Biol. Ret:.(rg7S), 48,!0.509-559
509
BRCPAFI a8-a
THE PRODUCTION OF HORMONES IN HIGHER PLANTS
Bv A. R. SHELDRAKE
Departmentof Biochernistry,(Jniztersityof Cambridge
(Receizted
3 April rgn)
CONTENTS
I. Introduction
IL
III.
The
r.
z.
3.
4.
5.
6.
Sro
biochemistry of auxin production
Animals, fungi and bacteria
Higher plants
The biochemical control of auxin production
Other indole compounds
Bound auxin
The production of auxin by autolysing tissues
Sites of auxin production in higher plants
r. Coleoptile tips
z. Young leaves, shoot tips and buds
3. Senescent leaves
4. Dicotyledonous seedlings
5. Stems
6. Roots
7. Flowers, fruits and seeds
8. Cellular sites of auxin production
IV. Auxin production under pathological conditions
r. Fungal and bacterial infections
z. Animals
3. Viruses
4. Crown gall
V.
VI.
VII.
Environmental
Auxin
The
r.
z.
3.
4.
auxin
and lower plants
production of other plant hormones
Abscisic acid
Gibberellins
Cytokinins
Ethylene
VIII. The wound response
IX. The control of hormone production and distribution
X. Conclusion
XI. Summary
XII.
References
.
.
5ro
5ro
Srz
5r5
5r8
5rg
S2z
5"3
523
526
527
528
529
530
530
532
533
533
533
534
534
JJJ
$6
537
537
538
540
54"
542
5+4
545
s+6
548
nnr 48
5ro
A. R. Snnlonern
In orderto understandgro*thlr;)::ff""oilt:,1,
is necessary
to analysewhat are
in fact coherent,continuousprocesses
into a seriesof causesand effects.A great deal
has been found out about the effects of hormones on the control of growth and
differentiation in plants; this has provided some understanding of the causesof
developmentalchanges.But the production of hormones is itself an effect which
requires a causalexplanation.Something is known about the regions of hormone
production in the plant and the way in which hormonesmove from theseregions.But
very little is known about the cellular sitesof hormone production or about the way
in which hormone production is controlled. Physiologicalinvestigationsof hormone
production in whole organsor parts of organsdo not in themselvesshedmuch light
on these problems. Biochemicalinvestigationsof hormone production by tissuesor
homogenatesof tissuescontaining a mixture of cells provide information about the
biochemistryof hormoneproduction under experimentalconditions,but they do not
revealwhich cells producethe hormonesin,uizto.Unlessthe cellular sitesof hormone
production are known, it is almostimpossibleto understandhow hormoneproduction
within the plant is controlled at either a physiologicalor biochemicallevel.
The majority of this reviewis concernedwith the production of auxin, about which
a vast and confusingliterature hasgrown up over the last forty-five years.The production of the more recently discoveredhormones,the gibberellins,cytokinins,abscisic
acid and ethylene,will be consideredonly briefly. The results of biochemicaland
physiologicalinvestigationsof hormone production will be discussedin an attempt
to obtain a clearerunderstandingof the cellularsitesof hormoneproductionand of the
way in which hormoneproduction is controlled.The major conclusionto which this
discussionleads is that much of the hormone production in plants takes place as a
consequence
of cell death.
II. THE BIOCHEMISTRY
OF AUXIN PRODUCTION
There is a great deal of evidencethat the natural auxin of plants is indol-3yl-acetic
acid (IAA) (Thimann, 1969).Members of almosteverygroup of living organismsare
known to be capableof producing IAA; it is formed by numerousspeciesof bacteria
(Roberts& Roberts,1939;Stowe,1955;Wichner & Libbert, 1968)and fungi (Gruen,
1959);it hasbeenfound in a varietyof animals(Went & Thimann, rg37; Gordon &
Buess, ry6il and is producedin developingchick embryos(Robinson& Woodside,
r%7). Considerablequantities are excretedin human urine, which is one of the
sourcesfrom which IAA was first isolated.The IAA in human urine is not simply
derived from plant material in the diet, nor can more than a third of it be attributed
to auxin production by the microflora of the gut: the majority is actuallyformed in
the human body (Weissbach,King, Sjoersdma& Udenfriend, 1959).The rate of
human auxin production, expressedin terms that permit comparisonwith auxin
production in plants, is about 5-5o x ro-12g/*g/h. Coleoptile tips of Avena yield
that IAA
5o x ro-1' gl^glh (Went & Thimann, rg37). This comparisonemphasizes
production in plants should not be regardedas an isolatedbiochemicalphenomenon.
The production of hormonesin higher plants
5II
t. Animals,fungi and bactuia
IAA is producedin animalsas a consequence
of tryptophan catabolism.It is by no
meansthe major breakdownproduct: for example,the increasedexcretion of IAA
which follows the oral administrationof tryptophan to humansaccountsfor lessthan
o.ro/o of the tryptophan metabolized(Weissbachet al., ry59). The production of IAA
can occur as a result of the transaminationor the decarboxylationof tryptophan, but
the former is the predominantroute (Weissbachet al., ry59; Gordon & Buess,1967).
These reactionsyield indole pyruvic acid and tryptamine respectively(Fig. r). Indole
pyruvic acid canundergodecarboxylationto indole acetaldehyde,
which is alsoformed
by the action of amine oxidaseson tryptamine. IAA is producedby the oxidation of
indole acetaldehyde.
The production of IAA by bacteriaand fungi which occurs when tryptophan is
addedto the culture medium follows similar pathways.Although tryptophan degradation via tryptamine has been conclusivelydemonstrated,most bacteria and fungi
resembleanimals in the greater importance of the transaminationroute (Libbert,
Erdmann & Schiewer,rgTo).Again, it is important to bear in mind that IAA is only
one of severalpossibleproductsof tryptophan catabolism;for exampleindole pyruvic
acid is not only decarboxylated
but canalsobe reducedto indole lactic acid; and indole
acetaldehydeis not only oxidized to IAA but can also be reduced to tryptophol
(Fig. r). These substanceshave often been detectedin microbial cultures which are
degradingtryptophan(e.9.Kaper & Veldstra,1958;Rigaud,rg7oa, b). Only a small
proportion of the tryptophan supplied is converted to IAA: in cultures of Agrobacteriumturnefaciens,
for example,the maximum efficiencyis lessthan zo/o (Kaper &
Veldstra,1958).
The way in which indole pyruvic acid is convertedto IAA in animalsand microorganismsis not fully understood.Indole pyruvic acid is a rather unstablecompound;
in aqueous solutions, especiallyunder alkaline conditions, it breaks down spontaneouslyto give a number of different products,including IAA, indole acetaldehyde
and tryptophol (Bentleyet a1.,1956;Kaper & Veldstra,1958;Moore & Shaner,ry67).
Against this backgroundof spontaneousdegradationit is difficult to obtain evidence
for the participation of enzymes;and indeed there seemsto be no reasonto believe
that indole pyruvic acid doesnot decarboxylatespontaneously'in ohso,or that indole
acetaldehydeis not spontaneouslyoxidized.Enzymic oxidation or reduction of indole
acetaldehydemay alsotake place,and there is someevidencethat both occur (Kaper
& Veldstra, 1958; Rigaud, rg7oa, D).The relativecontributionsof enzymicand nonenzymic processesare, however,difficult to assess.Tryptophan can be convertedto
IAA inoitro by incubatingthe aminoacidwith a purified transaminasefromEscherichia
coli (Gunsalus& Stamer, 1955)in the presenceof a-keto glutarate(an amino group
acceptor)and pyridoxal phosphate(the co-factornecessaryfor transamination)but in
of any other enzymes(A. R. Sheldrake,unpublishedresults).This is hardly
the absence
surprisingin view of the instability of indole pyruvic acid, but it emphasizesthat, in
oioa, IAA could be produced simply asa consequenceof tryptophan transamination,
although the yields might be increasedby enzymescapableof decarboxylatingindole
32-2
A. R. Snrlonern
512
yt,
<\-fcu,CHco,n
\,4"/
Tryptophan
T ransaminay/
\carboxvlase
oHo
.I-^II
a\yn-cgrCuco,tr
V\"1 i i H
cH,dcortt
t-.----'--7
\An/
<'\--11-cH2cH2NH2
\,,\"/
H
Tryptamine
Indole pyruvic acid
Indole lactic acid
\/
4--t-T-.cH,cH2oH
<-\"/
HH
Tryptophol
=:==--;
\-\-/
L----- <-\---1cH,cHo
Indole acetaldehYde
t
Cl-'-r-cH2co2H
\A*/ H
IAA
Fig. r. Pathways of tryptophan degradation'
Conversely,the yieldsmight be diminpyruvic acid and oxidizingindole acetaldehyde.
to indolelactic acid and tryptophol'
compounds
these
ieduce
ishedby enzymeswhich
any other role in animals,fungi or
indeed
or
IAA is not known to play a hormonal
of tryptophan catabolism,
by-product
bacteria.It is perhupt b.tt tegardedas a minor
in influencingthe
importance
of
be
althoughfor organismspatholenic to plantsit may
responseof the plant to the pathogen.
z. Higher Plants
in
Numerous investigationshave shown that IAA is produced from tryptophan
rgTo;
plant tissues(for reviewsseeGordon, ry6r; Mahadevan,1964;Libbert et al',
results by
Wightman, 1973).Doubt was cast on the validity of some of the earlier
bacteria
epiphytic
circumstances
Libbert et al. tigOOlwho showedthat under some
The
tissues.
plant
could account for much of the auxin production by nonsterile
of
finding that sterile coleoptilesectionsof Atsenasathtawhich grew in the presence
&
IAA did not elongate when tryptophan was supplied (Winter, ry66; Thimann
a
as
tryptophan
against
evidence
Growchowska, r9OAl seemedto provide further
and
(1966)
al.
et
Libbert
precursor of IAA. In order to explain their results,both
which
fuir,t", (1966) proposedhypotheticalpathwaysof IAA synthesisfrom indole
that
did not involve tryptoph"tt. Wittt.r's conclusionswere basedon the assumption
synthesizeIAA in the sameway as other plant tissues'This
coleoptiletissues
"ot*"tty
The production of hormonesin higherplants
513
assumptionis not valid, for it has long been known that coleoptiletips have an anomalous auxin economywhich dependson a supply of auxin and/or 'inactive' auxins
from the seed;there is no evidenceto suggestthat de noooIAA production occursin
coleoptiles(Section III, r). Therefore the inability of sterile coleoptile tissues to
produce auxin from tryptophan in sufficientquantitiesto stimulate growth doesnot
support a generalargument againstthe role of tryptophan as a precursorof IAA. In
fact, sterile coleoptiletissuescan produce small quantities of IAA from tryptophan
(Libbert et al., 1968; Libbert & Silhengst, ry7o; Black & Hamilton, r97r). The
ineffectivenessof exogenoustryptophan as an auxin precursor in sectionsof.Aoena
coleoptilesappearsto be due to its rapid incorporationinto proteins,preventing any
significant increasein the intracellularlevels of free tryptophan (Black & Hamilton,
ry7r). There is good evidencethat other sterile tissuescan convert tryptophan to
IAA (e.g. Kulescha,gSz; Libbert et al. 1968;Sherwin& Purves,t969; Mitchell &
Davies, rgTz).Evidenceagainstthe hypotheticalpathwayof IAA synthesisfrom indole
without tryptophan as an intermediatehas been obtained by Erdmann & Schiewer
GgZr) and Black & Hamilton (r97r). Libbert et al. (tqZo) havenow concludedthat
IAA is, after all, formed from tryptophan in higher plants. Evidencefor this view has
continuedto accumulate(e.g.Gibson,Schneider& Wightman, rg72; Wightman, ry73).
The predominant way in which plant tissuescatabolizetryptophan is by transamination(Libbert et al., ry7o; Wightman, ry73). As in other organisms,the production of IAA occursas a result of the breakdownof indole pyruvic acid. There is some
evidencethalthis reaction can be catalysedenzymically,possibly by an oxidative decarboxylationanalogousto the oxidative decarboxylationof pyruvic acid to acetylcoenzymeA. This evidencedependson the stimulatory effects of thiamine pyrophosphate(Gordon, 196r; Moore & Shaner,1968)and lipoic acid (Gordon, ry6r),
which are co-factorsin other oxidative decarboxylations.There is also evidencethat
indole acetaldehyde,which can be detectedas an intermediatein the production of
IAA from tryptophan by the use of radioactivetracersand/or trapping agentssuch as
2,4-dinitrophenylhydrazine(Phelps& Sequira,1967;Khalifah, ry67; Wightman &
Cohen,r968; Moore & Shaner,1968;Gibson,Schneider&Wightman, rg72),maybe
oxidized enzymically.Aldehyde dehydrogenases
capableof carrying out this reaction
havebeendetectedin a variety of plant tissues(Rajagopal,1967;Wightman & Cohen,
1968; Gibson, Schneider& Wightman, r97z), althoughthe presenceof theseenzymes
does not in itself prove that they are normally involved in IAA production. It is at
present almost impossibleto assessthe relative contributions of enzymic and spontaneousreactionsin the formation of IAA from indole pyruvic acid in ahto.
In their recent review, Libbert et al. (tgZo) critically examinedthe evidegcein
favour of the formation of IAA from tryptophan via tryptamine and concludedthat
in higher plants the formation of tryptamine from tryptophan was unproven and
unlikely. However, there seemsto be no doubt that tryptamine can be detectedin
some,but not all, plant tissues(for referencessee Schneider,Gibson & Wightman,
rgTz). Severalhypothetical pathwaysto accountfor its formation without the involvement of tryptophan have been proposed(Libbert et al., rgTo\ but there is now persuasiveevidencethat somehigher plant tissuesdo in fact contain enzymescapableof
A. R. Snnronern
5r+
--dARLEY
TOMATO
Tryptophan
2opg
/
Tryptophan
/
transanlrnase /
44/
V
Indole pyruvic acid
\
Tryptophan
12 1tg
Tryptophan
\"'o;*ttu'"
Tryptamine
2 us.
/'-
Tryptophan /
transamnaseT/
I
I Indole acetaldehyde
dehydrogenase
I
t10
J
IAA
0.012pg
\ecarboxylasc
Indole ldctic uri63:s:=;Indole pyruvic acid
0.005pg
\
Tryptophol*=;Indole
Tryptophol
dehydrogenase
t7J8
Tryptophan
\
/\
/
Indole acetaldehyde
/\
Tryptamine
/"'
acetaldehyde
I tndo," acetatdehyde
I dehydrogenase
IAA
0.05pg
Fig. z. Pathways of IAA. formation. The numbers below the names of the indole compounds
represent 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 tryptophan to tryptamine (Sherwin, rgTo; Gibson, Schneider& Wightman, ry72; Gibson,Barrett& Wightman, rgTz; Wightman, rg73).Some,but not all,
plants contain amine oxidaseswhich can convert tryptamine to indole acetaldehyde.
The most intensive investigationof these enzymeshas been carried out with pea
(Pisamsatioum)tissues(Mann, 1955; Clark & lVlann, rySil but unfortunately this is
a specieswhich doesnot containtryptamine(Schneideret al., ry72).Gibson,Schneider
& Wightman (1972)have shown that in tomato (Lycopersicon
esculentum\
and barley
(Hordeurnoulgare)tissues,both of which normally contain tryptamine, tryptophan
can be convertedto tryptamine and this can in turn be convertedto IAA. Enzymes
capableof forming IAA from tryptophan via indole pyruvic acid are also present.
These authors have estimatedthe relative activities of the enzymesinvolved in the
degradationof tryptophan and also the naturally occurring amounts of tryptophan
and its degradationproducts in thesetissues.Their results (Fig. z) indicate that the
tryptamine pathway is relativelyunimportant. Further resultsfrom the samelaboratory haveconfirmedthat in tomato shootsthe primary pathway for the production of
IAA 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,known to animal physiologistsas serotonin (Schneideret al.,
r97z).In some,for examplein the stinginghairs of nettles (Collier & Chesher,1956),
it has a role in defenceagainstanimals;in othersits function is unknown. In animals
5-hydroxytryptamine is formed by the decarboxylationof 5-hydroxytryptophan
(Udenfriend, Titus, Weissbach& Peterson,1956)but in plants it is probably formed
by the hydroxylationof tryptamine (Gibson, Schneider& Wightman, rg72).Tomato
tissuescontainaboutfive timesmore 5-hydroxytryptaminethan tryptamine(Schneider
et al., rgTz).
The production of hormones'in higher plants
5r5
Tryptophan, in common with a number of other amino acids, can be degraded
in oitro by reaction with phenolsunder alkaline conditions; the mechanismappears
to involvean oxidativedeaminationby the quinonesformed by the oxidationof phenols
such as catechol(Gordon, 196r). The incubation of catecholwith tryptophan at high
pHs, and under less alkaline conditions in the presenceof phenolase,leads to the
production of small amountsof IAA (Gordon & Paleg, 196r; Gordon, ryfi; Whitmore &Zahser, t964i Wheeler& King, 1968).Gordon & Paleg(196r) suggested
that
this route of IAA production is probably of little significancein oiao becauseof the
compartmentalizationof the substratesin living cells, but they stressedits possible
importancein maceratedplant tissuesand during extractionprocedures.This point
has also been emphasizedby Whitmore & Zahner (1964).
Wightman (rgZl) hasshownthat when labelledphenylalanineis suppliedto tomato
shoots,a number of breakdownproducts including phenylpyruvic acid and phenylaceticacid are produced.The major pathwayof phenylalaninedegradation,like that
of tryptophan, is by transamination;indeed the sametransaminaseis probably involved.Phenylaceticacid hasweakauxin activity and occursnaturallyin tomatoshoots
(Wightman, ry73).
3. The biochemicalcontrol of auxin production
Plant tissuesconverttryptophan to auxin with efficienciesas low as,or lower than,
those found in animals,bacteriaand fungi. In short-term experiments,IAA rarely
accountsfor even as much as o.r o/oof the tryptophan supplied.Tryptophan is converted to auxin by auxin-requiring tissue cultures and can substitute for auxin as a
growth substanceif supplied at concentrationsabout a hundred times higher, implying an efficiencyof conversionof about r o/o(Kulescha,rgsz). None of the enzymes
thought to be involved in the production of IAA from tryptophan havehigh specificities. For example,a purified ' tryptophan transaminase'from Phaseolus
aureushas.a
higher activity with alanine,leucine,methionine,arginine,lysine, phenylalanineand
tyrosinethan with tryptophan (Treulson, rgTz). A purified tryptophan decarboxylase
from tomato shootsis much more specificfor tryptophan, but this enzymeis of little
importancein the production of IAA (Wightman, tg73). An amine oxidasefrom pea
seedlingscapableof oxidizing tryptamine to indole acetaldehydealso oxidizesa wide
rangeof monoamines(Mann, 1955).And there is no evidenceto suggestthat enzymes
which are able to oxidize indole acetaldehydeto IAA are specificfor this substrate.
The unspecificnature of theseenzymes,the probability of spontaneousbreakdownof
intermediatessuch as indole pyruvic acid and the low efficienciesof IAA production
from tryptophan suggestthat in plants, as in other organisms,IAA is formed rather
unspecificallyas a by-product of tryptophan catabolism.It would be misleadingto
think of the reactions leading to the production of IAA as a biosynthetic pathway
directly comparableto the efficient and specific enzyme pathways involved in intermediary metabolismor in most biosyntheses.A consequence
of this view is that the
control of IAA production from tryptophan is not likely to involve any very specific
regulatorymechanismsat the enzymiclevel.
Severalattempts to explain the control of IAA production in this way have been
516
A. R. Snnronerp
made,but they arenot persuasive.Rajagopal& Larsen(tgZz) purified an enzymefrom
non-sterileAztenacoleoptiletissueswhich catalysedthe conversionof indole acetaldehydeto IAA. They found that the best preparationsunder optimal conditionshad
a turn-over number of six moleculesof indole acetaldehyde/min/enzyme
molecule
and suggestedthat this extremely sluggishactivity might be of decisiveimportance
in the control of IAA biogenesis.This conclusionseemsimprobable if only for the
reasonthat coleoptiletissuesdo not normally synthesizeIAA. For similar reasons,
the findingsthat gibberellicacid enhancesthe growth of non-sterilecoleoptilesections
in the presenceof tryptophan (Sastry & Muir, 1965)and that gibberellic acid has a
promotive effect on the decarboxylationof tryptophan by homogenatesof coleoptile
tissues(Valdovinos& Sastry, 1968) do not support these authors' conclusionsthat
auxin biosynthesisis regulatedby gibberellic acid. These attemptsto demonstratea
direct effectof gibberellicacid on auxin biosynthesisstemmedfrom observationsthat
auxin production was enhancedin organsstimulatedto developby the applicationof
gibberellins. For example, tomatoes stimulated to develop parthenocarpicallyby
gibberellic acid produceauxin (Sastry& Muir, rg6l). But so do tomatoesdeveloping
parthenocarpicallyafter other chemicaltreatments(Section III, 7), indicating that
auxin is probably produced as a consequenceof fruit development.Similarly, the
enhancedauxin production by dwarf peasand Helianthu.splants stimulated to grow
by gibberellins(Kurashai & Muir, ry62) and in rosetteplants of Centaureastimulated
to bolt by gibberellins(Kurashai & Muir, 1963)seemslikely to be a consequenceof
the developmentalchangesbrought about over a period of daysrather than as a direct
effect of gibberellicacid on auxin biosynthesis.It is thereforedifficult to evaluatethe
significanceof the finding that enzymepreparationsfrom pea tissuespretreatedwith
gibberellicacidproducedmore 'ether-insolubleauxin'from tryptophan than controls
(Muir, ry6+).Valdovinos & Ernest (1966) found that homogenatesof plant tissues
which had beenpretreatedwith gibberellicacid in a detergentsolution releasedmore
uCOz from
[r-1aC]tryptophanthan controls; but even if this effect was due to the
gibberellicacidratherthan the detergent,its relevanceto the control of IAA production
is far from clear.
The widespreadoccurrenceof enzymescapableof catabolizingtryptophan with the
consequentproduction of IAA in animals,fungi, bacteriaand higher plants suggests
that the major factor controlling the production of IAA is the availability of tryptophan; many cells and tissuesdo not produce IAA in significant quantities unless
exogenoustryptophan is supplied. But all cells capableof protein synthesismust
contain a pool of free tryptophan. Therefore it is probable that these endogenous
levels of tryptophan are normally too low for tryptophan breakdownto occur. This
suggeststhat the affinities of the tryphophan-activating enzymes (responsible for
charging specifictransfer RNAs) arehigher than the affinitiesof tryptophan-degrading
enzymesfor tryptophan. The few availabledata on Michaelis constants(i.e. the substrate concentrationat which an enzymeis half saturated)support this view: aminoacid-activatingenzymesgenerallyhaveK-s betweenr x ro-o and r x ro-4 ivr(Novelli,
aureashas a K*of 17 x ro-3 M
1967),while the tryptophan transaminaseof Phaseolus
accordingto Gamborg& Wetter (tg6:) and 3'3 x ro-4 wraccordingto Treulson (tgZz).
The production of hormones'in higher plants
5r7
Tryptophan transaminaseand tryptophan decarboxylasefrom tomato shoots have
K-s of 5 x ro-a vI and 3 x ro-s rvrrespectively(Gibson, Barrett & Wightman, ry72).
The pathwaysof amino-acidbiosynthesishave been studied most extensivelyin
micro-organisms.In general,they are controlled by feed-backinhibitions whereby
the amino acid which is the product of the pathway inhibits an enzyme,or enzymes,
involved in its production, often at the beginning of the pathway; this inhibition
occurswhen the concentrationrises abovea certain level (Umbarger, ry6g). Similar
control mechanismshave been found to regulateamino-acid biosynthesisin higher
plants (Miflin & Cave, rg72; Miflin, rgn). In micro-organisms,tryptophan biosynthesisis regulatedby the inhibition of anthranilatesynthetase,an enzymeat the
beginning of the pathway, by tryptophan (Umbarger, 1969).There is evidencethat
tryptophan is synthesizedby a similar pathway in higher plants (Delmer & Mills,
1968)and it seemsreasonableto assumethat the control mechanismmay be similar.
Tryptophan biosynthesismust be regulatedin such a way that levels of tryptophan
sufficient for protein synthesisare maintained,but theseconcentrationsmust be too
low for tryptophan degradationto occur under most circumstances;otherwise all
plant tissueswould produceauxin all the time.
It is conceivablethat auxin productioncould be regulatedbya changein this control
mechanism such that higher concentrationsof tryptophan were synthesized.But a
simpler way in which the tryptophan levelscould be elevatedis by the degradationof
proteins.The free amino-acidpoolsin plant tissuesrepresentonly a small proportion,
often lessthan So/o,of the total amino acidswhich can be releasedby the hydrolysis
of the proteins(Allsop, 1948;McKee, 1958).In living cells,where there is a steady
turnover of proteins, proteolysispresumablycontributesto the steady-statepools of
free amino acids.But when net protein degradationoccurs,for examplein senescent
leaves, the levels of free amino acids are elevated considerably(Chibnall, r939i
McKee, 1958).
The amountsof freetryptophanin plant tissuesusuallylie in the rangeof ro-5o trglg
fresh weight (Schneideret al., rgTz). Higher amountsare found in shoot tips, young
leaves,senescentcotyledons(Nitsch & Wetmore, tg5z) and senescentleaves(Kim &
Rohringer,tg6g),all of which aresitesof auxinproduction(SectionII). Thesemeasurements give no information about the cellular distribution or intracellularlocalization
of tryptophan. It is thereforeimpossibleto deducethe intracellular concentrations.
The elevatedtryptophanlevelsin senescent
leavesand in the cotyledonsof seedlings
canbe explainedasa resultof protein degradation.The elevatedamountsof tryptophan
per unit weight in shoot tips and developingleavescould in part reflect the higher
ratio of cytoplasmto cell wall material in young tissues,and in part be explainedby
the net protein breakdownwhich occursduring vasculardifferentiation:xylem cells
and most fibresundergoa completeautolysisasthey differentiate,and partial autolysis
occurs in differentiatingsievetubes.
Many o-amino acids are convertedby plant tissuesto their N-malonyl derivatives
(Rosa& Neish, 1968)by what appearsto be a detoxificationmechanism.An analogous
conversionof o-amino acids to their N-acetyl derivativesoccurs in yeast (Zenk &
Schmitt, 1965).Malonyl-o-amino-acidconjugatesoccur naturally in a wide range of
-r-
A. R. SHnronern
5r8
H
Glucobrassicin
{
I
ll
\,A*/
Ascorbic z
acid
4/
Ascorbigen
ll
ii
3-Hydroxymethyl
indole
+Glucose
+so12+scN-
I
J
<i----"-cH,----A
tlt
ilililt
V-*/ H H
\*A/
<t\--T-.cH,co2H
\A"/
3,3'-diindolylmethane
*formaldehyde
Fig. l. The degradation of glucobrassicin
H
IAA
(after Gmelin,
1964\.
plants (Rosa& Neish, 1968)and malonyl-o-tryptophanhasbeendetectedin a number
of vegetativetissuesand fruits (Good & Andreae, rgST; von Raussendorf-Bargen,
ry62; Zenk & Scherf, rg6l). Apple (IlIaluspumila) fruits contain about o.z trglg fresh
weight (Zenk& Scherf,rg6:) and tomato shootsabout o.Spglg fresh weight (Good &
Andreae, rg1il.This compound appearsto be quite stable in plant tissues(Good
& Andreae, ry57). It is possiblethat in autolysingtissuesit could be hydrolysed,
releasingfree o-tryptophan which can be broken down by some plant tissuesto IAA
(e.9. Kim & Rohringer, 1969).However,the amountsof malonyl-o-tryptophanare so
small comparedwith the amountsof L-tryptophanin plant tissuesthat it is unlikely to
be of any significancefor auxin production.
4. Other indole compounds
A number of plants havebeenusedfor centuriesasa sourceof indigo dye, which is
formed by the oxidation and polymerization of indoxyl. Indigofera and Polygonum
tinctoriumcontain indoxyl-p-o-glucosideand woad (Isatis tinctoria) containsindoleB-o-5-ketoglucuronicacid (Stowe,Vendrell & Epstein, 1968).Indoxyl is releasedfrom
these compoundsby hydrolysis.Little is known about the indole metabolismof the
plants which produce thesecompounds,but there is no reasonto supposethat they
are precursorsof auxin or play any part in growth control.
A variety of mustard oil glucosides,or glucosinolates,are found in the Cruciferae,
Resedaceae,Tropaeolaceaeand certain other families. They appear to be stable
in oivo, but when tissuesare crushedor otherwisedisruptedthey rapidly break down
under the influenceof the enzymemyrosinase,which is probably confinedto special
cells (Virtanen, 1965).This reactionis of culinary importancein the preparationof
mustard and is also responsiblefor the releaseof the compoundswhich give plants
The production of hormonesin higher plants
519
such ascress(Lepidiumsatirrum)and Tropaeolwntheircharacteristicflavours(Virtanen,
1965).Brassicaand a number of other generacontainthe indole-glucosinolates
glucobrassicin and neoglucobrassicin.These compounds are present in both roots and
shootsin quantitieswhich canaccountfor up to lo/o of the dry weight (Gmelin, W64;
Virtanen, 1965; Elliott & Stowe, ry7r). Either spontaneouslyunder acid or alkaline
conditions,or as a result of the action of myrosinase,glucobrassicinbreaksdown to
form a variety of indole compounds(Fig. 3), the nature of the products being determined by the conditionsof the reaction (Gmelin, lq,6+;Virtanen, 1965). One of the
breakdown products, 3-hydroxymethyl indole, dimerizes with the elimination of
formaldehyde,but if ascorbicacid is present reacts with it spontaneouslyto form
ascorbigen.The pH optimum of this reaction is 5 (Schraudolf & Weber, ry69).
Indole acetonitrile is produced enzymically by myrosinase,but only if the pH is
below 5'z (Schraudolf& Weber, 1969).Reportsof the occurrenceof indole acetonitrile and ascorbigenin tissuesof Brassicawere made before the discovery of glucobrassicin,but thesecompoundsare now known to have arisenas extractionartifacts.
Little or none can be detected after extraction proceduresthat minimize the breakdown of glucobrassicin(Kutddek& Prochizka, ry64; Schraudolf & Bergmann,
r965).
Indole acetonitrileforms indole acetamidespontaneouslyunder acidic conditions;
indole acetamidecan be hydrolysedto IAA. The conversionof indole acetonitrileto
IAA is also effectedby the enzymenitrilase which is present in some, but not all,
plant tissues (Thimann, 1953). Indole acetonitrile can, if suppliedto tissueswhich
contain nitrilase, act as an auxin precursor; but there is no evidencethat it has any
such role in oizto.The hypothesisthat indole acetonitrilewas involved as an intermediatein auxin production (e.9.Wightman, tg6z) dependedon the belief that it was
of natural occurrencein speciesnow known to contain glucobrassicin.
The functions of glucobrassicinand neo-glucobrassicin,
like those of many other
secondaryplant products, are unknown. There is no evidencethat under normal
conditions they act as auxin precursorsor are involved in the regulation of growth.
But since they break down when tissuesare crushedor damaged,it is possiblethat
auxin could be produced from glucobrassicinunder pathologicalconditions.Gmelin
(rg6+) has suggestedthat a processof this sort might explain the existencein the
cabbagefamily of growth abnormalitiessimilar to those induced by exogenousauxin.
Some support for this view comes from the finding that in club-roots of Brassica,
causedby the pathogenPlastnodiophera
brassicae,indole acetonitrile is present,whereas it is not detectablein uninfected roots (Tamura, Nomoto & Nagao, rgTz).
5. Bound a:uxin
It has long been known that animalspossessa variety of detoxifying mechanisms
which involve the formation of amino-acid or sugar conjugates.Benzoic acid, for
example,is conjugatedwith glycine to form benzoylglycine (hippuric acid) which is
then excretedin the urine. In humansand chimpanzeesglutaminecomplexesare also
formed (Thierfelder & Sherwin, r9r4). A wide rangeof compoundsincluding phenylacetic 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 (Weissbach et 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 unphysiologically large amounts of IAA results in the formation of IAA complexes whose 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) is 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,ry62; Stidi,ry6+).
'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), suggesting that 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 level 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 IAAaspartate 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 considerable physiological importance under these circumstances. A number of dicotyledonous 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 seedsthere is at first a large increase in 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 both
free and bound auxin; the levels of free auxin decline as the seed matures (Avery,
Berger & Shalucha, rg4z; Hemberg, 1958; Hamilton, Bandurski & Grigsby, 196r).
The production of hormonesin higher plants
52r
About half the bound auxin of maize seedsconsistsof low molecular weight esters
which 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)suggested
that the bound auxin formed during seed developmentservesas a source of free
auxin in the germinatingseed.It is on the auxin releasedby the hydrolysis of these
auxin 'precursors' that the vicarious auxin economy of the coleoptiletip depends
(SectionIII, r).
Auxin is not the only hormonewhich is convertedto a bound form. Glucosidesof
abscisicacid (Milborrow, rgTo) and gibberellic acids occur naturally in plants. The
latter are formed during seed developmentfrom free gibberellic acids; these, like
auxin, are liberated by hydrolysisfrom their bound forms in germinating seedsand
embryos(Barendse,Kende & Lang, 1968; Barendse,rgTr; Dale, ry6g).
For many years the possible existence of auxin-protein complexes has been
shroudedin confusionand controversy.The releaseof auxin from proteinsby alkaline
hydrolysisor by proteolyticenzymeshasoften beentakenasevidencefor the existence
of protein-bound auxin. But resultsof this type can also be explainedby the release
of tryptophan, followed by the conversionof tryptophan to IAA. This conversion
takes place spontaneouslyunder alkaline conditions (Gordon & Wildman, rg$).
Auxin is producedby the alkalinedigestionof casein,but not of gelatinewhich contains negligible amounts of tryptophan (Gordon & Wildman, ry$). Wildman &
Bonner (rg+il found that auxin was releasedby the hydrolysisof spinachleaf protein
in quantities greater than they estimatedwould be produced by the spontaneous
conversionof tryptophan; they concludedthat an auxin-proteincomplexwas present.
However, Schocken(rg+g) showedthat Wildman & Bonner'sassumptionsabout the
rate of spontaneousproduction of auxin from tryptophan were wrong. He re-investigatedthe productionof auxin from the spinach'auxin-protein' and found that similar
yields of auxin were obtainedby the hydrolysis of a number of animal proteins; the
amount of auxin producedwas roughly proportionalto the tryptophan content of the
protein.Wildman & Bonner'sevidencein favourof the'auxin-protein'alsodepended
on enzymicdigestions,but the incubationconditions(+8 h at 37 oC under non-sterile
conditions)make it probablethat much of the auxin detectedwas formed by microorganisms.
Winter & Thimann (1966) claimedthat part of the IAA which is immobilized in
coleoptile tissuesduring polar auxin transport is in the form of an auxin-protein
complex. The majority of labelled IAA present in the tissues at the end of the
transport period was found in the sedimentwhen ground-up coleoptiletissue was
centrifuged. Radioactivitywas releasedfrom this fraction by treatment with proteolytic enzymesor urea. However, the binding of IAA was very labile since IAA could
be recoveredby extraction with ether. These authors were apparently unawareof the
ability of IAA to partition into lipid membranesin a pH-dependentmanner (Hertel,
Thomson & Russo, rgTz) much in the sameway that IAA partitions into non-polar
solventsat pHs below about 5. Their experimentsinvolved either unbuffered solutions, or a comparison of various treatments in solutions buffered at different pHs.
A. R. SHnr-onexn
522
Their resultsmay thereforebe explicablein terms of a pH-dependentassociationof
IAA with membranes.
Siegel& Galston (rgSg) claimed to have demonstratedthe formation of an IAAprotein complex in aitro by plant homogenates.The binding of IAA to protein was
stimulatedby nucleotides.These resultsare now known to havebeendue to an artifact causedby the use of tri-chloro-aceticacid as a protein precipitant (Zenk, 1964).
Evidencefor the formation of an IAA-transfer RNA complex (Bendanaet a1.,1965)
has alsobeenrefuted (Davies, rgTr).In a recentpublicationfrom the samelaboratory
the formationof IAA-polysaccharidecomplexeshasbeenpostulated(Davies& Galston,
ry7r), but the evidencefor their existenceis indirect.
Zenk (tg6+) has provided the only convincing evidence for the existenceof an
IAA-protein complex. A small proportion of labelled IAA supplied to pea epicotyl
tissueswasfound to be bound to a protein fraction fromwhich it could not be removed
by treatmentwith acetone,dialysisor by exchangewith unlabelledIAA. Theseresults
suggestthat the IAA was chemicallybound to the protein. It is not known whether
this IAA-protein complexhas any physiologicalsignificance,or indeedwhether it is
formed at all under natural conditions.
In extractionprocedures,'free' auxin is taken to be the auxin obtainedafter short
periods in the cold, whereasthe additional auxin obtained after longer periods of
extractionhas been consideredto be due to the releaseof free auxin from a 'bound'
form (Bentley, 196r). This definition of bound auxin is confusing.In some cases,
for example in the extraction of cerealseeds(e.g. Hemberg, 1955)and coleoptiles
(Wildman & Bonner, 1948)this bound auxin representsgenuineIAA complexessuch
as the lAA-inositol compounds.The free auxin which can be liberated from the
'bound auxin' of Brassicatissues(e.g. Avery, Berger
& White, 1945) is probably
formed as a consequenceof the breakdown of glucobrassicin.In other casesthe
'bound auxin' representsIAA produced from
tryptophan during extraction. The
addition of proteolytic enzymesto tissuesleadsto larger yields of IAA during extraction (Skoog & Thimann, r94o; Thimann, Skoog & Byer, ry42) probably because
more tryptophan is releasedfrom proteins.No auxin production occurswhen boiled
tissuesare subjectedto prolongedextractionwith ether (Thimann & Skoog, r94o).
6. Theproductionof auxin by autolysingtissues
The continuedproduction of auxin by plant tissuesasthey autolyseduring extraction with etheris probablydueto the continuedreleaseof freetryptophanfromproteins
and its subsequentdegradation.But it is not only during ether extractionsthat autolysis results in auxin production. Yeast,plant and rat liver tissuesproduce auxin as
they autolysein oitro (Sheldrake& Northcote, ry68a). fncreasesin the amount of
auxin of up to a hundredfold can occur within z+h.
The production of auxin by autolysing tissues in aitro suggeststhat autolysing
tissueswithin the plant might alsoproduceauxin. But it would be almostimpossible
to duplicate in the test tube the complex sequenceof changesthat occur during
the autolysisof a differentiating xylem cell, to take only one example.The pH of the
different cellular compartments may change as differentiation proceedsand as the
The production of hormonesin higherplants
523
intracellular membranesbreak down; the concentrationsof tryptophan would be
expectedto changeas it is releasedfrom intracellular compartments(e.g. plastids)
and the amount producedby proteolysiswould be affectedby the changingcompartmentalization of proteolytic enzymesand the changingpH in which they are operating.
These factors would also affect enzymes which break down tryptophan. It is also
probable that some of the substancesreleasedas the cell autolyseswould be metabolized by adjacentliving cells.
The techniquesof biochemistry are at presenttoo crude to investigatein any detail
changesof this type as they occur in ztizto.From a biochemicalpoint of view it seems
probable that autolysing cells would produce auxin; but biochemicalinvestigations
alone cannot reveal the extent and significanceof auxin production by such cells
within the intact plant. The problem must now be consideredat the physiological
and anatomicallevels.
III. SITES OF AUXIN
PRODUCTION
IN THE PLANT
With the exceptionof senescentleavesand coleoptiletips, practically all the sites
of auxin production in the plant are in regionsof meristematicactivity. This correlation between auxin production and meristematicactivity has led to the widely, if
implicitly, acceptedhypothesisthat auxin is producedby meristematiccells. A very
different hypothesisof auxin production has recently been proposed,according to
which auxin is produced as a consequenceof cell death (Sheldrake& Northcote,
1968a, b). The production of auxin by autolysingtissuesin oitro and by senescent
leavesin vivo indicatesthat dying cells can produceauxin; the sitesof auxin production in the plant can be explainedby the associationof regionsof meristematicactivity
with the cell deathswhich occur as nutritive tissuesregressand as vasculartissues
differentiate.In the following sectionsthe sitesof auxin production will be discussed
with thesehypothesesin mind.
The best reviews on the production and distribution of auxin in the plant are by
Siiding (tg;z, 196r) but these,like many of the publicationsin this field, are in
German.This important and interestingliteratureseemsto be little known in Englishspeakingcountries.
Investigationson auxin in plants have dependedeither on the use of extraction
techniques,'free' auxin being taken asthat obtainableby short periods of extraction
in the cold, or by the trapping of auxin diffusing from plant patrs in agar. The disadvantageof the diffusion method is that it dependsnot only on the amount of auxin
in the tissuebut also on the auxin transport system; the disadvantageof extraction
is that it may give a distorted picture of the amount of auxin availableas a hormone,
since some may be immobilized or compartmentalizedwithin the tissue.
r. Coleoptiletips
Auxin was clearly identified as a plant hormone in the classicalinvestigationsof
Went (1928)on the growth and tropisms of the coleoptilesof grassseedlings.Since
that time the coleoptilehas continuedto occupy a central position in 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 few
hours 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 substancesfrom 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, mostly
IAA esters (Section II, 5). Pohl (t935, r936) showed that a depletion of the amount
of auxin in the seed resulted in a decline in coleoptile growth. He suggestedthat 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 to the coleoptile tip where it was converted 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-sterile
conditions. 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 precursor. There is, however, no evidence that it does so inoizto. Raadts & Soding (tgSZ)
found that both IAA and a labile substance which could undergo spontaneous conversion to IAA could be detected in coleoptiles. Chromatographic investigations of
diffusates from coleoptile tips by Ramshorn (r955), Bohling (rgSg) and Shen-Miller
& Gordon (1966) also showed that, in addition to IAA, at least one other compound
with auxin activity in the coleoptile extension bioassay was present. This compound,
called P by Shen-Miller & Gordon, is not transported in the polar auxin transport
The production of hormonesin higher plants
525
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 treatment 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 even
declined. 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 presence of 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 in
the 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, including 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
substances moving 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 substances
accumulate 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 in the xylem from the seed to
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 extension 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 xylem
in the extreme tip of the coleoptile is asymmetrical (Thimann & O'Brien, 1965). Such
an asymmetric accumulation of auxin would account for the autonomous curyature
rnn 48
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 estersfound in suchlargequantitiesin the seed.
Perhapscompoundssuch as P detectedafter chromatographyin ammoniacalsolvents
representdegradationproducts of IAA esters,formed either during the extraction
proceduresor as a result of ammonolysis.
The production of auxin from 'inactive' auxin in the coleoptiletip was referred to
in the earlier literature as auxin actioation,a term which emphasizedthe difference of
this processfrom the de nouo synthesisof auxin which takes place in other parts of
plants. However in more recent, progressivelysimplified, accountsof this classical
work, the production of auxin in coleoptiletips is describedas auxin synthesis(e.g.
Bonner& Galston,rgsz; Leopold, ry6+).The vicariousnatureof the auxin economy
of coleoptiletips is thus obscuredand this hasresultedin considerableconfusion.For
example,the fallaciousconclusionsof Winter (1966) referredto on p. 5 13 depended
on the assumptionthat the coleoptilewasa typical site of auxin biosynthesis.Problems
of this sort disappearwhen it is rememberedthat there is no evidencefor the de novo
synthesisof IAA by coleoptilesin oioo.
z. Youngleaaes,shoottips and buds
Avery (rgSS)was amongthe first to recognizethe generalpatternof auxin production by developingdicot leaves;in hisstudieson Nicotianahefoundthat "auxin is
present only in growing leavesand that its concentrationis roughly inversely proportional to the age of the leaf". Similar results havebeen obtainedwith leavesof
Phaseohn(Shoji, Addicott & Swets, r95r; Humphries & Wheeler, ry6+; Wheeler,
1968), Solidago(Goodwin, 1937),Aster (Delisle, rg37), Coleus(Jacobs& Morrow,
rg1il and fronds of Osmunda(Steeves& Briggs, 196o).Auxin is producedthroughout
the period of leaf developmentin the basal meristematicregion of the leaf of the
unifoliate dicot, Streptocarpuswendlandii,while very little is present in the mature,
apical parts of the leaf (Hess, 1958).Developing monocot leavesalso contain more
auxin in the basal meristematicregion than in the rest of the leaf (van Overbeek,
r938).
Leaf developmentinvolves both meristematic activity and vasculardifferentiation.
Jacobs& Morrow (tgSZ) found a close correlation between auxin production and
xylem differentiation in Coleusleavesand interpreted this as showing that the differentiation of xylem is controlledby auxin; but the resultscould alsoindicatethat auxin
is produced as a consequenceof xylem differentiation: theseinterpretationsare not
mutually exclusive.DevelopingNicotianaleavescontainmore auxin in the veins than
in the lamina (Avery, 1935)which might indicate that it is producedin the differentiating vasculartissue.
It is well known that relativelylarge amountsof auxin are producedby shoot tips
(Thimann & Skoog,r934i du Buy & Neurnbergk,1935;Delisle,ry37; S<iding,1938;
Eliasson,rg6g) and developingbuds (Czaja,rg3+; Zimmerman, 1936; Sdding, rg37;
Avery, Burkholder & Creighton, rg37; Gunckel & Thimann, 1949; Diirffiing, 1963).
The production of hormonesin highn plants
527
leaoes
Table r. Theproductionof auxinby smescent
Species
Phaseolus uulgais
Age of attached leaves
(days)
r9
33
40
4o (shrivelled
leaves)
Free auxin content,
pg IAA equivalents/kg
fresh weight
e)
631
, q 6|
33zl
Author
Wheeler(t968)
Days after detachment
of leaves
Cucurbita pepo
o
II
Phaseolus aulgaris
o
Aaena satiaa
6
o
+
o.sI
50
,;\
1
,17
Conrad (rq6S)
Sheldrake&
Northcote (r968c)
The tissuesused in all these investigationsincluded not only the meristem but also
the submeristematicregion and young leavesand therefore contained both meristematic cells and differentiating vasculartissue.
3. Smescentleaoes
The production of auxin by young, developingleavesdeclinesas the leavesmature
(seeabove).Shoji et al. (r95 r), on the basisof a singlemeasurementof the auxin level
concluded that a further decline occurred
in senescentleavesof Phaseoluszsulgares,
during leaf senescence.This conclusion appearsto have been widely accepted,
especiallyby workers on leaf abscission(e.g. Addicott, rgTo). But there is now considerable evidencethat auxin is produced as leavessenesce.This effect has been
observedin Bryophyllum mmaturn(Raadts, tg6z), Cannabis(Conrad, ry62), Cucurbita
(Dcirffiing, 1963),Phaseolus
aulgaris(ShelPePo(Conrad, 1965),Acer psatdoplatanus
drake & Northcote, 1968c; Wheeler, 1968),Aoena sativa (Sheldrake& Northcote,
(Chua, ry7o) andPrunuscerasus
(Kaska,r97z).The increases
1968c), Heaeabrasiliensis
in auxin levels are large, often from 30- to a roo-fold (Table r). The treatment of
resultsin a suppression
detachedleaveswith kinetin, which retardstheir senescence,
of auxin production (Conrad, 1965). The production of auxin during senescence
could account for the findings that Bryophyllurnplants contain high levelsof auxin in
the autumn (Raadts,ry62) and that auxin levelsincreaseconsiderablyin plantskept in
darknessfor protractedperiods (von Guttenberg& Zetsche,1956).
The hydrolysis of proteins which occurs in senescentleaves results in elevated
levels of free amino acids (Chibnall, rg3g) including tryptophan (Commoner &
Nehari, 1953;Pearse& Novelli, 1953;Lihdesm?iki,1968;Kim & Rohringer,1969).
The degradationof tryptophan and the consequentproduction of IAA could occur
by transaminationand possibly,in somespecies,to someextent by decarboxylation;
systemcould alsobe of someimportancein cellsin an advanced
the phenol/phenolase
33-2
528
A. R. Sunrpnern
stateof disintegration.But the relativecontributionsof thesepathwayshave not been
investigated.
Measurementsof the auxin in senescentleavesof Acer pseudoplatanus
and several
other speciesin the autumn revealedthat the highestlevelswere presentin leavesthat
were actually falling from the trees (A.R. Sheldrake,unpublished results). This
auxin is thereforeof no importancefor the rest of the plant. But it seemspossiblethat
some of the auxin produced in senescentleavescould play a part in the control of
abscission.Numerous investigationshave shown that the application of auxin to
leavescould therepetiolesretardsabscission(Addicott, rgTo).The auxin of senescent
fore have an abscission-retardingeffectaslong as the transport systemby which auxin
moved down the petiole to the abscissionzonecontinuedto function. This might be
of considerablephysiological significance: leaves do not generally abscind until
is well advancedand it is this delay in abscissionwhich enablesnitrosenescence
genousand other compoundsto be translocatedback into the stem. Auxin produced
in the senescentleaf might help to retard abscissionuntil the transportsystemsthemselvessenesced.Thus the relatively small amountsof auxin which can be collected
by diffusion from the petiolesof senescentleaves(Bcittger,ry7o) reflect a decline of
the auxin transport systemrather than a declinein the amountof auxin in the leaves.
4. Dicotyledonousseedlings
Auxin is producedin the cotyledonsof seedlingsof Lepidium(van Overbeek,rg3z),
Raphanu.r(van Overbeek, 1933), Lupinus (Navez, 1933) and Phaseolus(Wheeler,
1968).The production of auxin in cotyledons(or in the endospermof seedlingswith
of the breakdownof reserve
endospermousseeds)can be explainedas a consequence
materials.The auxin production of cotyledonscould also be regardedas analogous
to the production of auxin by senescentleaves.The shoot tips of seedlingsare sites
of auxin production (Soding, rg1z), as are shoot tips in general.
The demonstrationthat the growth of grass coleoptilesis controlled by auxin
moving basipetallyfrom the tip led to numerousattemptsto explain the growth and
tropisms of dicotyledonousseedlingsin an analogousmanner. fn some species,e.g.
(van Overbeek,1933)it was found that the removal of the cotyledonsand
Raphanu.s
the shoot tip inhibited the growth of the hypocotyl. But in other speciessuch as
Lupinus(Dijkman, r934i Jahnel, r%il and Hel:ianthus(du Buy & Neurnbergk, rg32)
decapitation had little or no effect on the growth of the hypocotyls. The attempt to
explain all growth in terms of auxin meant that these results were interpreted by
postulatingthat a diffuseproduction of auxin took placein the growing regionsthemselves(Jahnel,rg37; du Buy & Neurnbergk,1935; Sciding,1952,196r). However,
Jost (r94o) concludedfrom his studieson beanepicotylsthat auxin alonecould not be
responsiblefor the control of growth and suggestedthat a secondhormone was also
involved. It is now known that gibberellins have striking effects on the growth of
stems(Cleland, ry69). A widely usedbioassayfor gibberellinsdependson the stimulation of hypocotylgrowth by thesehormones(Frankland& Wareing, 196o).The recent
demonstrationthat gibberellins becomeasymmetricallydistributed in shoot tips of
Helianthu.ras a result of geotropic(Phillips, tgTza) and phototropic (Phillips, rgTzb)
The production of hormonesin higherplants
529
stimulationand the finding that stemgrowth in this speciesis controlledby gibberellin
rather than auxin (Phillips, rgTza) mean that much of the early work on growth and
tropisms in seedlings(on which the auxin theory of tropismsis based)must be reconsidered.In the light of thesefacts it no longer seemsnecessaryto supposethat auxin
is produced in the elongatingregionsof seedlingstems.
5. Stems
Auxin is present in the basal meristematic region of growing monocot stems
(Schmitz, 1933) where both meristematicactivity and vascular differentiation are
taking place. Mature stems of monocots(in which no secondarythickening occurs)
containlittle or no auxin (Schmitz,1933).The growth of maturenodescanbe resumed
as a result of geotropicstimulation; in sugar cane,the intercalaryzonegrows from a
few millimetres to more than a centimetrein a wedge-shapedway when the stem is
placed horizontally. A marked increasein auxin occurs during this process(van
Overbeek, Olivo & de Vasquez, 1945). It is interesting to note that these authors
concludedthat rather than being a cause,auxin wasproducedas a consequence
of the
geotropicresponse.Auxin production has also been observedin geotropicallystimulated nodesin other grassspecies(Schmitz, 1933).Unfortunately in thesereports no
information was provided about the anatomicalchangeswhich accompaniedthe geotropic reactionand the associatedproduction of auxin.
Auxin is produced in secondarilythickening dicot stems (Zimmerman, $36;
Sciding,ry37, 1938,rg+o; Jost, ry+o; Allary, 1958;Hatcher, 1959;Diirffiing, 1963i
Sheldrake& Northcote, 1968&).The site of auxin production is the cambial region
itself (Stiding, 1937,1938,rg+o; Ddrffiing, 1963).In young leaves,developingbuds,
etc., a direct investigation of the cellular sites of auxin production has not been
possible; but the cambial region has the advantagefrom an experimentalpoint of
view that the tissuescan be separatedeasily,first by stripping off the bark and then
by u selectivescrapingof tissuesfrom the outside of the wood and the inside of the
bark. In this way Sciding (1937, 1938, r94o) showed that much more auxin was
present in the cambium and its young derivativesthan in the mature phloem and
xylem tissues.Becausehe consideredit intrinsicallyimprobablethat the differentiating
vasculartissuescould produce auxin, he concludedthat the auxin was produced by
the cells of the cambium itself. However, if the undifferentiated cambial cells are
separatedfrom the young, differentiatingphloem and xylem tissuesand their auxin
contents are analysedseparately,the highest amounts are found in the differentiating
xylem cells,lessin the cambium and leastin the phloem (Sheldrake,rgTra). Unless
there is a radial movement of auxin against a concentration gradient (which seems
unlikely), these results suggestthat auxin is produced in the differentiating xylem
tissue rather than in the cambium.
The same conclusion has been reached by
different experimental approach.
Segmentsof tobacco internodes maintained in "sterile culture produce auxin and
continue to do so for many months. This production of auxin is associated
with continuing secondarythickening; if cambial activity is eliminated, auxin production
ceases.In experiments with separated,regenerating bark tissue it was found that
530
A. R. Snnlonern
auxin production dependedon xylem differentiation,indicatingthat auxin is produced
as a consequence
of xylem differentiation(Sheldrake& Northcote, 1968D).The possibility that someauxin might alsobe producedin differentiatingphloem tissuewas not
eliminated.
The continuing activity of the cambium in isolatedstem segmentsin the absence
of exogenoushormones(Jost, t893, rg+o; Sheldrake& Northcote, 19680)indicates
that the system is self-catalysing.Auxin is known to be involved in the control of
cambial activity and vasculardifferentiation; it is also produced as a consequenceof
these processes.Some of the auxin produced in the cambial region is transported
basipetallyby the polar auxin transport system.In isolatedstem segmentsthis leads
to considerablyenhancedcambial activity towards the basalend of the explant and
alsoto the formation of a basalcallus(Jost, rg4o; Sheldrake& Northcote, 19680).By
contrast, tobacco stem segmentscultured in the presenceof.2,3,5-tri-iodo benzoic
acid, a specificinhibitor of polar auxin transport,exhibit pronouncedcambialactivity
all along the explants,resulting in the production of serriedranks of tracheidswhich
can exceedby severaltimes the amount of xylem originally present (Sheldrake&
very strikingly the 'positive feedback'inherent
Northcote, 1968b). This demonstrates
in the cambialsystem,which under normal circumstances
must be'damped'to some
extent by the removal of auxin by polar transport.
6. Roots
Auxin is produced in isolatedroots cultured in oitro (van Overbeek,1939),but in
the intact plant it is alsotransportedfrom the shoot into the roots (McDavid, Sagar
& Marshall, ry72) where it may be conjugatedor destroyed.In Lensroots little auxin
is found in the tip itself; most is found about 5 mm behind the tip (Pilet & Meylan,
1953) where xylem differentiation is taking place. Similarly, in Zea roots the tip
containslessauxin than the region behind the zone of elongation,and nearly all the
auxin is found in the stele rather than the cortex (Greenwood,Hillman, Shaw &
Wilkins, ryn). The sourceof this auxin could be the differentiatingvasculartissues,
althoughsomeof it could havebeentransportedacropetallyin the stelarregion. Other
sites of cell death where auxin could be produced are the differentiatingvascular
tissuesin the cambialregion of secondarilythickening roots, the regressingroot hairs
and the root cap. The latter two may be of particular importanceunder non-sterile
conditions.
Roots are very sensitiveto exogenousauxin which is often present in soils in concentrationssufficientto affectthem considerably(SectionV). Therefore environmental
as well as endogenousauxin could be important in the control of root growth under
natural conditions.
7. Flowers,fruits and seeds
Auxin is producedin considerablequantitiesin developingflower buds; its production declines as 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 earliest stagesof flower developmentare not known, but it seemsreasonableto
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 receptacle have been demonstrated in the strawberry (Nitsch, r95o). But although
hormones formed in the developing seeds influence the development of the fleshy
parts of the fruit to a striking extent in some species, in others the developing fruit
tissues may be more or less autonomous from a hormonal point of view. This must be
the case in 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 influences such as exposure to cold (Nitsch, r95z). Once fruit
development has been initiated it proceedsmore or less normally, although parthenocarpic 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 demonstrated 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 parthenocarpically developing tomatoes are found in the tissues of the central axis and partitions (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
tissues cultured in aitro require auxin; if dividing cells produced it, these tissueswould
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
tissues adjacent to them; and it is clearly unable to explain the production of auxin by
senescentleaves.This hypothesis would 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
The production of hormonesin higherplants
533
significantextent; and that autolysisresultsin increasedlevelsof tryptophan. Some
of the tryptophan releasedby autolysing cells may be convertedto auxin by adjacent
living cells; it is for this reasonthat I have referred to auxin production as taking
place as a consequenceof cell death,rather than simply in the dying cells themselves,
although thesemay well be the major site of tryptophan degradation.With the possible
exception of geotropically stimulated grassnodes, all the known sites of auxin production in plants are sites of cell death.
The sites of cytolysis within plants which, accordingto this hypothesis,are likely
to be sitesof auxin production are: regressingnutritive tissues(the tapetum, nucellus,
endospermand cotyledons),the dying cells of senescentleaves,possiblydying root
hairs and root-capcells,differentiatingxylem cells,most differentiatingfibres, possibly
differentiating sievetubes, and differentiating cork cells. Auxin is probably also produced by damagedand 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 enzymes of the host cells themselves or of the pathogen.
r. Fungal and bacterial infections
Tissues infected by bacteria or fungi usually contain considerably elevated levels of
auxin (e.g. Wolf, 1956; Gruen, 1959; Sequeira, 1965;Kim & Rohringer, 1969) and
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,it is possible that tryptophan released by 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 responseto 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 less stationaryanimalswhich live entirely enclosedwithin the plant tissues.
Animals whose posterior parts remain outside the plant do not produce galls (Mani,
1968).There is evidencethat gallsinducedby Meliodogynenematodescontain elevated
levelsof amino acids and auxin (Dropkin, 1969);it seemsprobablethat auxin may
also be produced in other galls. However, the wide diversity of galls suggeststhat
hormonesother than auxin are also involved; the balanceand sequenceof hormones
releasedmust be characteristicof the gall-inducingspecies.
3. Viruses
Unlike fungal, bacterial and animal pathogens,viruses do not sustain themselves
by digestingthe contentsof the host cells but rather by perverting the metabolismof
the cells to their own ends.Thus amino acidswithin the cells are used directly in the
synthesisof viral proteins. Tryptophan is found in the protein of many plant viruses
(Fraenkel-Conrat,r968); its utilization in infectedcells might be expectedto lower
rather than raisethe levelsof free tryptophan. This is indeed known to be the casein
virus-infectedpotatotubers(Andreae& Thompson, r95o). It is thereforeinteresting
to note that tissues infected with viruses generally contain less auxin than normal,
uninfectedtissues(S<iding,196r; Sequeira,i963).
4. Crowngall
In many species,tumours develop in the vicinity of wounds infected by virulent
strains of the crown gall bacterium, Agrobacteriumturnefaciens.
The proliferation of
the tumour cells continues even if the bacteria are killed, showing that a permanent
transformation, analogousto animal cancer,has taken place. The actualtransforming
(Beardsley,
agentmay be a virus, carriedasa temperatebacteriophagebyA. tumefaciens
r97z). Sterile tumour tissue continuesto proliferate when grafted into healthy plants
and can alsobe cultured in ztitroon a simple medium containingsugarand salts.These
facts indicate either that crown gall tissuesdo not require auxins and other growth
factors that are necessaryfor the growth of normal callus tissues,or that they have
acquiredthe ability to producethem. The evidenceis in favour of the latter explanation
(for reviewssee Beardsley,rgTz; Wood, r97z). Non-sterile(Link & Eggers, r94r;
Dye, Clark & Wain, 196r) and sterile(Kulescha,r95z) crown gall tissueshave been
shownto produceauxin. But they do not containunusuallyhigh levelsof amino acids
in general(Lee, r95z) or tryptophanin particular(Henderson& Bonner, rg5z).
All existing theoriesof crown gall seemto assumethat the tissuescontain a more or
populationof tumour cells (e.g.Braun, 196z; Wood, rgTz),Braun
lesshomogeneous
(tqS8) has proposedthat the systemssynthesizinggrowth hormonesare'activated'
in the transformed cells. An 'activation' of the auxin-synthesizingsystem would
presumably involve an increasedability of the cells to convert tryptophan to auxin
and/or an increasedsynthesisof tryptophan. But in fact crown gall tissues neither
show an enhancedability to convert tryptophan to auxin (Kulescha, r95z) nor do
they contain more tryptophan than normal callustissues(Henderson& Bonner, rgsz),
It is therefore necessaryto question Braun's assumptions.
Perhapscrown gall tissuescontain a more or lessstablemixture of cells,sometrans-
The production of hormonesin higher plants
535
formed, others normal. Even 'clones' grown from single cells can contain a mixture
of normal and transformedcells,sincereversionis known to be possible(Braun, 1959).
The differencesbetweenpartially and fully transformed tissues(Braun, r96z) could
be explainedon this basisin terms of the former having a lower propoftion of transformed 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,for example,if at any given time some of the transformed cells died, with
the consequentproduction of auxin and also, possibly, cytokinins (Section VII, 3).
Crown gall tumours do in fact contain considerablenumbers of dead and dying cells;
it has often been observedthat cell divisions occur in crown galls around necrotic
areasor zonesof tracheiddifferentiation(Robinson& Walkden,r9z3 ; Banfield,1935;
Manignault, 1953i Therman, 1956;Kupila, 1958).No comparablehistologicalstudies
of crown gall tissues cultured in zsitrohave been published, but in all the cultures
which I have examineddeadcells are quite common. The tumourous transformation
viewed in this light would not involve an 'activation' of the auxin synthesizingsystem
in all the cells but would lead to auxin production by causinga more or lessconstant
percentage of the cells to die. This could be thought of by analogy with lysogenic bacterial cultures where at any given time a minority of the cells are killed by
bacteriophageswhich in the other cells are integrated with the genome and remain
latent.
The autonomy of 'habituated' tissues which arise spontaneouslyfrom normal
callusesafter more or lessprolongedperiodsin culture might be explicablein a similar
way. But in the absenceof any quantitative data on cell death within these tissues,
or indeed within normal callustissues,the hypothesesadvancedabovecan be no more
than speculative.
V. ENVIRONMENTAL
AUXIN
r94o; Stewart&
Auxin hasbeendetectedin a wide varietyof soils(Parker-Rhodes,
Anderson, tg4z; Hamence, rg4+, 1946; Whitehead,1963; Sheldrake,r97rD). It is
producedfrom tryptophanby many soil micro-organisms(Roberts& Roberts,1939)
and is found in the highest amounts in soils rich in decaying organic matter. The
auxin content of the soil representsan equilibrium betweenproduction and destruction (Parker-Rhodes,r94o; Hamence,1946)and is presumablyalsoaffectedby factors
such as rain and leaching,
The elongationof root hairs can be stimulatedby extraordinarily low concentrations
of auxin: r x ro-4 pgllis sufficient to bring about a significant effect (Jackson,196o).
The growh of roots themselvesis also sensitive to exogenousauxin, often being
inhibited by concentrationsin excessof about to pgll (Whitehead, 1963).Thus roots
grown in this order of auxin concentrationhave a more densecovering of root hairs
which are also longer (Ekdahl, 1957).The concentrationof auxin in the soil solution
usuallylies in the rangeof r-5opg/l (Whitehead,r963; Sheldrake,ry7tb). Theseare
averagevalues; it should be rememberedthat the soil is made up of many microenvironmentswhere locally higher or lower concentrationsmay be present.
The effects of auxin on roots may well be of adaptive significance;the relatively
536
A. R. Ssnronarn
high concentrations of auxin associated with decaying organic matter could cause the
absorptive area of the growing roots to be increased where nutrients are most readily
available. For similar reasons, environmental auxin may also be important for bryophytes which grow in close association with their substratum. Auxin above about
rc pgll induces rhizoids in liverworts ; a variety of substrata supporting the growth of
bryophytes have been found to contain concentrations of auxin in this range (Shel-
drake,ry7rb).
VI. AUXIN AND LOWER PLANTS
Ideas derived from the study of the hormonal role of auxin in vascular plants have
been extrapolated to algae, bryophytes and fern gametophytes on the assumption that
auxin is synthesized in meristematic cells. But attempts to explain apical dominance
and the control of growth in these plants in terms of auxin may be based on a false
analogy if auxin is produced in higher plants as a consequence of cytolysis. Demonstrations that non-vascular plants are able to form auxin from exogenous tryptophan (e.g.
Libbert et al., 1966; Ahmad & Winter, 1969) do not prove that they normally produce
auxin or that auxin is a hormone in these plants.
Small amounts of auxin have been detected in a variety of algae (Conrad & Saltman,
ry62), but the use of non-sterile material renders these results equivocal since auxin
can be produced by micro-organisms epiphytic on algae (Libbert et al., ry66). Several
reports that IAA supplied in ethanolic solutions enhances the growth of algae have
been shown to be due to the ethanol rather than the IAA (Bach & Fellig, 1958; Street
et al., tg58). Other investigations of the effects of IAA have involved the use of
unbuffered solutions where the relatively high amounts cf IAA used may have
lowered the pH and thus affected the algae in a rather unspecific way (e.g. Jacobs,
r95r). In short, there seems to be no convincing evidence that auxin has specific
effects on algae and still less that it is an endogenous hormone.
The evidence for auxin as an endogenous hormone in bryophytes and fern gametophytes is equally unconvincing. There is no persuasive evidence that it is produced
in sterile tissues of these plants and attempts to demonstrate that apical dominance
and other inhibitory phenomena are controlled by auxin have involved the use of
high and probably toxic concentrations of IAA (for references see Sheldrake, ry7rb).
The only well-established positive response to auxin which occurs at low concentrations is the stimulation of rhizoid development in liverworts. This can be explained
in terms of a reaction to exogenous, environmental auxin (Section V).
This response may have evolved in the liverworts as an adaptive reaction for the
absorption of nutrients, with auxin acting as a messengerof decomposition and decay.
It could perhaps provide a clue for the understanding of the evolutionary origin of
auxin as a hormone in higher plants (Sheldrake, ry7rb). If the precursors of the
vascular plants had evolved a similar response, auxin would have been in a suitable
position to become an endogenous hormone when cell death and auxin production
within the plant became an integral part of development with the evolution of the
vascular system. Seen in this light, the response of roots and root hairs to environmental auxin might represent an evolutionarily primitive characteristic which has
been adapted and retained.
The production of hormonestn higlter plants
VII. THE PRODUCTION
J5t
OF OTHER PLANT HORMONES
This subject will not be discussedat such length as the production of auxin, partly
becausethe literatureis more recent,smallerand lesstangled;partly becauseseveral
recentreviewsare available:on 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 concentrateon thoseaspectsof the literature that have a bearing on the understandingof
the sites of productionof thesehormones:aswith auxin, it is only when the cellular
sites of synthesisare known that a clearer understandingof the control of hormone
production will becomepossible.
In the light of the evidencein favour of auxin production as a consequenceof cell
death, it seemsworth consideringthe possibility that other hormonesmight also be
formed as a result of cytolytic processes.There is no a priori reasonwhy they should
be; but converselythere is no a priori reasonfor assumingthat they are synthesized
in living cells. In the following sections I have attempted to weigh up the evidence
for and againstthesepossibilities.On balanceit seemsprobablethat abscisicacid is
synthesizedby living cells; that the synthesisof gibberellin precursorsoccurs in
living cells, but that the final oxidative reactionsnecessaryfor the production of these
of cell death; that cytokininsthough prohormonesmight takeplaceasa consequence
duced by living cells, at least in root tips, are also formed by dying cells; and that
ethylene is produced as a consequenceof cell damageand cell death.
t. Abscisicacid
Two pathwaysof abscisicproductionhavebeenproposed:by direct synthesisfrom
mevalonateor by the oxidativebreakdownof carotenoids(Addicott & Lyon, 1969).
The photo-oxidationof violaxanthin resultsin the production of xanthoxin, a naturally
occurring compound closelyrelatedto abscisicacid and with similar biological activity
(Taylor & Burdon, rgTo).
Abscisic acid has been found in a wide range of plants and in a variety of tissues
(Milborrow , 1967,1968).It is presentand probablyproducedin the shootsof seedlings
(Teitz & Diirffiing, 969), in young leavesand buds (Milborrow, 1967)and in developing fruit tissue (Rudniki, Pieniazek& Pieniazek,1968; Rudniki & Pieniazek,
r97o; Ddrffiing, r97r; Davis & Addicott, rgTz). A striking increasein abscisicacid
occurs in leavessubjectedto water stress(Wright & Hiron, ry72) and mature leaves
of treesproduceabscisicacidin responseto short-dayconditions(Phillips& Wareing,
leaves(Chin
rg59). Abscisicacid is producedin considerablequantitiesin senescent
fruit tissues(Rudniki,
& Beevers,r97o; Biittger, r97o) and in ripening and senescent
Machnik & Pieniazek,1968; Goldschmidt,Eilati & Goren, tgTz;Davis & Addicott,
rg72).
Taylor & Smith (tg6l) suggestedthat abscisicacid is producedin,uiaoby the photooxidative breakdown of carotenoids,especiallyin senescentleaves.This hypothesis
at first sight seemsto provide a plausible explanationfor the increasedabscisicacid
538
A. R. Sunronerc
production which occurswhen etiolatedtissuesare exposedto light (Wright, r968).
However, light brings about many changesin etiolatedtissuesand there is no reason
to supposethat the influence of light on abscisicacid production is direct. There is in
fact no evidencefor the production of abscisicacid from carotenoidsin aioo; and the
availableevidenceseemsto be againstit. In pea seedlingstreated with gibberellic acid
no relationship could be found betweenabscisic-acidproduction and changesin the
amounts of carotenoid pigments ('Iietz & Diirffiing, 1969). There is evidence that
abscisicacid is producedde noztoin ripening strawberries,rather than by the breakdown of carotenoids(Rudniki & Antoszewski,1968).The direct synthesisof abscisic
acid from mevalonatehas been demonstrated in wilting leaves (Milborrow, r97z)
and in this case again the evidence is against carotenoids acting as precursors.
Abscisicacid is producedby senescentleaves(Chin & Beevers,r97o) and ripening
fruits (Rudniki, Machnik & Pieniazek,1968) in the dark. Thus although the photooxidation of carotenoidsmay result in the formation of growth inhibitors under some
production
it doesnot appearto be the major pathwayof abscisic-acid
circumstances,
in the plant, even in senescenttissues; the predominant route of abscisic-acidproduction in aioo may well be the direct one. This de noao synthesisof abscisicacid
presumablyoccursonly in living cells.
z. Gibberellins
Gibberellinswerefirst isolatedfrom fungi; they arealsoproducedby somebacteria
(Vandura, 196r) and have been detectedin 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 synthesisof kaureneprevent the production of gibberellins
(Dennis, Upper & West, 1965) and when applied to higher plants act as growth
retardants.The conversionof kaureneto gibberellic acidsinvolves a seriesof oxidative
reactionswhich are little understood(West & Fall, tgTzi West, 1973).
Gibberellins are produced in young, developingleavesof.Phaseolus(Humphries &
Wheeler, ry64), Helianthus(Jones& Phillips, r966) and Taraxacum(Fletcher,Oegema
& Horton, 1969).The levelsof gibberellinsin mature leavesare low. In Helianthus
shoot tips, gibberellin production occurs in the young leavesrather than in the
meristemitself (Jones& Phillips, 1966).Gibberellinshavebeendetectedin the xylem
sap of a number of species(e.g.Phillips & Jones,r964; Skene,1967)indicating that
they are producedin roots; and the productionof gibberellinsby root tips+caps of
Helianthushas been directly demonstrated(Jones& Phillips, r966). There is indirect
evidencethat they are producedin the cambialregion. The applicationof auxin to
woody stems induces cambial development and tracheid differentiation; but for
normal vessel differentiation another hormone is necessary.This second hormone
was shown by Rehm (tg:6) and Jost (tg+o) to be formed not only in young
leavesbut also as a consequenceof 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, suggestingthat it was in fact gibberellin. Gibberellins are
The production of hormonesin higherplants
s39
formed in considerablequantitiesduring seeddevelopment(Cleland, 1969),by far
the largest amounts being produced as the nucellus and/or endosperm degenerate
(Corcoran& Phinney,r 962; Jackson& Coombe, 1966;Luckwill, Weaver& MacMillan,
1969; Chacko, Singh & Kachru, r97o). In developingseedsof Echinocystisthe great
majority of the gibberellin is found in the nutritive tissuesrather than in the embryo
(Corcoran & Phinney, 196z). As seed developmentproceedstowards dormancy,
free gibberellins are converted to bound forms, as glycosidesof gibberellic acids
(Barendseet al., 1968; Barendse,r97r; Sembdneret al., ry72). fn a number of
dicotyledonousspecies,free gibberellins have been shown to be releasedfrom these
bound forms after germination of the seeds;and at least the early stagesof seedling
developmentare independentof de noztogibberellinsynthesis(Barendseet al., 1968;
Dale, r969). Similarly, the 'production' of gibberellinsby newly germinatedbarley
embryosmay be due to their releasefrom a bound form (Cohen& Paleg,1967).The
releaseof free gibberellinsfrom a bound form hasalsobeenshownto occur in etiolated
wheat leavessoon after an exposureto red light (Loveys & Wareing, ry7r).
Taken as a whole, these observationswould seem to exclude the possibility that
meristematictissuesare major sites of gibberellin production: the apical meristem of
Helianthus does not produce gibberellins; the meristematic embryonic tissues of
developing seeds contain relatively little gibberellin and the apparent production
of gibberellins by germinating embryos can be explained in terms of a releasefrom a
bound form. Growing cells in general also seem unlikely to be sites of gibberellin
synthesis,otherwisethesehormoneswould not be a limiting factor for the growth of
stems.The only featurewhich all the sitesof gibberellin production havein common is
the presenceof dying cells; in root caps,in regressingnutritive tissuesand in differentiating vasculartissue. In this connexionit is interestingto note that gibberellinsare
produced at or near the wounded surfacesof potato and Jerusalemartichoke tuber
tissuesshortly after cutting (Rappaport& Sachs,r967; Kamisaka& Masuda, 1968;
by thesedata is that
Bradshaw& Edelman,1969).The simplesthypothesissuggested
gibberellins are produced as a consequenceof cytolysis. This suggestionmay seem
both surprising and improbable from a biochemicalpoint of view. But it is not necessary to supposethat gibberellinsare synthesizedfrom mevalonatein dying cells; more
immediate precursorssuch askaureneor kaurenoicacid could alreadybe presentand
then only the final oxidativereactionsnecessaryfor the conversionof thesecompounds
of cell death.This hypothesisappears
to gibberellinsneedtakeplaceasa consequence
gibberellin
levels decline rather than increase
to be in conflict with the finding that
in senescentleaves(Fletcher et al., t96g; Chin & Beevers,r97o) and fruits (Goldschmidt et al., ry72) but perhapsin thesecasesthe cells do not contain the necessary
precursors.
Fungal cultures do not producegibberellins in significantquantitiesuntil the phase
of exponentialgrowth has ceased.There is, however,someevidencethat gibberellins
are produced in the stationary phase metabolically rather than autolytically, since
autolysis within these cultures does not become apparent until a rather later stage
(Borrow et al., tg55; Jefferys, r97o). It is difficult to assessthe relevanceof these
findings to gibberellin production in higher plants, but they perhapsweakenthe case
540
A. R. Snoronexr
for thinking that gibberellins are produced as a consequenceof cell death, or at least
of autolytic processes.
3. Cytokinins
A diphenyl urea isolated from coconut milk has cell-division-stimulating activity
(Shantz & Steward, 1955); and cell division factors, thought to be nicotinamide
derivatives,were isolated from severaltypes of cell cultured 'in oitro (Wood, Braun,
Brandes& Kende, l969). Thesecompoundshavenow beenre-identifiedaspurinones
(Wood, r97o). With these exceptions,the naturally occurring cytokinins are adenine
derivatives,found in plants as free basesand as ribosidesand ribotides. The sameor
closelyrelated compoundshave been found in the transfer RNA (IRNA) of animals,
bacteria,fungi and higher plants(Skoog& Armstrong,rgToi Kende, l97r). There is
strong evidence,summarizedby Kende (rg7t), that cytokinins in tRNA are synthesizedby the attachmentof an isopentenylgroup, derivedfrom mevalonate,to adenine
in preformed IRNA, that plant tissuesrequiring an exogenoussupply of cytokinin
as a growth factor are capable of synthesizing cytokinins in IRNA and that free
cytokinins are not involved in any direct way in the formation of cytokinin nucleotides in tRNA. But while the cytokinins of IRNA are not derived from free
cytokinins, the free cytokinins could be derived from the cytokinins in IRNA by
hydrolysis (Sheldrake& Northcote, 19686).Hydrolysatesof IRNA from animals,
micro-organismsand plants are activein cytokinin bioassays
(Bellamy,r966; Skoog
et al., 1966;Letham & Ralph, 1967).
Cytokininsare found and probably producedin developingfruits and seeds,particularly in the nutritive tissues(Steward& Shantz,1959;Skoog& Armstrong, ryJo)t
in germinating seeds(Barzilai & Mayer, 1964),in young leavesand buds (Engelbrecht,
r97r), developingflower petals(Mayak,Halevy &Katz, tgTz), roottips + caps(Weiss
& Vaadia,1965;Short & Torrey, ry7za) and in the cambialregion(Bottomleyet al.,
1963; Nitsch & Nitsch, 1965).Although the hydrolysisof IRNA with the releaseof
cytokinin ribotides, ribosidesand free basesmight take place in living cells, it would
be almost inevitable in dying, autolysing cells. The tissuesin which cytokinins are
produced contain dying cells, either in regressingnutritive tissues,in root capsor in
differentiating vascular tissues; in the latter caseRNA breakdown takes place not
only as xylem cells and fibres differentiate and die, but also during sieve-tubedifferentiation which involves the loss of the nucleus, ribosomesand most cell organelles
(Northcote & Wooding, t968).
The production of cytokinins by autolysing cells might lead to an apparently
paradoxicalsituation in senescentleavessince these hormones are known to retard
leaf senescencein many species.Nevertheless,there is evidencethat a cytokinin is
produced in senescentleavesof Populusand Acer (Engelbrecht, ry7r). fn senescent
leaveswhich contain low levelsof cytokinins, any cytokinin production which occurs
may be maskedby a rapid destruction and metabolismof the hormones(Srivastava,
1968).There is evidencethat cell-division-stimulatingsubstancesare produced in the
yellowing regions of slowly senescingcherry laurel leaves; Godwin Qgz6) observed
zonesof cell division on the outskirts of the yellowing regions, resemblingthose that
:;:fi:fr,T"l':
""0::,#:r::::of
hormones
inhigher
ptants
j't":F*evr",,,,s"1i
,lT[',:1;"rl$lll*ll$***ii*:i:H,'"x'.:
r.i'i..iv,"""f;.jl1**'..i'."f.?l,:I"+#,iJ,"'.,
tmmediatety
adjacent*
species remain
is impliedby t
j#l:1t".r
reaves
of cherry laurol' n;;;^r.-""'s rnat the regions
rilx:
iiir:;1:*i*l*$'{t+}l}i#H,T1ff
"ri.'i""'
].,r"."^i.::_""
areas
point
'o'ir,".*o,o;;"r;i:l
enislandst'u l' a';^i*!";;
1;$,;;;';['iJ:lJ'TJ[fffiTjJlsion1(Motn".,
l?#[ili;T d*ffi :ffi
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:
"
ffi${*f*il*?liffi
i#i'ii*H*::rii
j:i::'*r,,i",,-,ii"it
.::":F,,,:.:
:,'*:"*.",*1,f
:r:*i:r,{:"".,;JilT,'#H,rj;Ei*;;4;ni,:j'y
"'T'I:;#i'.",;:lT:Tf
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$'f;
ffiilT:,:Tff
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numbers
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if a criticalIevel
5r.':J::".""'iif'::!fr",1;i**ifi"*i,TT'l*
p'*:***#lr,nt"rt'"f;Jt:it,1rJffi
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kin"''nNa.
in;;; i;,h::ltli:
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;;;ru-;;g;;;ffi
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";,ffi
;';:ffiIt";:ilI:"^,1'+'Fi#td*":IIII#:'#T#"?:?
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quantitative
34
investigat
_-_,vyrwrurrns
are releasedfrom
the IRNA
"::r."rjr.a*
542
A. R. Snnronexr
micro-organisms,decayingorganicmatter, etc., in the soil. Presumablythey are also
degradedin the soil; but perhapsthe levelsof environmentalcytokinins might be
sufficient to influenceparts of plants sensitiveto exogenouscytokinins. For example,
the induction of buds on mossprotonemataby low levelsof cytokinins(Bopp, 1968)
could representan adaptiveresponseto environmentalcytokinins analogousto the
adaptiveresponseof liverwortsto environmentalauxin (Sheldrake,rgTrb).
4. Ethylene
Severalpossibleprecursorsof ethylenein the plant havebeen proposed(Mapson,
1969),but the most probableis methionine(Yang & Baur, r97z). Methionine at the
C-terminal end of peptidesproducedby proteolysiscan also act as an ethyleneprecursor (Demorest & Stahmann, rgTr).Ethylene can be releasedfrom methionine
non-enzymicallyby the action of hydrogen peroxide in the presenceof certai.nmetal
ions,and,'inaitro, by the actionof peroxidase,
but the enzymicmechanismof ethylene
production in aivo is not yet known (Yang & Baur, ry72; Lbeles, ry72).
A burst of ethyleneproductionoccurssoonafter plant tissuesare wounded(Burg,
quantitiesareproducedin diseasedtissues(Williamson,r95o).
ry62) andconsiderable
Under normal conditionsethyleneis producedby senescingleaves(Morgan, Ketring,
petioles(Rubinstein& Abeles,1965),senescingflower
Beyer& Lipe, rgTz),senescing
petals (Mayak, Halery & Katz, rgTz), ripening fruits (Burg, ry62) and germinating
seeds(Spencer& Olson, 1965).It is alsoproducedin young, growing tissues(Burg,
1968)in which vasculardifferentiationis taking place.The applicationof toxic compoundssuch as coppersulphateto plant tissuesleadsto'stress-induced'ethylene
production (Abeles & Abeles, ry72). Ethylene production is also induced by the
administrationof unphysiologicallyhigh concentrationsof auxin (e.g. Chadwick &
Burg, 1967;Burg & Burg, 1966)probablyas a result of non-specificdamage(Muir &
Richter, rg72). A rise in ethylene production follows the irradiation of fruits or
vegetativetissues(Pratt & Goeschl,1969).In all thesecasesethyleneproductioncould
or death.
be explainedas a consequence
of cell damage,senescence
Ethyleneproductionis increasedwhenColeus(Abeles& Gahagan,r968) and tomato
(Leather, Forrence& Abeles,r97z) plants are placedhorizontally.In the latter case
the plantswererotatedon a clinostat;the increasedethyleneproductionwastherefore
of the geotropicresponse.It seemspossiblethat these
unlikely to be a consequence
ethyleneproductionas
observationscould be explainedas instancesof stress-induced
a result of literal, physical stress.
Ethylene is produced in the soil by micro-organisms(Lynch, ry72) and some soils
contain sufficient quantities to affect the 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). In
many cases it 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 hormonesin higherplants
543
studies showed that the wound responsewas influenced
by substancesreleasedby the
damaged cells which he called wound hormones,
or more generally necrohormones.
The more pronounced wound. responsein the neighborrrhoJd
of vascular bundles was
interpreted by Haberlandt (rgra) to be due to;leptohormones,difiusing
from the
vascular tissues. cell division around dead or dyinj
ceils has been observed in many
other situations, for example in tissue cultures (Jones,
Hildebrand, Riker & wu, r96oi
'genetic'
crown gall tumours (section IV,
tumours of Nicotiana(Hagen, Gunckel &
4),
sp1t19*, 196r) and around necrotic areas infected
with viruses (Esau, r93g).
cell division around wounded areas usually ceases
after a few days; the wound
responseis self-limiting. This shows that the dividing
cells do not themseiv.. p.od.r""
the necessarystimulus for cell division, but that
ceil division depends on the wound
stimulus. It is interesting to imagine what would
happen if the cells around the
wounded area possessedsome 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 new
wound response would take place around the dying
cells and then, after this new
wave of division, further cell death might ensue, and
hence further division, and so on.
The result would be an autonomous, tumourous tissue.
This is essentiallythe mechanism proposed for crown gall in Section IV,4.
. .Haberlandt Q9z8) suggested that necrohormones might be of importance in the
initiation of the periderm, which often occurs
below necrotic areas where hair cells
have died or where epidermal cells are disrupted
by the growth in circumference of
the stem' He was unable to explain the continued activity'of
the cork cambium once
it had been initiated. However, by a simpre extension
oi the necrohormone concept
this could be seen as a consequenceof the differentiation
of the cork cells, which die
as they differentiate' Similarly, the division of
the cells of the vascular cambium
adjacent to differentiating- xyl,em cells provides
a striking analogy to the wound
response. Thus the idea that hormone production
o".rrrr1, a consequence of cell
be arrived at independently ofa knowledge of the
chemical identity of the
*:l^:t
normones.
A-substance capable of inducing cell division in bean
pods was isolated by Bonner &
English (tqr8) and given the name traumatin. But the
bean-pod assayis very unspecific
and traumatin is inactive in other cell-division assays
(Fox, 1969); it i, prorutty oi
little importance in the wound response in most
tissues u.rd
in bean pods its
physiological significance is far from clear.
"rr",
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;; ,o
gibberellins
(sectionvII, z), ethyrene
(bectionvII,
u.ri, frobubty]
1e
i.,ir*
(SectionVII, 3). The productionof .or.r"o, all of these4)
"yiot cells
iro.-orr". by wounded
could explain the effects of necrohormones; for example,
u .o-birrution of auxin and
cytokinins 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 seen as two aspects
oi the same phenomenon (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 compartmentalization of substrates and co-factors, on the control of enzyme synthesis at the
transcriptional and translational levels, and on feedback mechanisms involving allosteric enzymes. Factors such as these presumably control the production of 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 to understand, at least in general terms, 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 important 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 differentiation of vascular tissues, the regressionof nutritive tissues and the senescenceof 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. But
in 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 leaves, differentiating xylem cells, fibres
and cork cells undergo a progressive autolysis and disintegration as they die. To start
with they are living; at the end of these processesthey 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 seem very fruitful to pursue. However, this point is more interesting
when considering sieve tube differentiation, which involves a controlled, partial
autolysis. Many of the biochemical changesthat 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 cell
constituents takes place. Whole organelles such as mitochondria are broken down in
vacuoles,which can be regarded as lysosomes(Mathile, 1969). Thus some of the autolytic processesthat 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 stressinduced ethylene production. Hormone production as a consequence of cell death
could be seen as an extreme caseof hormone production by autolytic processeswhich
may occur to some extent in living cells.
The distribution of plant hormones depends not only on the amounts produced
The production of hormonesin higherplants
5+5
and on the sites of production, but also on their movement and destruction. Auxin
moves in a specific transport system basipetally in shoots and acropetally in roots
(Goldsmith, 1969). There is evidence that gibberellins (Jacobs, ry72) and abscisic
acid (Milborrow, 1968) can move basipetally in shoots. Both auxin and gibberellins
(Phillips, tg7za,6) become redistributed in tissues as a result of phototropic and
geotropic stimulation. Gibberellins (Phillips & Jones, 1964; Skene, 1967) and cytokinins (Kende, r97r) move from the roots to the shoots in the xylem. Little or no
auxin is found in the xylem sap of trees, which contains auxin-destroying enzymes
(Sheldrake & Northcote, ry68d). Gibberellins (Kluge, Reinhard & Ziegler, 1964) and
abscisic acid (Bowen & Hoad, 1968) have been detected in phloem sap. These movements account for the effects of hormones in regions remote from their sites of production; but it is probable that their movement over short distances by diffusion is
important for the control of processeswhich occur in the immediate environment of
hormone-producing cells; for example, cell division and new xylem differentiation in
the neighbourhood of differentiating xylem cells (Sheldrake & Northcote, 19686).
Littie is known about hormone destruction and immobilization in oiao, although it
is clear that under some circumstances the conjugation of auxin, abscisic acid and
gibberellins is important in the regulation of hormonal levels. A great deal of work on
the destruction of auxin by oxidasesin plant homogenateshas been carried out (e.g.
Galston & Hillman, 196l) but its relevance to the destruction of auxin in oiao is
obscure. Indeed the extent to which endogenous hormones are actually destroyed
rather than conjugated, lost from the plant (e.g. hormones in abscinding leaves;
ethylene escaping by diffusion) or simply diluted by growth is not known.
X. CONCLUSION
All sorts of biochemical processestake place when cells die and autolyse. Proteins are
hydrolysed, releasing peptides and amino acids; amino acids are degraded to a variety
of different products; nucleic acids are hydrolysed to oligonucleotides, nucleotides,
nucleosides and free bases; lipid membranes disintegrate, releasing enzymes, substrates and salts that were previously localized within cellular compartments; oxidation reactions occur (for example in the browning of cut potato and apple tissues)
which would not take place in living cells. Autolysing cells must releasea great variety
of compounds into their immediate environment and it seems almost inevitable that
nearby living cells would be influenced in some way. Cells die as they differentiate in
all vascular plants; cells die in all organisms as a result of wounding and infection; cell
death occurs in animals in some types of differentiation (e.g. of keratinized skin cells)
and as cells are turned over in most mature tissues; many cancerous tumours in
animals contain considerable numbers of dead and dying cells; and several tissues or
groups of cells regressand die during the development of animal embryos. It is perhaps
significant that a number of substances with hormonal activity in animals are, like
IAA, breakdown products of amino acids: tyramine, tryptamine, 5-hydroxytryptamine
and histamine. The latter two are involved in wound and inflammation reactions.
Yet in spite of the widespread occurrence of cell death in the development of animals
546
A. R. Snnlpnexr
and plants, the biochemistry of dying cells has hardly been investigated at all; it is not
even mentioned in most textbooks of biochemistry. But it cannot be ignored. Haberlandt showed many years ago that, in plants, damaged and dying cells could produce
hormones; and it now seems likely that hormones are produced as a consequenceof
cell death during normal development. The evidence discussed above is strongly in
favour of the normal production of auxin occurring in this way; it seems probable
that autolysing cells can also be a source of 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 observed that the evidence for the production of hormones as
a consequenceof cell death is in many cases indirect and circumstantial. But hypotheses are guessesas to what might be the case rather 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 synthesized by living cells. The latter, however implicitly it is accepted, cannot be taken for granted. Only further research can
establishthe relative contributions of living and autolysing cells to hormone production
in plants.
XI. SUMMARY
r. Although much is known about the effectsof plant hormones and 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 in 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 is
the more important.
3. In higher plants auxin is also produced as a minor breakdown product of tryptophan, 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 probable importance of spontaneous,non-enzymic reactions in the degradation of tryptophan 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 Brassica and a number of other
The production of hormonesin higherplants
547
genera. It breaks down readily to form a variety of products including indole acetonitrile, 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 developing seedsand fruits. There is no persuasive
evidence for the natural occurrence of 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 in aitro.
8. During the senescenceof leaves proteolysis results in elevated levels of tryptophan. Large amounts of auxin are produced by senescentleaves.
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 esters and the conversion of
labile, as yet unidentified compounds, to IAA. There is no evidence for the de nozto
synthesis of 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 the cells break down. In shoot tips, developing leaves,secondarily thickening stems,
roots and developing fruits auxin is produced as a consequence of vascular differentiation; the differentiation of xylem cells and most fibres involves a complete autolysis 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 suggestedthat 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 they 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 that auxin is a hormone in non-vascular plants.
The induction of rhizoids in liverworts by low concentrations of auxin can be explained as a responseto environmental auxin.
r5. Abscisic acid is synthesized from 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 that they are also formed 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 substanceswhich stimulate cell division. It now seemslikely that the production of wound
hormones and the normal production of hormones as a consequenceof cell death are
two aspectsof the same phenomenon. 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 consequence of cell death depends on the control of cell death itself. Cell death is controlled by hormones which are themselves produced as a consequenceof cell death.
2r. In spite of the fact that dying cells are present in all vascular piants, in all
wounded and infected tissues, in certain differentiating tissues in 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 substancesreleasedby dying cells may also be
of physiological significance in animals.
This review was written
durins the tenure of the Roval Societv Rosenheim Research Fellowshio.
XII,
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