Biochem. Physiol. Pfla nzen 176, 218-227 (1981)
Indole-3-Acetic Acid and its Metabolism
in Root Nodules of Pongamia pinnata (L.) PIERRE
B. K. SINHA and P. S. BASU
Department of Botany, University of Burdwao, Golapbag, Burdwan, West Bengal, India
Key Term Ind ex : root nodule, leguminous plants; indoleacetic acid, indoleacetic acid oxidase,
peroxidase, methyleneoxindolc reductase; Rhizobium spec., POllgamia pinnata.
Summary
The IAA content of root nodules of different leguminous pla.nts was estimated colorimetric ally.
The nodules from the plants of Mimosaceae contained no detectable amount of lAA. Among the
members of Papiiionaceae, there was a large variation in the amount of IAA in the nodules; those of
Potlgamia pilHlutu(L.) PIERRE contained quite a large amount. More tha.n 50% of the IAA became
oxidized during extraction by the endogenous oxidizing enzymes. This co uld be checked by addition
of inhibito rs of the enzymes during extraction. The lAA was metabol ized in the nodules, which was
evidenced by the presence of the IAA oxidizing enzymes in the nodules. The micro-symbiont lRhizobill 1ll sp.) produced an appreciable amount of IAA in the culture when su pplied with I..-tryp to J)han.
Introduction
The root nodules of some leguminous and non-leguminous plants are known to
contain appreciable amount of IAA. Among non-leguminous plants, DULLAART (1970a)
found that root nodules of Alnus glutinos{I contained substantially more IAA than
roots. He also showed (1968) that IAA content of root nodules of eyeas eircinales did
not differ significantly from that of the roots. The nodules of bean, soybean, pea and
lupin were found to contain extractable auxin (LINK and EGGERS 1940 ; DULLAART
1967, 1970b). The lAA level in nodules of pea was estimated to be 40-60 times higher
than in roots (PATE 1958). The auxin contained in nodule tissue was reported to remain in free condition (THmANN et al. 1942). Opinions differ regarding the presence
of IAA-metabolic enzymes, e.g., IAA oxidase, in the nodules (WAGENKNECHT and
Bumus 1950 ; DUl.LAAllT 1970b).
The purpose of this communication is to present some data regarding content,
metabolism and production of IAA in nodules of Pongami{l pinnata, a leguminous tree,
as most of the previous information was mainly in respect of the allllual herbs. The
results of a survey of the lAA and IAA-oxidase content in some other leguminous
plants are also presented here.
Materials and Methods
materials
Roots a.nd root nodules of Potlgamia pilllla ia (L.) PIERltE, Crotala.ria rdusB L., CliiQria lerna lea L.,
M'imosa. plIdica L., Acacia arabica WlLLD. were used.
Pla/lt
IAA .Metabolism in Root Nodules of Pongamia
219
Extraction of IAA from root nodules
The method used for extraction of IAA was a modification of the method specified for the purpose
by different authors (DULL-U.R'I' 1967; HAMILTON et 301. 1961; STOESSL and V"~NNIS 1970). Known
weight of washed, healthy and cut open root nodules were preincubated for 2 h in 20 ml of 10- 3 M
EDTA and then crushed with 70 ml of chilled 80% ethanol in cold in presence of the inhibitor of
same concentration and kept at 4 °0 with occasional stirring for 1 h. The solution used for pre~
incubation was utilized during extraction. The extract was filtered tlrrough cloth and centrifuged
to make it clear. It was evaporated at 28 °0 until the water solution remained, to which equal volume of 1 N NaHC03 solution was added and was acid ified ,\ith H2 S04 to pH 3. The mixture was
extracted four times, each time with equal volume of peroxide free diethyl ether. The pooled ether
extract was evaporated at 37°C and the residue was dissolved in 95 % ethanol which was used for
colorimetric estimation of lAA (GORDON and WEBER 1951) using standard curve prepared from
authentic IAA (Sigma Chemical Co., U.S.A.). In some extraction EDTA was omitted or replaced
by 2,2'-dipyrid yl or chI orogenic acid or a mixture of the two.
Biollssay of tlte exlracted l . .lA
For bioassay, the extracted IAA from the nodules was purified by 'fLO on silica gel G developing
with 5 % isopropanol and isopropanol: ammonia: water (8: 1: 1) running side by side with authentic
IAA and checking the concentration by colorimetric method after TLC. Straight growth measurement of wheat (Triticum vulgare, cv. Sonalikil,) coleoptiles were utilized for bioassay of IAA, by the
method described earl ier (B.\su and TULl 1972 a). The length of the coleoptile segments was measured
with a fine scale (1 liivn. = 0.39 mm) watching through ,\ simple microscope (x 10 magnification).
Sig nifi cant differences between mean val ues were analysed by the method of DUNCAN (1955). In
each case growth of coleoptile sections were greater at 1 % level of significance than in the control
sect ions.
Extraction a.Jld estimation of I AA oxidtlse
Healthy nodu les (5 g) wcre crushed with 20 ml of cold 0.02 II K H2 PO" buffer, pH 0.3. The extract
was filtered through doth and centrifuged at 8,000 rpm in cold and the snpernatlmt was used as
crude enzyme extract.
The IAA oxidase was estimated as done by other authors (GALSTON and DALB 'ERG 1954; SACHER
1962 ; SEQUEntA 1964) with little modification. The reaction mixtu re co nsisted of 1 ml each of 1 m:U
2,4-dichlorophenol, 1 mM l\InCI 2 , :2 mM IAA, enzyme extract (di luted if necessary) a.nd 6 ml 20 roM
citrate buffer, pH 4.5. The rcaction mixture was incubated a.t 30°C for one hour with occasional
stirring. The residual] AA was estimated by Salkowski reagent (GORDON a.nd WEBER 1951). IAA
oxidase from root was extraded and estimated in a sim.ilar way. The protein content in the enzyme
extract was estimated by the method of LOWRY ct i~l. (1951).
Extraction atlll assay of peroxidase
The enzyme was extracted ll.nd assayed following KAR and MISURA (1976). Nodul es or roots were
erushed with 50 mM phosphate buffer, pH 6.8. The extract was centrifuged at 8,000 rpm in cold and
was used as crude enzyme extract. The activity of the enzyme was estimated by the amount of
purpurogallin formed from pyrogallol which was ralculated from a standard curve prepared from
a.uthentic purpurogallin (Sigma Chem iCitI Co., U.S.A.). The assa.y mixture (5 1ll1) contained 125,umole
phosphate buffer, pH G.8, 151lmoie pyrogallol, 50,amole H2 0 2 and 1 ml of the 20 times diluted enzyme extract. The re,\ction was stopped aftcr 2 min by the addition of 0.5 ml of 5 % H2S0~ and
cstimation WI\S done spectrophotometri caliy at 420 nm.
Extractiott atld assay of mcthylclleoxiJldole reductase
The enzyme was extrMted a.nd assa.yed following lIoYED ,\l1d WILLIAMSON (1967). Roots or nodules (5 g) were extracted in cold in 20 ml of 10 m:\l phosphate buffer, pH G.5. The extract was centrifuged in cold r\,t 8,000 rpm and was llsed as ('TIlde enzyme extract.
13. K
220
SINHA
and P. S. BAS U
The assay mixtnre (4 ml) co ntained 1.3 ml of 0.1 i\[ phosphate buffer, p H 6.5, 1..15 ml distill ed
wa.ter, 1ml of 1 x 10- 3 M methylene oxindole so lution, 0.1 tnl of t he enzyme extract and 0.10 011
of NADPH (1 mg/ml). Ox idation of NA DPH was measured spettropho to metriC'.ally by dC('fcase in
the nbsorbancy at 340 nm.
Preparation of methylene QxitJdote
3-llromooxindole-3-acetic acid was prepa.red by readion of IAA wit h N-bromOSI1Ctilli lllide
(HI NMAN a.nd BA UMAN 1964). This compound in water is rapidly converted to a-methylene oxilldole
which was lI sed for assa.y of meth ylene ox indol e redu ct ase.
Culture of Rhizobium 8p. and production of 1A11 by the orgallis'lll
The microsymbiont was isolated from the root nodules of P.1)i/ltiata and was identified to be a.
Rh izobium sp. Identifi cation of the microorganism was by fo llowing the methods given in the Manual
of Mi cro b iolog j c,~ 1 Methods (Society of American Bacteriologists). The organi sm was grow n in broth
of inorgani c sa lts (S IDDIQ UI and n ,\ NER J}:E 1975) in which 0.1 % Casamino aeids were added and
NaCI was replaced by CaCI 2 • To stud y the production of IAA by the organism, soluti on of trypto·
phan or other chemicals was :\dded to the med ium during inoculation. The organi sms were grow n in
50 1111 medium co ntained in 250 mlErlenmeyer flasks and incu bated at 30 °C on a rota-r y sb:Lker.
Gro wth was measured turbidimetrically in an EEL (Evans Electroselinium Ltd. , England) colorimeter at 540 nm \Ising a 16 mm cuvette. The IAA prod uced in the medium was estimated co lorimetricall y ( GORDOO a.nd W EBER 1951) a.fter centrifuging down t he cell s.
SlalisticltL analysis
Sta.tistical analysis of the results were determined by the methods as described by PA NSE and
SUKIIAT>lE (1967).
Results
The IAA contents in roots of all the plants studied were far below the detectable
level Crable 1). The nodules of M. pudica and A. arabica contained no detectable
amount of IAA. But appreciable amount of IAA was found in the nodules of the other
plants, ranging from 2.1/.1g to 206p.g per gram of nodules (Table 1 and 2). The minimum
amount was found in O. ternaJea and the maximum amount was found in P. pillllOta,
O. retusa contained an intermediate amount of 93/.1g 01 lAA per gram of nodules.
Table 1. Variation in IAA COllteflt and Level of l ilA-oxidase it, foots and root 1Jodules.
The experimental pro cedure is described in the text
N.D. ~ Not Detect.ble
Pla-nts
Ctiioria tcrnatea L.
Crotalaria fetusa L.
j f imosa pudica L.
Acacia arabico
WlLLD.
Critic.l difference (CD)
p ~ 0.05
JAA content
lAA-oxidase
("g/g fresh ",eight) (pg of lAA converted / mg of prote in/h)
Protein conte nt
(mg/g fresh weight)
Nodule
2.1
93.1
N.D.
N.D.
14.75
Root
N.D.
N.D.
N.D .
N.D.
Nodu le
Root
Nodu le
Root
10.2
153.3
111.9
51.5
25.0
13.4
15.0
18.5
21.2
7.5
14.3
6.1
44.0
N.D.
N.D.
5.87
8.93
0.91
EG
1.08
221
IAA Metabolism in Root Nodules of POl1gltfnia
Table 2. l tlcrellse ill tlt e lI1nOtWt of lAA extract from root 'liodules of P. pinnatll USI IIg different chemicals
durillg e:draction.
Th e experi mental procedure and co ncentration o[ the chemica.ls used are described in the text.
Nodules were extracted [or one hOllr with occasional stirring except when mentioned otherwise.
By simil:~r extnwtion proced ure, with or without inhibitors , no detecta.ble IAA was round in the
roots of t he pla.nts
Amount of IAA
extractable
(I'g lAA{g of
nodule)
Chrmil'als used
No trea,tmellt, extnwted
i mmediately (within 2 min)
No treatme nt
Chlorngenic a(·ill
2,:t-dipyrillyl
Chlorogenic :wid :md
Net increase I) in lAA
extracte d
(/Ig lAA{g of
nodule)
6B
95
13:!
38
72
1tii
:!1O
115
~06
111
2,:!'-tlipyric\yl
EDTA
CD
7.18
P - 0.05
I) Increase calculated wit h reference to the set'ond row of the b\ble.
Table 3. Levels of enzymes related to lAA-tnetabolisUl £'1 P. pitiliula.
The ex perimental procedure is described in the text.
Pla.nt part
Nodul e
Root
CD
P
=
(Ill; IA A converted/h)
( 11lg
Pero xi dase
)lurpmogallin
formed)
~lethylene oxindole
reductase (Del'rease in
.~bsorbaney .~t 340 nm)
Per 100 mg Per mg
fresh we ight prutein
Per 100 mg 1'er mg
fresh wcight. prot.ein
Pcr 100 lUg Per IIIg
fresh weight protein
I AA oxida.se
:t7li.9
10S.0
~.24
U:U1
IOG.S
O.7H
U 3
4.41i
U'!
0.29
0.11
0.086
0.89
(l.~~
(I.~I
0.08
O.OO:'!6
U.015
0.05
Lowest activity of IAA oxidase was found in the nodules of C.lernatea (Table 1)
and highest activity was observed in P. pinnata (Table 3). Roots of all the plants studied however, contained appreciable amount of IAA oxidase though the IAA content
was not detectable in any of them. In fact, roots of all the plants studied contained
more IAA oxidase than the nodules when measured as I'g of IAA converted per rug of
protein per hour ('rable 1 and 3).
222
B. K.
SIX H A
and P. S.
BASU
150,----------------,
130
g, 110
c
o
w
'u6
" 90
cr
Concentration ILog,. Ml
Fig.l. Effect of ill/th,,,ti, 1.4.1 (e)
coleoptilcs.
a"a
U .1 purified from "odules (0)
011
elongation of ,,'heat
The experimental proceuure is described in the text. The concentration of IAA from nodules wa.s
caleu lu.ted by colorimetric estimatinon using a stantlanl curve prepa.red from authentic IAA.
Root nodules of P. pinnata showed presence of high level of IAA, which amounted
to 95,Ltg of IAA per gram of nodule (Table 2). When the e!l<traction was done immediately (within 2 min) instead of allowing the e!l<traction time for 1 h, the amount was
reduced to 63,Ltg showing incomplete enraction. In presence of chi orogenic acid, the
amount of e!l<tractable IAA went up to 133,Ltg. The net increase was by 38,Ltg per gram
of nodules i. e., by 40 %. In a similar e!l<periment, in presence of 2,2'-dipyridyl, the amount
of IAA was 167,Ltg showing a llet increase by 72,Ltg per gram of nodules. The increase
was about 76%. Presence of chlorogen ic acid and dipyridyl together during extraction
increased the amount of enraeted IAA to 210,Ltg. The net increase was 115,Ltg per gram
of nodules, which was actually more than double (121 %) than in the control. Almost
similar result was obtained using EDTA during extraction (Table 2).
The authenticity of the c!l<tracted IAA was checked (a) by the colorimetric test
which was dcscrihed by GORDON and WEBER (1951) as a colour reactions for IAA having
high specificit.y and (b) by bioassay which was comparable with results for authe.ntic
IAA (Fig. 1).
The level of three IAA-metabolising enzymes, IAA oxidase, peroxidase and methyleneoxindole reductase were studied in root and root nodulrs of P. pinna/a.. Levels of
all the enzymes were much higher in the nodules than in the roots when the lewis of
the enzymes were estimated per 100 mg of fresh weight (Table 3). But when estimated
IAA :Metabolism in Root Nodules of Pongamia
223
160~------------------~
:: 120
~
E
7
~
c
5
"0>
"-
0
..J
W
w
£
~
3
0
~
Cl
40
~~'~------~2-------3~!---'1?
ConcentratiOtlimg/mll
Fig. 2. Production-of 1 AA by Rhizobium sp. in culture medium supptimented uith DL-tryptophan
(0) L-tryptophan(.).
The experimental procedure and medium composition arc described in the test. Growth in presence
of DL-tryptophan (£;). L-tryptophan (.) was measured after 120 h of incubation. Bars indicate the
standard errors.
per mg of protein, the difference between the levels in roots and in the nodules came
down. This was due to the fact that the nodules always contained much more protein
than the roots.
The Rhizobium sp. obtained from the nodules of P. pinnata was found to produce
IAA in the culture medium when supplied with tryptophan. L-Tryptophan was found
to be more suitable precursor of IAA production than DL-Tryptophan evidenced by
higher level of IAA production (Fig. 2). In presence of L-Tryptophan, the auxin production increased proportionately up to 110 pg IAA/ml by 2 mglml of the amino acid,
then proceeded towards plateau with higher concentration of the precursor. In spite
of the difference in IAA production by L-Tryptophan and DL-Tryptophan, the bacterial growth had no difference when those were added in the medium to study the production of IAA (Fig. 2).
An attempt was made to increase the IAA production by the Rhizobium sp. by manipulation of the nature of the cell wall and membrane of the organism. Chemicals
like EDTA or antibiotics like bacitracin and penicillin were used for this purpose. The
chemicals were used in sublethal level so that the bacterial growth was not hampered.
The results as presented in Table 4 show that the antibiotics failed to increase the
IAA production. But in presence of 5"g/ml EDTA in the medium, the production was
increased from 103"g/ml to 160 "g/ml with a net increase of 57 "g/ml of IAA without
hampering the bacterial growth. EDTA of 10 "g/ml was inhibitory to the cells as both
the IAA production and growth became inhibited.
224
ll. h.
SINH ,\
and P. S. B,\su
'fable 4. Effect on grou:th (lila release of JA A produced by Rhizobium sp. by different chemicals af(ecttl1g
its nature of cell tl'll H.
The procedure is des(;ribed in the te xt. The concentration of L-tryptopha.n was 2 mg/ml in a.1l the
cases
Chemicals
Control
llacitmtin
Batitracin
Batitf<ltin
Penicillin
Peni cillin
Penir.iJlin
EDTA
EDTA
EDTA
1) S.E.
Concen-
Production of
tmtion
(I'g/ml)
IAA (I' g IAA /ml
of medium)
1.0
0.5
U.t
l OU I V/ml
50 IU lml
20 1U1", 1
10.0
=
110.0
87.6
97.0
105.0
95.0
107.n
107.i)
47.0
0.0
liiO.O
1.0
100.0
±S.K')
Growth
± S.E.
(EEL Unit)
0.1
6.8
~. S
0.5
6.2
2.4
G.O
4.4
6.2
6.2
3 .)
5.8
8.1
G.G
0.7
G.1
liJi
5.7
G.8
G.8
O.lS
0.17
O.lS
O.lS
0.16
0.23
0.2
0.15
0.2G
o.J
Standa.rd Error.
Discussion
The level of IAA in the nodules of the plants studied revealed some interest iug
findings. The nodules of thc monbers of MimosarC(le, M. pudica and A. ambica, o.ontained no detectablc amount of IAA. But appreciable amount of IAA was found in
the nodules of membcrs of Papili o nace ~e which showed some relation with the habit
of tIlC plants. The minimum amonnt was found in C. ternaiia which was a creeping
shrub, an intermcdiate amollnt was 101llld in the undershrub, C. reiusa and the maximum amount was found in P. pinnala which was a large trcc. The IAA content in the
root of all the plants st udied was far below the detectable level. This result was in
accord with the data obtained by others (LINK and EGGERS 1940 ; PATE 1058; ]) "LJ.AART 1n07) s}lOwing that the roots cont ained less amount of IAA than the nodules .
The root nodules of Vicia. {ara and Lupinll s /uieu s arc reported to contain 0.4 ftg and
0.25 - 0.4f1g JAA per gram Ofll0dules respectively (WHEELER et al. 1979). The papilionaceous plants studied here showed much larger amount of IAA in the nodules.
Add ition of EDTA during extraction of IAA increased the amount of the extractable
auxin . Thi s was a result of an attempt to increase the extractable IAA from nodules
of P. pin nata using different chemicals dUl"ing extraction. There was a possibility of
oxidation of the endogenous IAA during e,,-(raction by the IAA oxidizing cnzymes, IAAoxidase and perolCidase. To prevent this possibility> specific inhibitors of the enzymes
were used during extraction from P. pir/nata (Table 2). In presence of chlorogenic acid,
1In inhibitor of IAA oxidase (RAlllN alHl KT.EIN 1957), the amount of ext.raetable IAA
went upto 40 % . In a similar experim ent, in the presence of 2,2'-dipyridyl, the amount
of IAA was increased by 76 %. Dipyridyl was expected to inhibit the perolCidase by
lAA Metu.bolism in Root Nodules of Pongamia
225
chelating Fe+' ions required for its activity. In an attempt to inhibit both the IAA
oxidizing enzymes during extraction by using both the inhibitors together, the extractable IAA was found to increase by an amount which was equal to the sum of the
increase by these chemicals when used alone. Almost similar results were obtained
using a general chclator of metal ions, EDTA, during extraction (Table 2). EDTA was
used during extraction of IAA from all other plants (Table 1). Extraction for 1 h was
sufficient to extract completely the IAA present in the nodules (SINHA and B~su
1980).
Estimation of IAA oKidase in the nodules of these plants (Table 1 and 3) COI1firmed the presence of the enzyme. Lowest activity of IAA oxidase was found in the
nodules of the plant which contained lowest amount of IAA (Table 1) and the activity increased consistently with the increase in the IAA content. The nodules of the
members of Mimosaceae contained no detectable amount of IAA oxidase, which also
contained no IAA. Roots of all the plants studied, however, contained appreciable
amount of IAA oxidase though the IAA content was not detectable in any of them.
The data presented here go against the findings of WAGENKNECHT and BURRIS (1950)
who found no IAA oxidase present in the root nodules.
The auxin present in nodule tissue remains in free condition (THIMANN and SKOOG
1942) which is transportable unchanged from the nodules to other plant parts (BAUMA
1970; WHEELER et al. 1979). The data presented here show definitely that at least a
part of the IAA present in the nodules is metabolised within the nodules. The nodules
of P. pinnata were found to contain high levels of the IAA degrading enzymes, IAA
oxidase, peroxidase and methylene ox indole reductase (Table 3). Both the IAA oxidase
and peroxidase can oxidise IAA, through two different pathways, to methylene oxindole
(HIN}{AN and LANG 1965; BASU and TULl 1972b), which is believed to be an intermediate for some growth promoting action of IAA (TULl and MOYED 1969; BASU and
TULl 1972a). Metnyleneoxindole may be degraded to metnyloxindole by the enzyme
methyleneoxindole reductase (BASU and TULl 1972b).
Attempts for finding out how the IAA in the nodules is synthesized revealed that
the Rhizobium sp. from P. pinnala is able to produce IAA in a culture medium, which
supplied with tryptophan. The organisms prefer L-Tryptophan than DL-Tryptophan
for this production. It is possible that the endophytic organism is directly responsible
for the high IAA content of the nodules. Some of the earlier workers also reported about
this character of Rhizobium sp. (sec DULLAART 1970b).
SHlBUKAWA et al. (1969); BIRNB AUM and DE~fAIN (1969) and many others reported
that permeability of the cells played an important role in the accumulation of amino
acids in the medium, which is produced by the cells. HATTORI (1965) reported that
artificially increasing tnc pcrmeability of the cells with antibiotics like penicillin, the
amino acid accumulation could be significantly stimulated. In a similar attempt to
increase the production of IAA in the mediulll, it was revealed that the antibiotics
interfering with the cell waH synthesis, penicillin and baeitraeill, had no effect on the
release of IAA by the Rhizobium sp. from P. pinnata. 011 the other hand, a 50 % inorease in the release of IAA was obtained with EDTA.
226
B.
K. SINHA
.nd
P.
S.
BASU
Acknowledgement
This investigation was supported by a resea.rch grant from CSIR, New Delhi.
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Receit'ed December 12, 1979; in revised form September 17, 1980.
Authors' address: B. K SINHA and P. S. BASU, Department uf Botany, University of Burdwan.
Golapbag. Burdwan 713104, West Bengal, lndia.
16 Bioehcm . Physiol . Pflanzen, Bd. 176