Journal of Experimental Botany, Vol. 55, No. 398, pp. 791±801, April 2004
DOI: 10.1093/jxb/erh086 Advance Access publication February 27, 2004
RESEARCH PAPER
The auxin conjugate 1-O-indole-3-acetyl-b-D-glucose is
synthesized in immature legume seeds by IAGlc synthase
and may be used for modi®cation of some high molecular
weight compounds
Anna Jakubowska* and Stanislaw Kowalczyk
Nicholas Copernicus University, Institute of General and Molecular Biology, Department of Biochemistry, ul.
Gagarina 9, 87-100 TorunÄ, Poland
Received 17 July 2003; Accepted 16 December 2003
Abstract
Immature seeds of some dicotyledonous plants contain IAGlc synthase catalysing the synthesis of 1-OIAGlc. This enzyme activity is comparable with 1-OIAGlc synthase activity investigated earlier in liquid
endosperm of Zea mays. Polyclonal antibodies
against maize 1-O-IAGlc synthase cross-react with
partially puri®ed 1-O-IAGlc synthase from immature
pea and rape seeds. Single immunoreactive bands
were observed at a locus corresponding to 45.7 kDa
and 43.7 kDa from pea and rape enzyme preparations, respectively, unlike that from the 50 kDa molecular mass of the maize enzyme. It was also
observed that some high molecular weight compounds of pea seeds are labelled in vivo by [14C] IAA,
and unlabelled 1-O-IAGlc inhibits that labelling. In
immature pea seeds 43±49.8% of the IAA-modi®ed
high molecular weight compounds, obtained after
ultracentrifugation, was found in the soluble fraction
and 50.1±57% in the insoluble fraction. Ester-linked
IAA accounted for about 6±9% and 38±45.6% in soluble and insoluble material, respectively, estimated
after hydrolysis in 1 N NaOH. Enzymatic hydrolysis
of IAA-labelled high molecular weight compounds
gives free IAA and compound(s) corresponding to
IAGlc isomers. These results suggest that 1-O-IAGlc
synthesized in legume seeds may be used for the
modi®cation of some high molecular weight compounds.
Key words: High molecular weight indole-3-acetic acid
conjugates, IAA-glucose synthase, indole-3-acetic acid,
indole-3-acetic acid glucose, legume plants.
Introduction
Indole-3-acetic acid (IAA) is the most abundant naturally
occurring auxin involved in the control of plant growth and
development. It is well established that IAA can occur
either as the hormonally active free acid or in bound forms
in which the carboxyl group is conjugated to sugars and
myo-inositol via ester linkages or to amino acids or
peptides via amide linkages (Cohen and Bandurski, 1982;
Bandurski et al., 1995; Normanly, 1997; Slovin et al.,
1999; Bartel et al., 2001; Ljung et al., 2002). IAA
conjugates have been identi®ed in a number of plant
species, ranging from liverworts to angiosperms (Sztein
et al., 1995, 2000). A limited number of species have been
studied, including maize, Arabidopsis, tomato, bean,
soybean, Scots pine, rice, oat, chestnut, and poplar,
although the most extensively studied have been seeds of
maize, bean, and soybean (Slovin et al., 1999). Maize
kernels contain primarily ester-linked conjugates, including IAA-glucose, IAA-myo-inositol, IAA-myo-inositol
glycosides, and a large cellulosic glucan conjugate that
together represent about 97±99% of the total IAA in the
seed endosperm (Bandurski and Schulze, 1977; Cohen and
Bandurski, 1982; Bandurski et al., 1995; Slovin et al.,
1999). Esteri®ed IAA is also the predominant conjugate in
* To whom correspondence should be addressed. Fax: +48 56 611 44 72. E-mail: [email protected]
Abbreviations: IAA, indole-3-acetic acid; 1-O-IAGlc, 1-O-(indole-3-acetyl)-b-D-glucose; IAInos, indole-3-acetyl-myo-inositol; IAGlc synthase, UDPglucose:indole-3-acetate glucosyltransferase; IAInos synthase, 1-O-(indole-3-acetyl)-glucose:myo-inositol indoleacetyl transferase.
Journal of Experimental Botany, Vol. 55, No. 398, ã Society for Experimental Biology 2004; all rights reserved
792 Jakubowska and Kowalczyk
seeds of rice which contain approximately 62±70% esterlinked IAA (Bandurski and Schulze, 1977; Hall, 1980) and
in the liquid endosperm of horse chestnut (Domagalski
et al., 1987). Moreover, Percival and Bandurski (1976)
described an IAA ester glucoprotein fraction, isolated from
oat seeds, that accounted for about 80% of the IAA found
in this tissue. In the early stages of bean seed development
ester-linked IAA represents approximately 35%, and free
IAA about 40%, of the total IAA pool (Bialek and Cohen,
1989). The level of esteri®ed and free IAA declines rapidly
during seed maturation, so that in fully mature seeds the
ester-linked IAA represents about 13% of the total IAA
pool, and only 6% is free IAA. It is noteworthy, that in
seeds harvested at full maturity, IAA is conjugated to
several polypeptides and proteins that approximate 80% of
the total IAA pool (Bialek and Cohen, 1986, 1989; Walz
et al., 2002). Soybean seeds have become another
dicotyledonous plant material in which essentially all
small molecular mass IAA conjugates have been identi®ed
and assayed. Quantitative evaluation indicates that conjugates with aspartate and glutamate are the predominant
IAA constituents present in these seeds (Cohen, 1982;
Epstein et al., 1986).
The entire complement of IAA conjugates has also been
determined in vegetative tissues of several plant species. It
was shown that ester conjugates of IAA constitute up to
80% of the IAA in the shoots of young dark-grown maize,
and IA-myo-inositol represents about 19% of these compounds (Pengelly et al., 1982; Chisnell, 1984). In tomato
pericarp, IAA undergoes conjugation to yield both IAAglucose and amide-linked IAA, and the preferential
formation of either IAA-glucose or amide-linked IAA
conjugates depends on the ripening stage of the fruit
(Catala et al., 1992; Iyer et al., 1997). Henbane cells,
tobacco explants, and tobacco leaf protoplasts produce
mainly auxin-aspartate and auxin-glucose conjugates
(IAA- or NAA-conjugates) (Caboche et al., 1984;
Delbarre et al., 1994; Oetiker and Aeschbacher, 1997;
Smulders et al., 1990). Arabidopsis, the model dicotyledonous plant, is also able to form the ester-linked IAA
conjugates that constitute approximately 8±10% of the
total IAA pool (Tam et al., 2000). In addition, analyses
have shown the presence of IA-alanine, IA-leucine, IAaspartate, IA-glutamate, and IA-glutamine, although, in
total, the small molecular mass conjugates together make
up only about 2±3% of the conjugate pool in the whole
È stin et al., 1998; Barratt et al., 1999; Barlier et al.,
plant (O
2000; Kowalczyk and Sandberg, 2001; Tam et al., 2000).
Because amide-linked IAA conjugates constitute
approximately 90% of the IAA pool, the high molecular
weight covalent complexes, apparently IAA-proteins, may
represent the major form of auxin in Arabidopsis, and in
other dicotyledonous plants as well.
Despite efforts from several laboratories there has
been no success in purifying the enzyme from plant
tissues catalysing the synthesis of low or high
molecular weight amide-linked conjugates. The enzyme
responsible for the synthesis of IAA-e-L-lysine in the
plant pathogen Pseudomonas savastanoi was studied
and the gene for this enzyme has been cloned (Glass
and Kosuge, 1986; Roberto et al., 1990). Recently,
Staswick et al. (2002) suggested that IAA-amino acid
conjugates can be produced via an adenylation intermediate. On the other hand, a comparatively large
amount of information is now available on the
biosynthesis of the IAA-ester conjugates in immature
kernels of Zea mays. Studies by Michalczuk and
Bandurski (1980, 1982) indicated that the formation of
the acyl alkyl acetal 1-O-(indole-3-acetyl)-b-D-glucose
(IAGlc) by indole-3-acetylglucose synthase (reaction 1)
is the ®rst step in the series of reactions leading to the
IAA±ester conjugates found in maize:
IAA+UDP-glucoseÛ1-O-IA-glucose+UDP (reaction 1)
UDP-glucose:IAA glucosyltransferase catalysing this
reaction has been extensively puri®ed from corn endosperm and polyclonal antibodies have been produced
(Kowalczyk and Bandurski, 1991; Leznicki and
Bandurski, 1988). Szerszen et al. (1994) have used
antibodies to select a cDNA clone for the IAGlc synthase
from a maize library. Recently, an Arabidopsis gene
encoding UDP-glucosyltransferase that forms 1-O-IAglucose was identi®ed (Jackson et al., 2001). Moreover,
the stimulation of IAGlc synthase gene expression by
auxin in maize coleoptiles has been observed (Kowalczyk
et al., 2002).
In immature kernels of Zea mays the energetically
unfavourable synthesis of 1-O-IA-glucose is followed by
an energetically favorable transacylation of the IAA
moiety from 1-O-IA-glucose to myo-inositol (reaction 2):
1-O-IA-glucose+myo-inositolÛIA-myo-inositol+glucose
(reaction 2)
The synthesis of IA-myo-inositol (IAInos) was observed
for the ®rst time in vitro in the seeds of maize (Michalczuk
and Bandurski, 1980, 1982), and the transferase catalysing
this reaction was partially puri®ed and characterized (Kesy
and Bandurski, 1990). Recently, a six-step procedure was
described for the puri®cation of an electrophoretically
homogenous IAInos synthase displaying similarity to the
serine carboxypeptidase-like acyltransferases family
(Kowalczyk et al., 2003).
In this report, it is indicated for the ®rst time that 1-OIAGlc synthase activity is present in immature seeds of
some dicotyledonous plants. Partially puri®ed 1-O-IAGlc
synthase from immature pea and rape seeds cross-reacts
with polyclonal antibodies against maize 1-O-IAGlc. It is
also observed that some high molecular compounds of pea
seeds are labelled in vivo by [14C] IAA.
Synthesis of 1-O-IAGlc in immature legume seeds 793
Materials and methods
Plant material
Plants used for analysis were grown under ®eld conditions, during
the summers of 2002 and 2003. The young pods with immature seeds
were harvested and the seeds were selected according to the length of
their long axis. The pods of pea (Pisum sativum) were harvested at
different times of seed development until full seed maturity. For
enzyme puri®cation the immature seeds were frozen immediately
after harvest and were stored in freezer bags at ±20 °C.
Chemicals
Chemicals were obtained from Gibco (USA), ICN (USA), Merck
(Germany), and Sigma (Germany). [2¢-14C] IAA and D-[U-14C]
glucose were from Amersham (UK). 1-O-IAGlc was synthesized by
Dr Antoni LezÂnicki using the methods of Jakas et al. (1993), IAAmyo-inositol was chemically synthesized according to the method of
Nowacki et al. (1978). 1-O-IAGlc synthase and IAInos synthase
preparations were puri®ed from immature corn endosperm according to the methods of Kowalczyk and Bandurski (1991) and
Kowalczyk et al. (2003).
Tissue homogenates for 1-O-IAGlc synthase activity assay
1 g of seeds or vegetative tissue from selected plants was
homogenized at 0±4 °C in a glass homogenizer with 1 ml of 50
mM TRIS-HCl buffer, pH 7.6 containing 2 mM EDTA, 2 mM 2mercaptoethanol, and PMSF (100 mg ml±1). The homogenates were
centrifuged at 10 000 g for 10 min, and enzyme activity was assayed
in the supernatant ¯uid.
Puri®cation of 1-O-IAGlc synthase from immature pea seeds
A 200 g portion of frozen pea seeds was homogenized with 200 ml of
50 mM TRIS-HCl buffer, pH 7.6 containing 2 mM EDTA, 2 mM 2mercaptoethanol, and PMSF (100 mg ml±1) using a mortar and pestle,
and then a Polytron homogenizer. All puri®cation steps were
performed at 4 °C. The homogenate was centrifuged at 16 000 g for
15 min (step I). To the resultant supernatant, 45% (w/v) polyethylene
glycol 6000 in 25 mM TRIS-HCl, pH 7.6, containing 2 mM EDTA
and 2 mM 2-mercaptoethanol was added slowly, with stirring, to
obtain a 15% (w/v) ®nal concentration of polyethylene glycol. The
solution was stirred in ice for 30 min, and after centrifugation for 60
min at 22 000 g a clear supernatant ¯uid was obtained (step II). The
supernatant ¯uid was applied to a DEAE-Sephacel column (2.5312
cm) equilibrated with 25 mM TRIS-HCl, pH 7.6 containing 1 mM 2mercatoethanol and PMSF (50 mg ml±1). The column was washed
with the same buffer until the A280 decreased to baseline. Bound
proteins were eluted with 80 mM NaCl and then with 150 mM NaCl
in the buffer. The fractions containing enzyme activity were pooled
(step III). The combined fractions were diluted with 10 mM TRISHCl, pH 7.6 containing 1 mM 2-mercaptoethanol and PMSF (50 mg
ml±1) and applied to a hydroxylapatite column (133 cm). The
column was washed with equilibrating buffer (25 mM TRIS-HCl,
pH 7.6, 1 mM 2-mercaptoethanol and PMSF 50 mg ml±1) and
proteins were eluted with 1 mM phosphate (K) buffer, pH 7.4
containing 1 mM 2-mercaptoethanol and PMSF (50 mg ml±1), and
then with 10 mM phosphate (K) buffer. The active fractions were
pooled and concentrated by ultra®ltration using an Amicon Dia¯o
YM-10 ®lter (step IV). TSK-Gel Toyopearl DEAE-650M column
(133 cm) was equilibrated with 25 mM TRIS-HCl buffer, pH 7.6
containing 1 mM 2-mercaptoethanol and PMSF (50 mg ml±1). The
combined fractions were applied to the TSK-Gel column after a 53
dilution to decrease the phosphate concentration. The column was
washed with the buffer, and the enzyme was eluted with a linear
gradient of 0±150 mM NaCl with a total volume of 100 ml. Fractions
containing IAGlc synthase activity were pooled and concentrated
(step V).
Qualitative assay of IAA-amino acid, IAA-disaccharide, and
IAA-glucose isomers synthesis
To examine formation of the IAA±amide conjugates or IAA-esters
with disaccharides, fresh immature seeds were transferred to 25 ml
Erlenmeyer ¯ask containing 10 ml of 10 mM phosphate (K) buffer,
pH 6.5, containing 25 mM IAA. The ¯ask was shaken for 12 h at
room temperature. The pretreated seeds were homogenized and
synthesis of IAA conjugates was tested in a total volume of 8 ml
containing 50 mM HEPES-NaOH buffer, pH 7.6, 1 mM 1-O-IAGlc,
2.5 mM MgCl2, and 5 mM amino acids (aspartate, glutamate or
leucine) or 5 mM disaccharide (melibiose, sucrose, and maltose), or
1 mM glucose and 0.1 mCi of D-[U-14C] glucose (speci®c activity
293 mCi mmol±1), respectively. The reaction was started by the
addition of 3 ml of homogenate and after a 2 h or 18 h incubation at
30 °C was stopped by drying a 4 ml aliquot on a Silica Gel 60 TLC
plate. The reaction products were separated by thin layer
chromatography (TLC) using as solvent ethyl acetate/methyl ethyl
ketone/ethyl alcohol/water (5:3:1:1, by vol.) (Labarca et al., 1965).
Indole compounds were detected by dipping the plate in the Ehmann
reagent (Ehmann, 1977), blotting and drying for 5 min at 100 °C.
The region corresponding to IAGlc isomers was scraped individually
from the plate and placed into the vial with 4 ml of scintillation ¯uid
EcoLite (ICN). The radioactivity was measured in a Wallac 1409
liquid scintillation counter.
Labelling high molecular weight compounds by [2¢-14C] IAA
0.5±2 g of immature pea seeds (3±4 mm) were rinsed in distilled
water and were then placed on a Petri dish ®lled with 1±3 ml 10 mM
phosphate (K) buffer, pH 6.5 containing 2% (w/v) sucrose, 1 mM
UDPG, 2.5 mCi of [2¢-14C] IAA (speci®c activity 50 mCi mmol±1),
and 25 mM IAA. In a parallel experiment, the incubation medium
contained in addition 0.5 mM unlabelled 1-O-IAGlc. Seeds were
incubated on a shaker at 25 °C in a dark room. After a 48 h
incubation, the seeds were rinsed many times with distilled water
and homogenized in 2±9 ml of 100 mM TRIS-HCl buffer, pH 7.6
containing 150 mM NaCl, 5 mM 2-mercaptoethanol, and 1 mM
unlabelled IAA. The homogenate was sonicated twice at 10 W for 30
s to disintegrate the protein bodies.
Determination of the high molecular weight IAA-conjugates
5 or 10 ml aliquots of the sonicated homogenate was spotted onto
Whatman 3MM or Miracloth ®lters (15 mm in diameter) followed
by immersion in ice-cold 10% trichloroacetic acid containing 0.5
mM unlabelled IAA. Filters were washed with the above solution
®ve times for 15 min each time, followed by 15 min in 95% ethanol
containing 1 mM IAA. Radioactivity on the dried ®lters was
determined by scintillation counting. The homogenate, obtained
after sonication, was ultracentrifuged at 60 000 g for 30 min. Solid
ammonium sulphate was added to the resultant supernatant to obtain
a 67% (w/v) saturated solution. The solution was stirred for 1 h and
was clari®ed by centrifugation at 15 000 g for 40 min. The pellet was
rinsed twice with 67% (w/v) saturated solution of ammonium
sulphate, and the precipitated proteins were then dissolved in 1 ml 25
mM TRIS-HCl buffer, pH 7.6, containing 1 mM IAA and passed
through a Sephadex G-15 column (1320 cm) equilibrated with 25
mM TRIS-HCl, pH 7.6. Cytosolic protein fractions were pooled and
concentrated by ultra®ltration using an Amicon Dia¯o YM-10 ®lter.
The pellet after ultracentrifugation was rinsed many times and
®nally resuspended in 25 mM TRIS-HCl buffer, pH 7.6. 10 ml of the
concentrated cytosolic proteins and insoluble material was spotted
onto Miracloth ®lters or 50 ml was placed into the vial with 4 ml of
794 Jakubowska and Kowalczyk
scintillation ¯uid EcoLite (ICN). The radioactivity was measured in
a Wallac 1409 liquid scintillation counter.
Alkaline and enzymatic hydrolysis of high molecular weight
IAA conjugates
The concentrated cytosolic proteins and insoluble fraction labelled
by [2¢-14C] IAA were hydrolysed in 1 N NaOH for 1 h at room
temperature in order to identify IAA-ester links. After hydrolysis, 10
ml samples were spotted onto Whatman 3MM paper or Miracloth
®lters followed by immersion in ice-cold 10% trichloroacetic acid
containing 0.5 mM IAA. Filters were washed, dried, and radioactivity was determined by scintillation counting. Alternatively, the
concentrated cytosolic protein pool as well as insoluble material was
hydrolysed enzymatically during the incubation at 30 °C. At the
appropriate time, 15 ml samples were separated on TLC plate and
indole-containing compounds were detected with the Ehmann
reagent or localized based upon the position of chemically
synthesized standards. The regions corresponding to free IAA,
IAGlc isomers, unidenti®ed compound (RF 0.2), and denatured high
molecular compounds were scraped individually from the plate and
the radioactivity was measured in a Wallac 1409 liquid scintillation
counter.
stopped by the addition of 1 ml 0.1 M NaOH, and the released pnitrophenol measured colorimetrically at 410 nm.
SDS-PAGE and western blotting
SDS-PAGE was performed according to the method of Ogita and
Markert (1979) in a Mini Protean II electrophoresis cell (Bio-Rad)
using 12% (w/v) resolving gel. The proteins used as molecular mass
standards were a 10 kDa Protein Ladder (Gibco). The separated
proteins were transferred to a nitrocellulose membrane in buffer
containing 50 mM TRIS, 380 mM glycine, 0.1% (w/w) SDS, and
20% (v/v) methanol. The positions of the protein markers were
visualized by staining with Ponceau S. The blot was incubated with
primary antibodies against IAGlc synthase puri®ed from maize
endosperm (Kowalczyk and Bandurski, 1991). The position of
IAGlc synthase was detected by an alkaline phosphatase-mediated
immunoassaying procedure using goat anti-rabbit-IgG antibodies
(Sigma), conjugated to alkaline phosphatase (Harlow and Lane,
1988).
Protein determination
Protein concentration was determined spectrophotometrically at 280
nm or by the Bradford method (Bradford, 1976) using g-globulin as a
standard.
Qualitative assay of 1-O-IAGlc synthase
A rapid and sensitive qualitative assay of enzyme activity was based
upon separation of the substrates and the reaction products by TLC.
1-O-IAGlc synthase activity was determined in a total volume of 8
ml containing 50 mM HEPES-NaOH buffer, pH 7.6, 7.5 mM UDPG,
4 mM IAA, 2.5 mM MgCl2, and 50 mM D-gluconic acid lactone (as
an inhibitor of b-glucosidase). Incubation was for 30 or 60 min at
30 °C, and the reaction products were separated by TLC using the
same standard conditions described above. Indole compounds were
detected using the Ehmann reagent. The RF of the various
compounds was IAA 0.83, 1-O-IAGlc 0.54, and IAInos 0.36.
Quantitative assay of 1-O-IAGlc synthase
The reaction mixture (100 ml) contained 50 mM HEPES-NaOH
buffer, pH 7.6, 7.5 mM UDPG, 4 mM IAA, and 0.05 mCi of [2¢-14C]
IAA (50 mCi mmol±1), 5 mM myo-inositol, 2.5 mM MgCl2, 0.25
mM 2-mercaptoethanol, and 20 ml of IAInos synthase partially
puri®ed from maize endosperm (Kowalczyk et al., 2003). IAInos
synthase is a trapping system, which converts ready hydrolysable 1O-IAGlc to the more stable IAInos. The reaction was started by the
addition of 20 ml of enzyme solution, and after a 15 or 30 min
incubation at 30 °C was stopped by the addition of 0.5 ml 50% (v/v)
2-propanol. Then 0.5 ml of the reaction mixture was transferred to a
2 ml bed volume DEAE-Sephadex (acetate) column in 50% (v/v) 2propanol. The uncharged products (IAInos and remaining 1-OIAGlc) not bound to the ion exchanger were eluted with 50% (v/v) 2propanol to make a total eluate volume of 5 ml. 1 ml was used for
radioactivity measurement in a Wallac 1409 liquid scintillation
counter.
Determination of b-glucosidase activity
During 1-O-IAGlc synthase puri®cation, b-glucosidase was also
monitored using p-nitrophenyl-b-D-glucopyranoside (pNPG) as the
chromogenic substrate. The activity was determined by measurement of the quantity of p-nitrophenol released. 50 ml of enzyme
solution was added to 50 ml 2.5 mM pNPG in 50 mM HEPES-NaOH,
pH 7.6. The reaction mixture was incubated for 20 min at 30 °C,
Fig. 1. TLC analysis of in vitro enzymatic synthesis of IAA
conjugates in Zea mays and Phaseolus vulgaris. The reaction mixture
(8 ml) contained 50 mM HEPES-NaOH pH 7.6, 7.5 mM UDPG, 4
mM IAA, 2.5 mM MgCl2, 5 mM myo-inositol, and 3 ml of whole
tissue homogenate of maize endosperm (lanes 1, 2), puri®ed IAGlc
synthase preparation from maize endosperm (lane 3), puri®ed
preparations of IAGlc with IAInos synthase from maize endosperm
(lane 4), whole bean seed homogenate (lanes 5, 6), and bean seed
homogenate with IAInos synthase puri®ed from maize endosperm
(lane 7). The samples were incubated for 30 min at 30 °C and the
reaction products were analysed in comparison with control samples
not containing UDPG (lanes 1, 5).
Synthesis of 1-O-IAGlc in immature legume seeds 795
Results
Detection of 1-O-IA-glucose synthase activity in
immature bean seeds
Though ester-linked IAA conjugates are the ubiquitous
form of hormone in different tissues of many plants (Sztein
et al., 1995, 2000), the enzymes catalysing synthesis of
these compounds have been well characterized only in
maize endosperm. It was assumed that ester-linked IAA
conjugates found in immature bean seeds are synthesized
in a manner similar to that of maize endosperm, and that
the synthesis of 1-O-IAGlc would be the ®rst step in this
pathway. In point of fact, the elementary qualitative assay
showed that in the reaction mixture containing UDPglucose, IAA, myo-inositol, and homogenate of liquid
maize endosperm, synthesis of 1-O-IAGlc and IAA-myoinositol occurs (Fig. 1, lane 2). Only 1-O-IAGlc is
synthesized when the partially puri®ed IAGlc synthase
from maize endosperm is used in place of the homogenate
(Fig. 1, lane 3), but IA-myo-inositol is the sole product
when the reaction mixture contains IAGlc synthase with
IAInos synthase partially puri®ed from maize endosperm
(Fig. 1, lane 4). Using the same reaction mixture and the
whole bean seed homogenate, the synthesis of a product
having an RF value the same as authentic 1-O-IAGlc
(Fig. 1, lane 6) was observed, but not the product that
corresponds to IAInos. 1-O-IAGlc, synthesized by been
seed homogenate, was identi®ed by comparing its RF value
with that of a synthetic standard. A de®nitive identi®cation
as 1-O-IAGlc was made when a partially puri®ed IAInos
synthase from maize endosperm was added to the reaction
mixture to convert the putative 1-O-IAGlc to IAInos. The
synthesis of IA-myo-inositol (Fig. 1, lane 7) demonstrates
that immature bean seeds really contain UDP-glucose:IAA
glucosyltransferase producing 1-O-IAGlc.
During the course of the previous experiments it was
observed that 1-O-IAGlc is readily hydrolysed by extracts
of different tissue at a rate so large that detection of the
IAGlc is not possible. In the present experiments it was
found that releasing free IAA from the chemically
synthesized 1-O-IAGlc is very rapid, supposedly as result
of high b-glucosidase activity present in the whole
homogenate of bean seeds (Fig. 2, lane 2). D-gluconic
acid lactone and castanospermine, potential b-glucosidase
inhibitors, signi®cantly inhibit IAGlc breakdown (Fig. 2,
lanes 4, 5). By contrast with 1-O-IAGlc, the IAInos
conjugate is not hydrolysed by bean homogenate (Fig. 2,
lane 7). These results indicate that quantitative determination of IAGlc synthase activity in crude homogenates is
possible provided that the synthesized 1-O-IAGlc is
immediately converted to IAInos using IAInos synthase
as a trapping system or that b-glucosidase inhibitors are
present in the reaction medium.
As shown in Fig. 2 (lanes 3, 4, 5), in addition to the
compounds identi®ed on the basis of the RF value or
Fig. 2. TLC analysis of in vitro enzymatic hydrolysis of 1-O-IAGlc
and IAInos by bean seed homogenate. The reaction mixture ((8 ml)
contained 3 ml whole tissue homogenate of bean seeds, 50 mM
HEPES-NaOH, pH 7.6, 2.5 mM MgCl2 (lanes 1, 2), and 1 mM
chemically synthesized 1-O-IAGlc (lanes 3, 4, 5), 50 mM D-gluconic
acid lactone (lane 4), or 40 mM castanospermine (lane 5), 1 mM myoinositol (lanes 6, 7). The samples were incubated for 30 min at 30 °C
except control samples (lanes 1, 6) and standard IAA, lane 8.
mobility of standards, an unidenti®ed new compound,
detectable by the Ehmann reagent, was observed during
incubation. This new compound is also produced during
incubation of homogenate with IAInos (Fig. 2, lanes 6, 7)
or homogenate alone (Fig. 2, lanes 1, 2). On the basis of
this result it is assumed that this compound is the peptide
containing IAA or tryptophan (positive reaction with the
Ehmann and ninhydrin reagent) accumulated as a result of
proteolytic degradation of some protein.
The distribution of 1-O-IAGlc synthase in different
dicotyledonous plants
Quantitative measurement of IAGlc synthase activity in
crude homogenate of some plants was based on the
measurement of uncharged reaction products, mainly
IAInos (not hydrolysed by tissue extract) synthesized
from 1-O-IAGlc in the presence of IAInos synthase
partially puri®ed from maize endosperm. Table 1 shows
activities (on a fresh weight basis) of the 1-O-IAGlc
synthase in crude homogenate of seeds or in vegetative
tissue extracts of the examined species. The enzyme
activity was assayed in immature seeds of 12 plant species.
Activity of 1-O-IAGlc synthase was found in ®ve of the
species examined as shown in Table 1. The highest activity
of 1-O-IAGlc synthase was found in very young Pisum
sativum, Phaseolus vulgaris (Leguminosae), and Brassica
796 Jakubowska and Kowalczyk
Table 1. The activity of 1-O-IAGlc synthase in different plant
species
The reaction mixture (100 ml) contained 50 mM HEPES-NaOH pH
7.6, 7.5 mM UDPG, 4 mM IAA, and 0.05 mCi of [2-14C] IAA (50
mCi mmol±1), 5 mM myo-inositol, 2.5 mM MgCl2, 0.25 mM bmercaptoethanol, 20 ml IAInos synthase preparation, and 20 ml
homogenate. Incubation was for 30 min at 30 °C and the reaction
was stopped by the addition of 0.5 ml of 50% (v/v) 2-propanol.
Enzyme activity was determined by measurement of [14C] IAA as
described in the Materials and methods.
Plant
Tissue
Enzyme activity
(nmol [14C] IAA
min±1 g±1 FWa)
Zea mays
Pisum sativum
Liquid endosperm
Seeds (3 mm)
Seeds (6 mm)
Seeds (mature, 24 h
after imbibition)
Vegetative tissue
(6-d-old seedlings)
Seeds (3 mm)
Pod (young)
Seeds (immature)
Seeds (immature)
Fruits (immature)
142.0
145.0
17.1
25.0
Phaseolus vulgaris
Lupinus polyphyllus
Brassica napus
Capsella bursa-pastoris
a
6.3
86.0
10.3
45.6
61.6
95.0
FW=fresh weight.
napus (Cruciferae) seeds, and Capsella bursa-pastoris
fruits. Immature pea seeds (3 mm in diameter) contain
IAGlc synthase activity (145 nmol IAGlc min±1 g±1 FW)
comparable with the activity of the enzyme present in
liquid maize endosperm (142 nmol IAGlc min±1 g±1 FW).
During development of pea seeds, the 1-O-IAGlc synthase
activity declines rapidly, reaching 8-fold lower activity in
the seeds that were 6 mm in diameter. Vegetative tissues of
Pisum sativum and young pods from Phaseolus vulgaris
display only a trace of 1-O-IAGlc synthase activity. 1-OIAGlc synthase activity has not been found in immature
seeds of watermelon, cucumber, tomato, and maple.
1-O-IAGlc synthase puri®cation from immature pea
seeds
Attempts to purify IAGlc synthase from pea seeds were not
entirely successful owing to the lability of partially
puri®ed preparation during column chromatography
(Fig. 3). Total activity in the homogenate increased 1.8times after PEG 600 precipitation, however, it drastically
dropped during the next chromatography steps. The ®nal
enzyme preparation displayed only a trace of 1-O-IAGlc
synthase activity (usually no more than 2% of the initial
activity in the homogenate) and still contained bglucosidase activity, as well as high hydrolytic activity
which breaks-down phosphoenolpyruvate (PEP). The
presence of this hydrolase in the ®nal preparation interferes
with the determination of 1-O-IAGlc synthase activity
based on a coupled enzyme assay.
Fig. 3. Puri®cation of 1-O-IAGlc synthase from immature pea seeds.
(A) Elution pro®les of IAGlc synthase activity from a DEAE-Sephacel
columnÐfractions were eluted with 80 mM KCl in equilibrating
buffer and then with 150 mM KCl in the same buffer; (B) elution
pro®le of proteins from a hydroxylapatite Bio-Gel HTP columnÐthe
active fractions eluted from a DEAE-Sephacel column with 80 mM
KCl in the 25 mM TRIS-HCl buffer, pH 7.6 were applied to a BioGel HTP column. Weakly retained proteins were removed by washing
a column with 25 mM TRIS-HCl buffer, pH 7.6, and the adsorbed
proteins were eluted with 1 mM (K) phosphate buffer, pH 7.4 and
then with 10 mM (K) phosphate buffer, pH 7.4; (C) anion-exchange
chromatography on TSK-Gel Toyopearl DEAE-650M of IAGlc
synthase adsorbed on a Bio-Gel HTP column. The enzyme was eluted
using a linear gradient of 0±150 mM NaCl in the buffer with a total
volume of 100 ml. b-glucosidase activity and activity of IAGlc
synthase were assayed as described in the Materials and methods.
Protein concentration was determined spectrophotometrically at 280 nm.
Synthesis of 1-O-IAGlc in immature legume seeds 797
Immunological cross-reactivity
Anti-maize (1-O-IAGlc synthase) antibody obtained previously by Kowalczyk and Bandurski (1991) was used in
the test for immunological cross-reactivity. Partially
puri®ed preparations of IAGlc synthase from corn liquid
endosperm and immature pea and rape seeds (after DEAESephacel chromatography step) were resolved on 12%
polyacrylamide gel. The separated polypeptides, after
being blotted onto a nitrocellulose membrane, were tested
for cross-reactivity with anti-IAGlc synthase antibody. As
shown in Fig. 4, a single immunoreactive band was
observed at a locus corresponding to 50 kDa in the case of
maize enzyme (Fig. 4, lane 1), as well as single
immunoreactive bands of 45.7 and 43.7 kDa with partially
puri®ed enzyme preparations from pea and rape, respectively (Fig. 4, lanes 2, 3).
Transfer of IAA from 1-O-IAGlc to IAA acceptor
The reversible reaction synthesis of 1-O-IAGlc is not
favoured energetically owing to the high energy of the acyl
bond, but it can be favoured by the relatively high levels of
UDP-glucose in the tissue and by the downstream synthesis of IAInos, di-O-(indole-3-acetyl)-D-glucose, and IA-dior trisaccharides (IAA-sucrose, IAA-oligosaccharides
from the raf®nose family) as indicated in maize endosperm
(Szmidt-Jaworska et al., 1997; Leznicki and Bandurski,
2001). It was expected that the synthesis of 1-O-IAGlc in
pea seeds would also be coupled to a downstream reaction,
but, as shown here, it is not coupled to the synthesis of
IAInos. A possible transfer reaction has been tested using
medium containing 1 mM chemically synthesized 1-OIAGlc and 5 mM disaccharides (melibiose, sucrose, and
maltose) or 1 mM [14C] glucose. The reaction was
conducted using an homogenate of immature pea seeds
that had been pretrated with IAA, as described in the
Materials and methods, and the reaction products were
separated by TLC. Using qualitative or quantitative ([14C]
glucose) determination of the expected products, the
synthesis of IAA-conjugates that contain an IAA-moiety
originating from 1-O-IAGlc was not observed (data not
shown). In these experiments, possible transferase activity
was also tested, which would transfer the IAA-moiety from
1-O-IAGlc to aspartic acid, glutamic acid, and leucine. The
reaction was conducted for 2 h and 18 h at 30 °C using the
immature pea seeds (4 mm in diameter) homogenate, and
the reaction products were separated by TLC.
Independently of incubation time, the formation of a new
IAA-conjugate corresponding to IA-aspartate, IA-glutamate, or IA-leucine was not observed (data not shown).
Labelling of high molecular compounds in pea seeds
by [2¢-14C] IAA
Bean seeds harvested at full maturity contain some
proteins and peptides to which IAA is covalently attached.
Fig. 4. Immunological cross-reactivity of IAGlc synthase. The
proteins from partially puri®ed plant preparations were separated by
SDS-PAGE using 12% (w/v) resolving gel, transferred to a
nitrocellulose membrane. Polypeptides cross-reacted with anti-maize
(1-O-IAGlc synthase) antibody were localized by alkaline
phosphatase-mediated immunostaining procedure: molecular mass
standards (line 0), liquid endosperm of Z. mays (lane 1), immature
seeds of P. sativum (lane 2), immature seeds of B. napus (lane 3).
On the basis of these data it was assumed that 1-O-IAGlc
produced in immature seeds can be used for posttranslational covalent modi®cation of some proteins. For
in vivo protein labelling, 0.5 g of immature pea seeds (3±4
mm) was incubated with labelled IAA as described in the
Materials and methods. In a parallel experiment, the
incubation medium also contained 0.5 mM unlabelled 1-OIAGlc. The results shown in Table 2 indicate that material
precipitated by TCA in ®lters contains [2¢-14C] IAA, and
that the presence of unlabelled 1-O-IAGlc in the incubation medium causes a 19.9±22.4% decrease of the total
IAA attached to TCA-precipitated material. These results
suggest that at least a part of the IAA bounded to high
molecular compounds originated from 1-O-IAGlc synthesized by 1-O-IAGlc synthase.
Alkaline hydrolysis of the high molecular IAA
conjugates
To establish the subcellular localization of these compounds, the homogenate from immature pea seeds incubated with [2¢-14C] IAA was ultracentrifuged and the
radioactivity was determined in the soluble fraction as well
as in the pellet containing the insoluble material. The
results of these experiments indicate that IAA is bound to
both of these fractions accounting for about 43±49.8% and
50.1±57% in the soluble and insoluble fractions, respectively (Table 3). Ester-linked IAA, contained both in the
soluble protein fraction precipitated with ammonium
798 Jakubowska and Kowalczyk
Table 2. Labelling of high molecular compounds by [2¢-14C]
IAA
Pea seed homogenate was prepared as described in the Materials and
methods. For [2¢-14C] IAA labelling determination, 5 or 10 ml
samples were spotted onto ®lters followed by immersion in ice-cold
10% TCA containing 0.5 mM unlabelled IAA. Radioactivity on the
washed and dried ®lters was measured in a liquid scintillation
counter.
High molecular
compounds labelled
by [14C] IAA
Homogenate
Experiment 1
Experiment 2
Total radioactivity (cpm)
Without 1-O-IAGlc
With 1-O-IAGlc
Table 3. The distribution of the IAA-labelled high molecular
weight compounds in the soluble protein fraction and
insoluble material
Pea seed homogenate was ultracentrifuged for 30 min at 60 000 g
and the radioactivity was measured in the soluble fraction as well as
in the pellet as described in the Materials and methods. The proteins
occurring in the soluble fraction and the insoluble material were
hydrolysed in 1 N NaOH in order to identify ester conjugates. 100 ml
samples were hydrolysed for 1 h at room temperature and the
radioactivity remaining on the ®lters after TCA precipitation was
measured as for Table 2.
Total radioactivity (cpm) in
48 115
31 750
37 337 (77.6%)
25 600 (80.1%)
sulphate and in insoluble material, was determined as IAA
released from the samples by hydrolysis for 1 h in 1 N
NaOH at room temperature. Following hydrolysis, 10 ml
samples were spotted onto Whatman 3MM or Miracloth
®lters and precipitated by TCA. As shown in Table 3, 38±
45.6% of the total radioactivity was released from the
insoluble fraction, while only 6±9% of the IAA was
released from the soluble fraction. Supposedly, this alkalilabile IAA is ester-linked with polysaccharides and
glycoproteins or ester-bound with the hydroxyl group
present in amino acid residues. The physiological role of
high molecular weight IAA conjugates is unknown,
although it has been proposed that these compounds can
be enzymatically hydrolysed to yield free IAA. On this
assumption, the enzymatic hydrolysis of high molecular
weight IAA conjugates was assayed in a mixture containing 50 ml of the concentrated soluble protein fraction and
50 ml of the insoluble fraction. After the appropriate time
of the incubation, 15 ml samples were separated on a TLC
plate and labelled compounds were localized, based upon
the position of standards. Each appropriate region was
scraped individually and radioactivity was determined in a
scintillation counter. As shown in Fig. 5A the formation of
the unidenti®ed product giving colour spots with the
Ehmann and ninhydrin reagents was observed, however
this compound was not [14C] IAA labelled. The accumulation of this supposedly tryptophan-containing peptide or
modi®ed tryptophan (lower mobility on TLC than
tryptophan) is signi®cantly inhibited by PMSF (results
not shown). It is of interest that radioactive compounds
increased during the incubation period correspond to free
IAA and IAA-glucose isomers (Fig. 5B). Based on these
data it is supposed that free IAA released from IAAlabelled high molecular weight compounds is a product of
enzymatic hydrolysis of ester- or amide-linked IAA, while
the IAA-containing compound, probably IAGlc isomers, is
a product of enzymatic hydrolysis of IAA-polysaccharides
or IAA-glycoproteins. These results imply that high
molecular weight IAA conjugates accumulated in great
Experiment 1
Before hydrolysis
After hydrolysis
Experiment 2
Before hydrolysis
After hydrolysis
Soluble fraction
Insoluble fraction
22 340
20 329 (91%)
28 957
15 752 (54.4%)
17 215
16 182 (94%)
17 340
10 750 (62%)
quantities in the cotyledons of seeds may be considered as
the major possible source of IAA required for the growth
of seedlings and support the hypothesis that these conjugates are the components of a system for homeostatic
control of IAA in plant tissues.
Discussion
The widespread distribution and high concentrations of
IAA conjugates in plant tissues suggest that these
compounds may play an important role in the metabolism
of IAA. The physiological signi®cance of the individual
conjugate moieties is yet to be determined, but now is
generally accepted that different conjugates play different
roles in IAA metabolism. The results obtained up to now
have suggested the general conclusion that ester conjugates predominate in seeds of monocotyledonous plants
(Cohen and Bandurski, 1982; Slovin et al., 1999). Most of
the knowledge on ester-linked IAA conjugates comes from
studies on maize. The synthesis of IA-glucose, followed by
transacylation to myo-inositol represents two potential
regulatory steps for the control of IAA concentration by
converting hormonally active free IAA into growthinactive IAA ester. Since all corn tissues hydrolyse IAglucose isomers and, more slowly, IAInos to free IAA, the
mechanism is interpreted as a shuttle to adjust the free pool
of IAA via the temporal storage of IAA esters.
Compared with maize, little is known about IAAconjugate synthesis in other plants. Until now there has
been no knowledge about the enzymes that synthesize
ester-linked IAA in dicotyledonous plants as well as the
enzymes catalysing the synthesis of IAA-amino acids and
IAA±protein conjugates. In this report, it is demonstrated
that immature seeds of some dicotyledonous plants possess
Synthesis of 1-O-IAGlc in immature legume seeds 799
Fig. 5. Enzymatic hydrolysis of high molecular [2¢-14C] IAA
conjugates. (A) The mixture containing 50 ml of concentrated
cytosolic proteins and 50 ml of the insoluble fraction was incubated at
30 °C for 0±4 h. After the appropriate time, 15 ml samples were
separated by TLC in the standard conditions. The elliptic regions
indicating the positions of free IAA, IAGlc isomers, unidenti®ed
compound, and denatured high molecular compounds were scraped
from the plate for the radioactivity measurement. (B) The radioactivity
level in the products of enzymatic hydrolysis of high molecular weight
IAA conjugates: denatured high molecular compounds (®lled circles);
free IAA (®lled triangles); IAGlc isomers (®lled squares).
high activity of 1-O-IAGlc synthase, comparable to the
activity that was previously investigated in liquid maize
endosperm. Western blot analysis demonstrated that
partially puri®ed pea and rape enzyme cross-reacts with
anti-maize (IA-glucose synthase) antibody. It is notable
that the molecular mass of both polypeptides is about 4 and
6 kDa smaller compared with the estimated molecular
mass of the maize enzyme (Kowalczyk and Bandurski,
1991; Szerszen et al., 1994). Attempts to purify IAGlc
synthase from pea seeds were not entirely successful
owing to the lability of the preparation during the
puri®cation procedures. 1-O-IAGlc synthase from immature pea, as well as from bean seeds, is extremely unstable
and complete loss of activity was found following
chromatographic separations. Partially puri®ed pea en-
zyme indicates the highest activity toward IAA, but
signi®cant activity (qualitatively determined) has also
been observed toward indole butyric acid and naphthaleneacetic acid. Though IAA is the preferred substrate for
pea enzyme in vitro, it is likely that in vivo 1-O-IAGlc
synthase may glucosylate either IAA or IBA depending on
the relative availability of substrates and the relative
compartmentalization of the enzyme and substrates.
It is of interest to elucidate what kind of reaction is
coupled with the synthesis of 1-O-IA-glucose which
pushes this energetically unfavourable synthesis. These
results indicate that, in legume plants (Pisum sativum,
Phaseolus vulgaris), the coupled reaction is neither the
synthesis of IAInos nor the transfer of the IAA moiety to
glucose or some disaccharides. Also, the in vitro experiments indicate that transfer of the IAA-moiety from 1-OIAGlc to some amino acids does not occur in the pea seed
homogenate. However, Delbarre et al. (1994) indicated
that tobacco leaf protoplasts incubated with labelled NAA
or IAA ®rst accumulate an auxin±glucose ester and only
then the auxin±aspartate conjugate. Because it is not
known to which downstream reaction the synthesis of 1-OIAGlc is coupled in pea seeds, attention has been given to
the synthesis of some high molecular weight IAA-conjugates. The ®rst such compound was found in bean seeds
when Bialek and Cohen (1986) isolated the hydrophobic
3.6 kDa peptide. Recently, a 35 kDa protein encoded by
the IAP1 gene has been isolated and cloned (Walz et al.,
2002). It was also observed that the presence of the most
abundant IAA-modi®ed proteins in bean seeds correlates
with a developmental period of rapid growth during seed
development and then they are rapidly degraded during
germination. Thus, the authors' idea was that 1-O-IAGlc
synthesized in immature legume seeds serves as a donor of
the IAA moiety which is then transferred to some proteins,
glycoproteins or some high molecular weight polysaccharides. Indeed, the results of in vivo experiments described in
the present paper demonstrate that some proteins and
perhaps other high molecular weight compounds of pea
seeds are labelled in vivo by [14C] IAA. Unlabelled 1-OIAGlc present in the incubation medium clearly inhibits
labelling of high molecular weight compounds The pools
of labelled compound were almost equal, both in the
soluble and insoluble fractions and accounted for 43±
49.8% and 50.1±57%, respectively. While 38±45.6% esterlinked IAA was present in the insoluble fraction, the
soluble protein fraction contained only 6±9% ester
conjugates as estimated on the basis of the hydrolysis in
1 N NaOH. It can be supposed that the amide-linked IAA
represents a signi®cant amount of IAA present in this
fraction. Moreover, IAA±protein conjugates enzymatically
hydrolysed in vitro yield free IAA and probably IAA±
glucose conjugate. It is very likely that the formation of the
high molecular weight IAA-conjugates is 1-O-IAGlc
dependent and that it is the downstream reaction to
800 Jakubowska and Kowalczyk
which the synthesis of 1-O-IAGlc is coupled in immature
seeds of legume plants.
Acknowledgement
The authors wish to thank Professor Robert S Bandurski
for his critical reading of this manuscript.
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