt"of wo,rrr?ing (Hemberg, ,9ai;; ,o1e

gibberellinssection II, z), ethyrenebection II, 4) u.ri, frobubty]"yiot

.,ir*(Section II, 3).The production f .or.r"o, allof thesero.-orr". by wounded ellscould explain the effectsof necrohormones; for example, u .o-birrution of auxin andcytokinins is known to stimulate cell division in a variety of tissues (F-ox,r969). The

production of wound hormones and the normal produition of hormones as a con_sequenceof cell death can be seenas two aspectsoi the samephenomenon (sheldrake& Northcote, ry68a).



5++ A. R. Snnlonarn

IX. THE CONTROL OF HORMONE PRODUCTION AND DISTRIBUTION

In living cells the control of biosynthesesdepends on the availability and compart-

mentalization of substrates and co-factors, on the control of enzyme synthesis at the

transcriptional and translational levels, and on feedback mechanisms involving allo-

steric enzymes. Factors such as hese presumablycontrol the

productionof hormones(e.g. abscisic acid) that are made in living cells. But beyond this vague and unspecific

statement, at present no more can be said.

On the other hand, it is possible o understand, at least n general erms, the control

of hormone production that occurs as a consequenceof cell death; this depends on the

control of cell death itself. The notion that compounds as mportant as hormones are

normally produced by dying cells may at first sight seem improbable on the grounds

that insufficient control would be possible. But this difficulty is illusory: the differentia-

tion of vascular tissues, he regressionof nutritive tissuesand the senescence f leaves

and other organs do not take place at random. And the biochemical changes in dying

cells occur in a definite and controlled sequence.

The idea that cell differentiation and cell death are controlled by hormones which

are themselves produced in dying, differentiating cells may appear paradoxical. Butin fact it is not at all surprising. Plant development is an autocatalytic process; the

control of growth and differentiation depends on the production of plant hormones;

hormone production must in turn be a consequenceof growth and differentiation.

The cells of nutritive tissues, of senescent eaves, differentiating xylem cells, fibres

and cork cells undergo a progressiveautolysis and disintegration as they die. To start

with they are living; at the end of these processeshey are dead; in between they are

dying. At exactly what stage they could first be said to be dying is a semantic question

which it does not seemvery fruitful to pursue. However, this point is more interesting

when considering sieve tube differentiation, which involves a controlled, partial

autolysis. Many of the biochemical changes hat occur in the early stagesof cell death

in other cells may take place during the differentiation of sieve tubes, although the

resulting cells are semiJiving. But even in parenchymatous cells a turn-over of cellconstituents takes place. Whole organellessuch as mitochondria are broken down in

vacuoles,which can be regardedas ysosomes Mathile, 1969). Thus some of the auto-

lytic processes hat occur on a large scale when a whole cell dies may be taking place

on a smaller scale within living cells. And a sublethal cytolysis may also occur in cells

which are damaged,but not badly enough to kill the cell. Haberlandt Qgzz) observed

cell divisions in damaged cells which he attributed to wound-hormone production

without cell death.

Many of the arguments advanced above in favour of hormone production by dying

cells would also apply to living cells in which a sublethal autolysis was taking place;

this might be of particular importance in the understanding of cytokinin and stress-

induced ethylene production. Hormone production as a consequence of cell death

could be seenas an extreme caseof hormone production by autolytic processeswhichmay occur to some extent in living cells.

The distribution of plant hormones depends not only on the amounts produced





546 A. R. Snnlpnexr

and plants, the biochemistry of dying cells has hardly been investigatedat all; it is not

even mentioned in most textbooks of biochemistry. But it cannot be ignored. Haber-

landt showed many years ago that, in plants, damagedand dying cells could produce

hormones; and it now seems ikely that hormones are produced as a consequenceof

cell death during normal development. The evidence discussedabove is strongly in

favour of the normal production of auxin occurring in this way; it seemsprobablethat autolysing cells can also be a sourceof cytokinins and ethylene; it is possible, too,

that gibberellins are produced as a consequence of cytolysis. Nevertheless, while

cytolysing cells may be important sources of these hormones, they may not be the

only sites of their production. Cytokinins, for example, are apparently made by living

cells in root tips.

The reader will have observedthat the evidence or the production of hormones as

a consequenceof cell death is in many cases ndirect and circumstantial. But hypo-

thesesare guessesas to what might be the caserather than statements of fact. The

alternative to the hypothesis that hormones are produced as a consequence of cell

death is the hypothesis that hormones are synthesizedby living cells. The latter, how-

ever implicitly it is accepted, cannot be taken for granted. Only further research can

establish he relative contributions of living and autolysing cells o hormone productionin plants.

XI. SUMMARY

r. Although much is known about the effectsof plant hormonesand their role in the

control of growth and differentiation, little is known about the way in which hormone

production is itself controlled or about the cellular sites of hormone synthesis. The

literature on hormone production is discussed n this review in an attempt to shed

some light on these problems.

z. The natural auxin of plants, indol-3yl-acetic acid (IAA) is produced by a wide

variety of living organisms. In animals, fungi and bacteria it is formed as a minor

by-product of tryptophan degradation.The pathways of its production involve either

the transamination or the decarboxylation of tryptophan. The transaminase route isthe more important.

3. In higher plants auxin is also produced as a minor breakdown product of trypto-

phan, largely via transamination. fn some speciesdecarboxylation may occur but is of

minor importance. Tryptophan can also be degraded by spontaneous reaction with

oxidation products of certain phenols.

4. The unspecific nature of the enzymes involved in IAA production and the prob-

able importance of spontaneous,non-enzymic reactions n the degradation of trypto-

phan make it unlikely that auxin production from tryptophan can be regulated with

any precision at the enzymic level. The limiting factor for auxin production is the

availability of tryptophan, which in most cells is present in insufficient quantities

for its degradation to occur to a significant extent. Tryptophan levels are, however,

considerably elevated in cells in which net protein breakdown is taking place as a

result of autolysis.

5. An indole compound, glucobrassicin,occurs in Brassicaand a number of other



Theproduction of hormonesn higherplants 547genera. It breaks down readily to form a variety of products including indole aceto-

nitrile, which can give rise to IAA. There is, however, no evidence to indicate that

glucobrassicin is a precursor of auxin in oioo,

6. Conjugates of IAA, e.g. IAA-aspartic acid and IAA-glucose, are formed when

IAA is supplied in unphysiologically high amounts to plant tissues. These and other

IAA conjugatesoccur naturally in developingseedsand fruits. There is no persuasive

evidence for the natural occurrenceof IAA-protein complexes.

7. Tissues autolysing during prolonged extraction with ether produce IAA from

tryptophan released by proteolysis. IAA is produced in considerable quantities by

autolysing tissues n aitro.

8. During the senescence f leaves proteolysis results in elevated evels of trypto-

phan. Large amounts of auxin are produced by senescent eaves.

9. Coleoptile tips have a vicarious auxin economy which depends on a supply of

IAA, IAA esters and other compounds closely related to IAA from the seed. These

move acropetally in the xylem and accumulate at the coleoptile tip. The production

of auxin in coleoptile tips involves the hydrolysis of IAA estersand the conversion of

labile, as yet unidentified compounds, to IAA. There is no evidence for the de nozto

synthesisof IAA in coleoptiles.

ro. Practically all the other sites of auxin production are sites of both meristematic

activity and cell death. The production of auxin in developing anthers and fertilized

ovaries takes place in the regressing nutritive tissues (tapetum, nucellus, endosperm)

as he ce lls break down. In shoot tips, developing eaves,secondarily hickening stems,

roots and developing fruits auxin is produced as a consequence of vascular differ-

entiation; the differentiation of xylem cells and most fibres involves a complete auto-

lysis of the cell contents; the differentiation of sieve tubes involves a partial autolysis.

There is no evidence that meristematic cells produce auxin.

r r. The lysis and digestion of cells infected with fungi and bacteria results in

elevated tryptophan levels and the production of auxin. Viral infections reduce the

levels of tryptophan and are associatedwith reduced levels of auxin.

r2. Crown-gall tissues produce auxin. It is suggested hat the crown-gall disease

may involve at any given time the death of a minority of the cells which produce

auxin and other hormones as hey autolyse; the other cells grow and divide in response

to these hormones.

r3. Auxinis producedin soils,particularlythose richindecayingorganicmatter, by

micro-organisms. This environmental auxin may be important for the growth of roots.

14. There is no convincing evidence hat auxin is a hormone in non-vascularplants.

The induction of rhizoids in liverworts by low concentrations of auxin can be ex-

plained as a response o environmental auxin.

r5. Abscisic acid is synthesizedfrom mevalonic acid in living cells. It is possible

that under certain circumstances, abscisic acid or closely related compounds are

formed by the oxidation of carotenoids.

16. The sites of gibberellin production are sites of cell death. It is possible that

precursors of gibberellins, such as kaurene, are oxidized to gibberellins when cells die.

17. Cytokinins are present in transfer-RNA (IRNA) of animals, fungi, bacteria and



548 A. R. Snnronern

higher plants. They are probably formed in plants by the hydrolysis of IRNA in

autolysing cells. There is evidence hat they are also ormed in living cells in root tips.

r8. Ethylene is produced in senescent,dying or damaged cells by the breakdown

of methionine.

19. It was shown many years ago that wounded and damaged cells produced sub-

stanceswhich stimulate cell division. It now seems ikelythat the

productionof woundhormones and the normal production of hormones as a consequenceof cell death are

two aspectsof the samephenomenon.Wounded cells can produce auxin, gibberellins,

cytokinins and ethylene.

zo. The control of hormone production in living cells is a biochemical problem

which remains unsolved. The control of production of hormones formed as a con-

sequenceof cell death depends on the control of cell death itself. Cell death is con-

trolled by hormones which are themselvesproduced as a consequenceof cell death.

2r. In spite of the fact that dying cells are present in all vascular piants, in al l

wounded and infected tissues, in certain differentiating tissues n animals, in cancerous

tumours and in developing animal embryos, the biochemistry of cell death is a subject

which has been almost completely ignored. Dying cells are an important source of

hormones in plants; some of the many substances eleasedby dying cells may also beof physiological significance in animals.

This review was written durins the tenure of the Roval Societv Rosenheim Research Fellowshio.

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