Download Auxins in the development of an arbuscular mycorrhizal

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Journal of Plant Physiology 162 (2005) 1210—1219
www.elsevier.de/jplph
Auxins in the development of an arbuscular
mycorrhizal symbiosis in maize
Dorothee Fitzea,1, Anne Wiepninga, Michael Kaldorfb,
Jutta Ludwig-Müllera,
a
Institut für Botanik, Technische Universität Dresden, Zellescher Weg 22, 01062 Dresden, Germany
Institut für Botanik, Universität Leipzig, Johannisallee 21, 04103 Leipzig, Germany
b
Received 23 July 2004; accepted 7 January 2005
KEYWORDS
Glomus intraradices;
Zea mays;
Arbuscular
mycorrhiza;
Auxin conjugates;
Indole-3-acetic acid;
Indole-3-butyric
acid;
Trifluoro-IBA
Summary
While the levels of free auxins in maize (Zea mays L.) roots during arbuscular
mycorrhiza formation have been previously described in detail, conjugates of indole3-acetic acid (IAA) and indole-3-butyric acid (IBA) with amino acids and sugars were
neglected. In this study, we have therefore determined free, ester and amide bound
auxins in roots of maize inoculated with Glomus intraradices during early stages of the
colonization process. Ester conjugates of IAA and IBA were found only in low amounts
and they did not increase in AM colonized roots. The levels of IAA and IBA amide
conjugates increased 20 and 30 days past inoculation (dpi). The formation of free and
conjugated IBA but not IAA was systemically induced during AM colonization in leaves
of maize plants. This implicated a role for auxin conjugate synthesis and hydrolysis
during AM. We have therefore investigated the in vivo metabolism of 3H-labeled IBA by
TLC but only slight differences between control and AM-inoculated roots were
observed. The activity of auxin conjugate hydrolase activity measured with three
different putative substrates showed a decrease in infected roots compared to
controls. The fluorinated IBA analog TFIBA inhibited IBA formation in leaves after
application to the root system, but was not transported from roots to shoots. AM
hyphae were also not able to transport TFIBA. Our results indicate complex control
mechanisms to regulate the levels of free and conjugated auxins, which are locally
and systemically induced during early stages of the formation of an arbuscular
mycorrhizal symbiosis.
& 2005 Elsevier GmbH. All rights reserved.
Abbreviations: ABA, abscisic acid; AM, arbuscular mycorrhiza; GUS, b-glucuronidase; IAA, indole-3-acetic acid; IAA-alanine, indole3-acetyl alanine; IAA-aspartate, indole-3-acetyl aspartic acid; IAA-glucose, indole-3-acetyl-b-D-glucose; IBA, indole-3-butyric acid;
IBA-alanine, indole-3-butyryl alanine; JA, jasmonic acid; TFIBA, 4, 4, 4-trifluoro-3-(indole-3-)butyric acid
Corresponding author. Tel.: +49 351 463 33939; fax: +49 351 463 37032.
E-mail address: [email protected] (J. Ludwig-Müller).
1
Present address: Biotechnologisches Zentrum, Tatzberg 47-51, 01307 Dresden, Germany.
0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2005.01.014
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Auxins and arbuscular mycorrhiza
Introduction
A majority of agriculturally important crop
species forms mutual symbioses with arbuscular
mycorrhizal (AM) fungi. The AM fungi can increase
growth and yield of many crops (Smith and Read,
1997). During colonization distinct structures (internal hyphae, arbuscules, vesicles) are formed by
the AM fungi within the host roots (Harrison, 1999;
Strack et al., 2003). From the symbiosis plants gain
minerals, such as phosphate, in exchange for
carbon (Smith and Gianinazzi-Pearson, 1988). For
mycorrhizal plants, also an enhanced drought
tolerance (Nelsen and Safir, 1982) and a better
resistance against plant pathogens (Dehne, 1982)
was described.
The complex mechanisms by which this plantfungal symbiosis is regulated requires a continuous
exchange of signals, which results in the proper
development of the symbiosis (Gianinazzi-Pearson,
1996). Plant hormones are signal molecules which
regulate many developmental processes in plants
(Davies, 1995) and are therefore suitable candidates to function in the colonization process
(Barker and Tagu, 2000; Ludwig-Müller, 2000).
First analyses of phytohormone levels in maize
colonized with an AM fungus used ELISA and
conventional bioassays. Measurements of abscisic
acid (ABA) showed considerably higher levels of
free ABA in AM-infected than in control roots,
whereas the amounts of zeatin riboside (with the
exception of a late growth phase) and IAA were
similar for infected and non-infected roots (Danneberg et al., 1992). The amount of ABA in the AM
fungal hyphae was at least one order of magnitude
higher than in maize roots without AM fungi (Esch
et al., 1994). Recent investigations on auxin
contents in roots of different plant species colonized with AM fungi have used gas chromatographymass spectrometry. For maize roots colonized by
Glomus intraradices no significant increase in free
IAA was found (Kaldorf and Ludwig-Müller, 2000),
confirming earlier results (Danneberg et al., 1992).
Other authors also did not find an effect of AM on
IAA levels in inoculated roots of leek (Torelli et al.,
2000) and tobacco (Shaul-Keinan et al., 2002),
whereas cytokinins and gibberellins changed during
colonization.
AM inoculated maize roots showed an early
increase in free indole-3-butyric acid (IBA) as well
as an increase in IBA synthesis (Ludwig-Müller et
al., 1997; Kaldorf and Ludwig-Müller, 2000). This
coincided with a significant increase in the percentage of lateral fine roots ca 10 days after inoculation. The phenotype of mycorrhizal maize roots
could be mimicked by IBA applied exogenously to
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non-mycorrhizal roots. Addition of trifluoro-IBA
(TFIBA), an inhibitor of IBA-induced root growth
and lateral root induction, simultaneously with IBA
resulted in a phenotype resembling that of untreated controls. In roots treated with TFIBA the
inoculation with AM fungi did not increase the
formation of fine roots. The TFIBA treatment also
reduced endogenous free IBA levels and the AM
infection rate in mycorrhizal roots (Kaldorf and
Ludwig-Müller, 2000).
Several possibilities for the regulation of IBA
content in AM inoculated roots can be envisioned:
(1) uptake and transport, (2) biosynthesis, and (3)
hydrolysis from inactive conjugates or regulation of
conjugate formation. To address these questions
we have investigated the amounts of auxin conjugates during AM colonization in more detail and
also examined their synthesis and hydrolysis. In
addition, we have used the IBA antagonist TFIBA to
study the localization of IBA/TFIBA action.
Materials and methods
Plant material and inoculation with AM fungi
Seedlings of maize (Zea mays L. cv. Alize) were
cultivated under sterile conditions in the light
(28 mmol m2 s1; Philips fluorescent tubes TL55
and TL32) at 23 1C as previously described (Ludwig-Müller and Epstein, 1991; Ludwig-Müller et al.,
1997). Seedlings were harvested after 6 days of
culture. For inoculation with AM fungi seedlings
were transferred to expanded clay (Lecatons)
substrate 7 AM inoculum after the root and
coleoptile tips were visible (3–4 days after sowing).
The AM isolate G. intraradices Schenck and Smith
INVAM Sy 167 was originally obtained from the
Institut für Pflanzenernährung, Universität Hohenheim, Germany. Inoculum production was as previously described (Schmitz et al., 1991). Plants
were grown in a greenhouse at 22–28 1C and a
photoperiod of 15 h light/9 h dark and watered with
tap water. The percentage of root length colonized
was determined by counting the fungal structures
(hyphae, arbuscules, vesicles) in infected roots
after staining with lactophenolblue as described
earlier (Schmitz et al., 1991).
Two-compartment system
Seeds were sown onto sterile filter paper and
after germination grown for 10 days. At this time
point, when roots and hypocotyls were clearly
visible, the seedlings were transferred to the first
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compartment of a two-compartment system (see
Fig. 5) filled with 100 cm3 expanded clay substrate
7 AM inoculum and cultivated as described above.
The roots were harvested at the appropriate time
points, washed thoroughly and dried between filter
paper. After recording the fresh weight, roots were
frozen in liquid N2 and stored at 80 1C prior to
analysis. The percentage of root length colonized
was determined by counting the fungal structures
as described above. Hyphae of G. intraradices were
isolated from the second compartment, which
contained quartz sand (a 1:1 mixture of sand with
particle size of 1–2 mm and 2–4 mm). The two
compartments were separated from each other by
a membrane (30 mm mesh size), which allowed
hyphae to penetrate into the sand compartment,
but no roots were able to grow into this side of the
system. Both compartments were watered on a
regular basis. For 2 weeks the system was treated
every second day with 60 ml tap water only on the
plant side. After this period the system was treated
once a week with 60 ml Hoagland solution (Eschrich, 1976) without phosphate, and the rest of
the time with 60 ml tap water. Watering was carried
out alternating between the plant compartment
and the sand compartment (hyphae). In addition,
every 4 weeks the plant compartment was treated
with 60 ml Hoagland solution containing 1 mM
K2HPO4. Isolation of hyphae was performed by
resuspending the sand of one compartment in 2 l of
sterile distilled H2O. The sand was then allowed to
sediment for 3 min and the supernatant, which
contained the slower sedimenting hyphae was
removed with a syringe attached to a rubber pipe.
The hyphal fraction was filtered over a 45 mm filter,
then washed from this filter with 100 ml distilled
H2O. The sedimentation and filtration procedure
was repeated and the purified hyphae were finally
resuspended in a small amount (5 ml) of sterile
distilled H2O. For TFIBA analysis hyphae were
frozen in liquid N2 and stored at 80 1C prior to
analysis. Hyphae were extracted with an Ultraturrax (Janke & Kunkel, Germany) at 9500 rpm by
adding 10 volume MeOH. After evaporating the
organic solvent, the extracts were purified over
NH2-colums and analyzed by HPLC as described
below.
Feeding of TFIBA
The synthetic analogue of IBA 4,4,4-trifluoro-3(indole-3-)butyric acid (TFIBA; Katayama et al.,
1995; Katayama and Gautam, 1996) was dissolved
as a stock solution in ethanol and diluted with
water before it was added to the plants. Final
D. Fitze et al.
ethanol concentration was o0.1%. In all experiments, an ethanol control was included, which did
not show differences to control plants without
ethanol treatment (data not shown). The plant
material was extracted and purified as described
for free IAA determinations. TFIBA was analyzed by
HPLC (Jasco BT 8100 pump) equipped with a
125 mm 4 mm Lichrosorb C18 reverse phase column (particle size 5 mm) and an UV detector
(280 nm, Jasco Uvidec 100-III) using an isocratic
system (52% MeOH: 48% aq. acetic acid). Flow rate
was 0.5 ml min1 and TFIBA eluted at Rt 7.2 min
under these conditions. The amount of TFIBA in the
sample was calculated using a standard curve.
Determination of free and bound auxins
The frozen plant material (ca. 0.2 g fresh weight
per analysis) was extracted and one third was
directly purified on NH2-columns (Chen et al.,
1988). To each sample 100 ng 13C6-IAA (Cambridge
Isotope Laboratories, Andover, MS, USA), and 100 ng
13
C1-IBA (Sutter and Cohen, 1992) were added. For
each sample three independent extractions were
performed. After elution from the column, the
samples were evaporated to dryness, directly
methylated with diazomethane (Cohen, 1984) and
resuspended in ethyl acetate for GC–MS analysis.
For the determination of auxin conjugates two
thirds of the extract were evaporated to the
aqueous phase and then half of it hydrolyzed (a)
with 1 N NaOH for 1 h at room temperature for ester
conjugates and (b) with 7 N NaOH for 3 h at 100 1C
for amide conjugates. After the incubation time
the samples were cooled to room temperature if
appropriate, the pH was adjusted to 3.5, the
samples were column purified on C18-columns
(Chen et al., 1988) and subsequently methylated.
GC–MS analysis was performed on a Varian Saturn
2100 Ion Trap MS system using electron impact
ionization at 70 eV, coupled to a Varian CP-3900 GC
equipped with an Varian CP-8400 autosampler
(Varian, Walnut Creek, CA, USA). For the analysis
2.5 ml of the methylated sample dissolved in 20 ml
ethyl acetate was injected in the splitless mode
(splitter opening 1:100 after 1 min) onto a Phenomenex ZB-5 column, 30 m 0.25 mm 0.25 mm (Phenomenex, Aschaffenburg, Germany) using He
carrier gas at 1 ml min1. Injector temperature
was 250 1C and the temperature program was 701
for 1 min, followed by an increase of 201 min1 to
280 1C, then 5 min isothermically at 280 1C. Methyl
esters of IAA and IBA eluted under these conditions
at 10.1 and 11.2 min, respectively. The settings of
the mass spectrometer were as described in
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Auxins and arbuscular mycorrhiza
Campanella et al. (2003a). For higher sensitivity
the mSIS mode (Varian Manual) was used to monitor
the diagnostic ions. The endogenous amounts of
free auxins were calculated by the isotope dilution
equation (Cohen et al., 1986). For the determination of IAA, the molecular and quinolinium ions of
the methylated substance at m/z 189/195 and 130/
136, respectively, were monitored (ions deriving
from endogenous and 13C6-IAA), for IBA the molecular and quinolinium ions at m/z 217/218 and 130/
131, respectively (ions deriving from endogenous
and 13C1-IBA). The levels for ester conjugates were
calculated by subtracting the amount of free IAA
from that after hydrolysis with 1 N NaOH, for amide
conjugates by subtracting the free plus ester from
the amount of total auxin after hydrolysis with 7 N
NaOH.
Metabolism of IBA
Metabolism experiments were carried out using
25 kBq [5-3H]-IBA (0.268 TBq mmol1, Nuclear Research Center, Dimona, Israel) per experimental
condition as previously described (Ludwig-Müller
and Epstein, 1993; Ludwig-Müller et al., 1997). The
3
H-IBA was purified by HPLC prior to the feeding
experiment. Control and AM inoculated maize roots
harvested at three different time points were
incubated for 3 h in 100 mM MES buffer, pH 6.0.
The extraction of labeled compound was performed
as described elsewhere (Ludwig-Müller and Epstein,
1993). The metabolites were separated on TLC
according to Ludwig-Müller et al. (1997). After
separation, the plate was sprayed with EN3Hances
(Du Pont, NEN) and incubated with an X-ray film for
8 days at 20 1C for autofluorography (LudwigMüller and Epstein, 1993; Ludwig-Müller et al.,
1997). X-ray films were scanned and relative pixel
density determined.
Auxin conjugate hydrolase activity
The enzymatic hydrolysis of different auxin
conjugates was determined as described elsewhere
for Chinese cabbage (Ludwig-Müller et al., 1996)
and wheat (Campanella et al., 2004). Since only
one IBA conjugate is commercially available, three
IAA conjugates were also used as substrates. For
IAA amide conjugates indole-3-acetyl alanine (IAAalanine) and indole-3-acetyl aspartate (IAA-aspartate), as an ester conjugate indole-3-acetyl-ß-Dglucose (IAA-glucose) and as IBA conjugate indole3-butyryl alanine (IBA-alanine) were employed. The
preparation of the enzyme extract and the reaction
conditions were as described by Ludwig-Müller
1213
et al. (1996). The reaction products IAA and IBA
were separated from the different auxin conjugate
substrates by HPLC (Ludwig-Müller et al., 1996).
Results
Analysis of auxin concentration and
distribution during AM formation
In previous reports we have demonstrated that
IBA but not IAA formation was induced during
colonization of maize roots with the AM fungus G.
intraradices (Ludwig-Müller et al., 1997; Kaldorf
and Ludwig-Müller, 2000). We have here extended
our analysis with focus on different conjugates of
both auxins IAA and IBA, including leaves in
addition to roots to differentiate between local
and systemic effects of AM colonization.
It was confirmed in an independent experiment
that free IBA formation was induced in roots during
early stages of AM infection (Fig. 1), while free IAA
levels did not change. Ester conjugates of IBA were
only detectable in one control root sample,
whereas ester conjugates of IAA were found in all
root samples with the exception of 30-day-old
control roots. However, ester conjugate levels did
not change significantly after AM inoculation (data
not shown). IAA conjugates with amino acids were
not found in 10-day-old roots, 20 dpi the amide
conjugates of IAA increased significantly in AMinoculated roots, while they dropped below control
levels 30 dpi. On the contrary, IBA amide conjugates were already found in AM roots 10 dpi,
whereas there was no difference between control
and inoculated roots 20 dpi. Contrary to IAA amide
conjugates, the IBA amide conjugates increased
30 dpi in AM roots. There was a continuous increase
in the percentage of intraradical structures such as
hyphae, arbuscules and vesicles (Fig. 1) during the
time period under investigation, whereas the
percentage of extraradical hyphae did not change
(data not shown).
Possible systemic changes of IAA and IBA levels in
leaves of control and AM-inoculated plants was
investigated at the same time points used for the
root analysis. Because AM fungi show growth
promotional effects, an increase in auxins may also
contribute to the growth of leaves. Neither free nor
total IAA levels were changed in leaves of AM
inoculated plants compared to controls (data not
shown). On the contrary, leaves of AM inoculated
plants showed an increase in free IBA formation 20
and 30 dpi (Fig. 2), which is the same time point
when the amount of IBA was enhanced in AM roots.
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1214
25
20
free
hyphae
vesicles
IBA (ng/ g Fresh wght)
% Mycorrhization
A
D. Fitze et al.
arbuscules
spores
15
10
5
conjugated
1800
1500
-TFIBA
1200
900
600
300
0
10
0
B
30
1200
1000
IAA (ng/g Fwt)
20
IBA (ng/ g Fresh wght)
10
amide
free
800
600
400
10
20
30
20
30
10
20
30
1800
1500
+TFIBA
1200
900
600
300
10
0
Age of plants after inoculation (days)
10
20
30
10
20
30
Figure 2. Analysis of free and total IBA during AM
colonization in leaves of maize plants. Data are mean
values of three independent determinations. ( ) Control
plants, ( ) plants infected with Glomus intraradices. The
experiment was performed without and with treatment
of plants with 10 mM TFIBA, which was added to the root
system of the plants.
C 450
IBA (ng/g Fwt)
30
0
200
400
20
free
amide
350
300
250
200
150
100
50
0
10
20
30
10
20
30
Age of plants after inoculation (days)
Figure 1. Mycorrhizal colonization rates (A), analysis of
free, and amide-bound IAA (B) and IBA (C) during AM
colonization in roots of maize plants. Data are mean
values of three independent determinations. B+C: ( )
control roots, ( ) roots infected with Glomus intraradices.
The levels of conjugated IBA were induced at
earlier time points in leaves from AM plants (10
and 20 dpi) compared to the roots (30 dpi). These
results show that colonization with G. intraradices
also influences the auxin content in the upper part
of the plant. Treatment of inoculated roots with
TFIBA reduced the IBA content to control levels
(Kaldorf and Ludwig-Müller, 2000). Here we show
that also the systemic increase in IBA is reduced
after TFIBA treatment (Fig. 2). This was mainly
observed for free IBA levels in leaves from plants
inoculated with Glomus, although a slight effect
was also observed for conjugated IBA.
Auxin metabolism and hydrolysis
Since the ratio of free to conjugated IBA changed
during development of AM-colonization, we inves-
tigated the IBA metabolism in control and Glomusinfected roots at different time points. 3H-IBA
was fed to roots for 3 h and the metabolites
determined by autofluorography (Fig. 3A).
Four different metabolites in addition to IBA could
be distinguished, but there were only little
differences between (a) control and infected roots
and (b) between the different time points.
Previously, two metabolites from control roots
were tentatively identified as ester conjugates
and one as amide conjugate (Ludwig-Müller et al.,
1997).
The induction of IBA conjugate hydrolysis
might also contribute to the increase in free IBA
in roots of maize inoculated with G. intraradices.
Therefore, the capacity of maize roots to hydrolyze
auxin conjugates was examined. No increase in
auxin conjugate hydrolase activity was observed
in AM-colonized roots when amide conjugates of
IAA or IBA with alanine and an ester conjugate of
IAA with glucose were used as substrates (Fig. 3B).
On the contrary, control roots showed higher
hydrolase activity than inoculated roots. In general, the rate of IBA-alanine hydrolysis was lower
than that of IAA-alanine and IAA-glucose. IAAaspartate was not a good substrate at all (data
not shown).
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Auxins and arbuscular mycorrhiza
1215
A
10
IBA
20
30 dpi
Rf value
0.98
0.84
0.76
0.69
control
0.51
0
control
AM
1000 2000 3000 0
1000 2000 3000 0
control
AM
1000 2000 3000 0
1000 2000 3000 0
AM
1000 2000 3000 0
1000 2000 3000
Relative intensities
B
nmol auxin released /min/mg
protein
18
16
IBA-alanine
IAA-alanine
IAA-glucose
14
12
10
8
6
4
2
0
10
20
30
10
20
30
10
20
30
Age of plants after inoculation (days)
Figure 3. In vivo metabolism of auxin conjugates (A) and in vitro hydrolysis of IBA and IAA conjugates (B) by maize roots
during colonization with the AM fungus Glomus intraradices. A. Metabolism of 3H-IBA in control (c) and Glomus
intraradices-inoculated (AM) roots at different time points (dpi ¼ days post inoculation) during AM-development.
Relative intensities are given for individual spots after scanning of the X-ray film. B. Enzyme extract from control ( )
and AM-inoculated roots ( ) of maize seedlings were incubated with three different possible substrates and the IAA/IBA
released enzymatically was determined by HPLC. Data are mean values of three independent determinations.
90
80
70
60
50
40
30
20
10
0
A
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16
Time (h)
TFIBA (ng/g fr wt)
Previously, it was shown that treatment of AM
inoculated roots with TFIBA, an IBA antagonist of
root growth in maize, resulted in the reduction of
AM structures, especially intraradical hyphae and
arbuscules (Kaldorf and Ludwig-Müller, 2000).
However, it was not clear whether TFIBA acted
locally or systemically. We have therefore conducted several experiments to investigate TFIBA
transport in maize roots and hyphae of G. intraradices.
TFIBA was taken up by the roots of maize
seedlings very rapidly (Fig. 4A) and the uptake
was saturable after ca. 10 h. However, TFIBA was
not transported in the plant, but it remained at the
site of uptake (Fig. 4B and C). Only the part of the
root emerged in the solution contained TFIBA,
whereas the following two root segments did not
contain any TFIBA. Although TFIBA is not transported in roots, it could probably be taken up by
hyphae of G. intraradices and transported from
hyphae to the plant. A two-compartment system
was used to answer this question (Fig. 5). TFIBA was
added to the hyphal compartment and after 6–70 h
incubation time the roots from the second compartment were extracted and analyzed for TFIBA.
To differentiate between hyphal transport and
Uptake (%)
TFIBA uptake and transport
50
C
B
40
30
20
10
3
0
2
1
1
2
3
Segment no.
Figure 4. Uptake and transport of the IBA derivative
trifluoro-IBA (TFIBA). A. Uptake was determined by
extracting the roots of maize seedlings after the
appropriate incubation time. TFIBA was determined by
HPLC. B+C. Transport of TFIBA (B) was measured by
extracting defined segments (C) of maize roots after 1 ( )
and 16 h ( ) and analysis of the amount of TFIBA by HPLC.
simple diffusion of TFIBA into the second compartment, the experiment included a control with not
inoculated plants, where TFIBA was added to the
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D. Fitze et al.
TFIBA
A
A
TFIBA
?
TFIBA taken up into
the roots (%)
Lecaton + Glomus
Quartzsand
+hyphae
-hyphae (control)
0.25
B
0.2
0.15
0.1
0.05
0
6
24
48
70
Incubation time (h)
Figure 5. TFIBA uptake and transport in maize roots and
hyphae of Glomus intraradices. A. Experimental setup
showing the two-compartment system used for the
transport experiment. TFIBA was added to the hyphal
compartment and the amount of TFIBA transported to the
roots was determined after different incubation times
with HPLC. The experiment was done with control plants
without hyphae in the second compartment ( ) and
inoculated plants, where hyphae of the AM fungus
developed in the second compartment ( ). For each
condition and time point three plants were independently assayed.
empty (hyphal) compartment before the plant
roots were analyzed at the same time points. In
both cases TFIBA was found in the roots of some
maize plants. Thus, TFIBA could diffuse into
several, but not all of the root compartments and
was there taken up by the roots, which came in
contact with the substance. This could explain
the irregular pattern of TFIBA uptake observed
(Fig. 5b). Nevertheless, a specific transport of
TFIBA by hyphae of G. intraradices can be ruled
out, as several of the AM colonized roots did not
contain any TFIBA.
Discussion
The role of plant hormones in AM symbiosis is far
from understood. It has been suggested that
phytohormones, such as IAA and cytokinins, released by the infecting fungi may contribute to the
enhancement of plant growth (Frankenberger and
Arshad, 1995). In addition, plant hormones may
play a role as signaling molecules during the
establishment of an AM symbiosis (Barker and Tagu,
2000, Ludwig-Müller, 2000). In ectomycorrhizal
symbiosis several examples are known where IAA
is produced by the fungal partner, and may result in
the increase of lateral roots and subsequently in a
stimulation of the formation of the symbiosis
(Karabaghli-Degron et al., 1998). A similar model
has been proposed for root colonization by AM fungi
(Kaldorf and Ludwig-Müller, 2000). Auxins, in
particular IBA, may facilitate the colonization of a
host by increasing the number of lateral/fine roots
during early growth phases and thus stimulating the
mycorrhizal root colonization. In accordance with
this hypothesis, Tisserant et al. (1996) found the
most active mycelium, characterized by fungal
enzyme activities, in newly formed lateral roots.
Besides the possibility that the increase in auxin
can result in a change in root morphology,
endogenous plant hormone formation may be
correlated directly with fungal structures. This
was recently reported for jasmonic acid (JA).
Colonization of barley roots by the AM fungus G.
intraradices led to elevated levels of endogenous
JA and its amino acid conjugate JA-isoleucine
(Hause et al., 2002). In situ hybridization and
immunocytochemical analysis of a JA-induced
protein revealed that its expression occurred
specifically in root cortex cells containing arbuscules. The authors suggested that the endogenous
rise in jasmonates might be related to the fully
established symbiosis rather than to the recognition
of interacting partners or to the onset of interaction (Hause et al., 2002). In our work, intraradical
hyphae, arbuscules and vesicles increased during
the investigation period (Fig. 1). However, in AM
colonized tobacco roots transformed with the
auxin-responsive promoter GH3 fused to b-glucuronidase (GUS) no clear correlation between AM
structures and GUS staining was found (F. Rehn and
J. Ludwig-Müller, unpublished results).
Different approaches were used to investigate
the role of IAA and IBA in AM symbiosis:
(1) measurement of endogenous auxin contents
locally and systemically, (2) analysis of auxin
conjugation/hydrolysis, (3) treatment of plants
with the IBA antagonist TFIBA. Since there were
no differences between AM and control roots in IBA
conjugate formation and hydrolysis of IBA conjugates in AM roots was even reduced (Fig. 3), neither
the regulation of IBA conjugate formation nor
hydrolysis contributes to the increase in free IBA
ARTICLE IN PRESS
Auxins and arbuscular mycorrhiza
formation (Kaldorf and Ludwig-Müller, 2000; this
study). The differences in the levels of IBA
conjugates during AM colonization could be explained by a degradation pathway as described for
IAA-aspartate in several plant species (Tuominen et
al., 1994; Östin et al., 1998). Whether certain IBA
conjugates may be subjected to degradation has
yet to be shown.
Auxin conjugate hydrolase activity was lower in
AM roots than in controls. The enzymatic hydrolysis
rate of IBA-alanine was below that for the two IAA
conjugates. It cannot be ruled out that the
hydrolysis of IBA conjugates other than IBA-alanine
contributes to the increase of free IBA levels in AM
roots. Recently, a gene from the IAR3 family of
auxin conjugate hydrolases was isolated from
wheat and the corresponding protein showed a
high substrate specificity for IBA-alanine and IBAglycine (Campanella et al., 2004). In the TIGR EST
database a large number of orthologs from maize
can be found (www.tigr.org, Campanella et al.,
2003b) with as yet unknown substrate specificity.
Spores and hyphae of G. intraradices did not
contain IBA (Ludwig-Müller et al., 1997; J. LudwigMüller, unpublished results), while spores but not
hyphae contained a minute amount of IAA. Therefore, uptake of IBA produced by AM fungi into the
host roots is very unlikely. Besides, the small
amount of IAA detected in AM spores may not be
sufficient to increase IBA synthesis because of
better substrate availability. The most likely
explanation for the higher amount of free IBA in
AM maize roots thus is increased de novo synthesis.
The shoots of AM plants showed an increase in
growth (Kaldorf and Ludwig-Müller, 2000) which
correlates with the increase in free IBA levels in
leaves. The latter could be explained by two
possibilities: (1) de novo IBA synthesis in leaves,
and (2) transport of IBA which is synthesized in AM
roots to the leaves of inoculated plants. While the
increase of IBA levels in leaves of inoculated maize
plants was very high (Fig. 2), the IBA synthetase
activity in leaves from inoculated plants was only
moderately increased by about 25% compared to
control plants (J. Ludwig-Müller, unpublished results). Therefore, at least part of the increase in
the amount of IBA is more likely due to transport
from the roots into the shoots. This hypothesis is
supported by experiments with TFIBA applied to
the roots of AM-inoculated plants, which resulted in
lower IBA levels in leaves. Previously, it was shown
that TFIBA treatment of AM inoculated maize roots
resulted in a decrease in IBA levels down to controls
(Kaldorf and Ludwig-Müller, 2000). On the contrary,
the transport of IBA from the shoots into the roots is
not very likely, since IBA synthesis is much higher in
1217
root tissue of maize compared to leaves (LudwigMüller et al., 1995). Further experiments with
auxin transport inhibitors should be carried out to
solve this question.
TFIBA is neither transported in hyphae nor in the
plant root and hypocotyl in our system (Fig. 5). This
indicates that the reduction in the number of
fungal structures previously described (Kaldorf and
Ludwig-Müller, 2000) is most likely due to a local
effect or mediated by mobile signals induced via
TFIBA. Scanning electron microscopy showed in
some cases slightly deformed hyphae after TFIBA
treatment (M. Ruppel and J. Ludwig-Müller, unpublished results), supporting the hypothesis of
local TFIBA effects. Probably TFIBA itself is an
inhibitor of IBA transport, because after application
of TFIBA to roots, not only root growth but also
shoot growth was inhibited (Kaldorf and LudwigMüller, 2000) and systemic IBA induction after AM
infection was suppressed in leaves (Fig. 2). In
addition, TFIBA inhibits IBA synthetase (Kaldorf and
Ludwig-Müller, 2000), thereby influencing IBA levels. Clearly, the complex scenario of auxin synthesis, conjugate formation/hydrolysis and transport
warrants further studies to elucidate their role for
increased auxin formation during early events of
AM colonization.
Acknowledgements
This work was supported by the Deutsche
Forschungsgemeinschaft (Lu500/5-1). 13C1-IBA was
a gift from Dr. Ellen G. Sutter, University of
California, Davis, CA, USA. TFIBA was kindly
supplied by Dr. Masato Katayama, National Industrial Research Institute of Nagoya, Japan. We thank
Silvia Heinze for technical assistance.
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