Plant Physiol. (1994) 105: 607-610
lncorporation of Label from 13C-, *H-, and 15N-Labeled
Methionine Molecules during the Biosynthesis of
2’-Deoxymugineic Acid in Roots of Wheat
Jian Feng Ma* and Kyosuke Nomoto
Suntory lnstitute for Bioorganic Research, Shimamoto-cho, Mishima-gun, Osaka, 618, Japan
The biosynthetic pathway of 2’-deoxymugineic acid, a key phytosiderophore, was investigated by feeding 13C-, *H-, and
‘5N-labeled methionine, the first precursor, to the roots of hydroponically cultured wheat (Triticum aestivum 1. cv Minori). The
incorporation of label from each methionine species was observed
during their conversion to 2‘-deoxymugineic acid, using ’H-, 15N-,
and 13C-nuclear magnetic resonance (NMR). i-[l-13C]Methionine
(99% 13C)was efficiently incorporated, resulting in 13Cenrichment
of the three carboxyl groups of 2‘-deoxymugineic acid. Use of D,L[‘5N]methionine (95% 15N)resulted in ”N enrichment of 2‘-deoxymugineic acid at the azetidine ring nitrogen and the secondary
amino nitrogen. When ~,~-[2,3,3-’H,-S-methyl-~H~]methionine
(98.2% ’H) was fed to the roots, ’H-NMR results indicated that
only six deuterium atoms were incorporated, and that the deuterium atom from the C-2 position of each methionine was almost
completely lost. [2,2,3,3-’H411-Aminocyclopropane-1-carboxylic
acid (98% *H) was not incorporated into 2‘-deoxymugineic acid.
These data and our previous findings demonstrated that only the
deuterium atom from the C-2 position of i-methionine was lost,
and that other atoms were completely incorporated when three
moleculesof methionine were convertedto 2’-deoxymugineic acid.
These observations are consistent with the conversion of i-methionine to azetidine-2-carboxylic acid, suggesting that i-methionine
i s first converted to azetidine-2-carboxylic acid during biosynthesis
leading to 2‘-deoxymugineic acid. Based on these results, a hypothetical pathway from L-methionine to 2‘-deoxymugineic acid was
postulated.
Gramineous plants respond to iron stress by secreting
mugineic acids, a group of ferric-chelating substances (Takagi, 1976; Takemoto et al., 1978). Until now, six of these
compounds from different gramineous plants have been
identified (Nomoto et al., 1987). In our previous report, two
related biosynthetic pathways for these mugineic acids were
described on the basis of feeding with ’H- and 13C-labeled
compounds (Ma and Nomoto, 1993). These pathways differ
after 2‘-deoxymugineic acid formation and are dependent on
the plant species. In oat, avenic acid A is biosynthesized from
2’-deoxymugineic acid by cleavage of the azetidine ring. In
barley, hydroxylation at the C-2’ position of 2’-deoxymugineic acid yields mugineic acid. Further hydroxylation at the
C-3 position produces 3-epihydroxymugineic acid in beer
barley and 3-hydroxymugineic acid in rye (Ma and Nomoto,
1993, 1994). However, a11 of these pathways share the same
steps from t-Met to 2’-deoxymugineic acid.
We found no significant differences in solubilization and
uptake of iron among different mugineic acids (Ma et al.,
1993). This suggests that 2’-deoxymugineic acid is the key
compound, because it is the first to be biosynthesized and
serves as a precursor for other mugineic acids. The biosynthesis of 2‘-deoxymugineic acid is also suggested as a key
step for plants resistant to iron stress. Therefore, elucidating
this pathway is helpful not only for isolation and purification
of the related enzymes but also for subsequent cloning of
genes encoding synthetic enzymes of 2’ -deoxymugineic acid.
Knowledge of the biosynthetic pathway between L-Met
and 2’-deoxymugineic acid is limited. From the results of
experiments in which t-[l-13C]Metwas fed to roots of barley
(Hordeum vulgare L. cv Minorimugi), elongation of Met units
was considered as a hypothetical pathway (Kawai et al.,
1988). However, this pathway has not yet been verified. An
in vitro study indicated that nicotianamine served as a precursor of 2’-deoxymugineic acid (Shojima et al., 1990); however, details of the pathway from t-Met to nicotianamine
remain to be elucidated.
In this study, feeding experiments with 13C-, ’H-, ”Nlabeled Met, the first precursor, were conducted using roots
of hydroponically cultured wheat (Triticum aestivum L. cv
Minori). From the observation of incorporation of label from
each Met molecule during its conversion to 2’-deoxymugineic
acid, a hypothetical biosynthetic pathway is proposed.
MATERIALS A N D METHODS
Plant Culture and Feeding
Wheat plants (Triticum aestivum L. cv Minori) were cultured hydroponically as previously reported (Ma and Nomoto, 1993). Briefly, 80 selected seedlings (7 d old) were transplanted into 3-L Wagner pots with continuously aerated
nutrient solution in an environmental chamber. Three-tenthsstrength Hoagland solution containing 1.5 m~ KN03, 1.5 m~
Ca(N03)*,0.6 m~ MgS04, and 0.3 m~ NH4H2P03was used
for the culture. Micronutrients were employed at the full
strength of the Hoagland solution [a
3.0
:
X 10-3 H3BO3, 5
Abbreviations: FAB, fast atom bombardment; nicotianamine,
2S,3’S,3”S-N-[N-(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl] azetadine-2-carboxylicaad.
* Corresponding author; fax 81-75-962-2115.
607
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Copyright © 1994 American Society of Plant Biologists. All rights reserved.
Ma and Nomoto
608
Table 1. lncorporation of ~-[l-'~C]Metinto 2'-deoxymugineic acid
(DMA) in wheat roots
was fed to roots that had
One hundred micromolar ~-[l-'~C]Met
been kept in a nutrient solution lacking iron for 11 d. The 13C-NMR
spectra (D20)were obtained using a 75.5-MHz spectrometer. The
calculation of the 13C enrichment was based on the line intensity
of the C-3 Dosition.
Carbona
Chemical
Shift
Signal Strength of DMA
Formed from
Unlabeled
~-ll-"ClMet
15661.7
12807.7
15561.9
1243.2
855.5
821.2
20.3
19.8
21.8
0.7
0.7
0.6
0.9
fold
PPm
c-4"
c -1
182.7
198.9
175.9
166.9
c-4'
174.8
c-3"
73.1
69.7
62.4
54.0
184.2
429.1
334.9
382.4
268.7
970.8
53.3
282.2
1046.1
47.0
348.0
1090.7
33.2
287.8
290.8
915.3
1062.1
1324.5
c-2
c-3'
c-4
c-1'
c-1"
c-2"
c-2'
c-3
a
27.3
23.9
"C Enrichment
of DMA
342.0
1.o
0.8
0.8
0.9
Plant Physiol. Vol. 105, 1994
NMR and FAB-MS Study
Samplles of 2'-deoxymugineic acid were dissolved in D 2 0
or HzO for NMR spectroscopy at 400 ('H), 61.3 ('H), or 75.5
MHz ("(3.The conditions for 'H-, 'H-, and l3C-1VMR measurement were the same as those previously reported (Ma
and Nomoto, 1993). Peaks in both the 'H and I3C: spectra of
2'-deoxymugineic acid have previously been assigned (Kawai
et al., 19138; Ma and Nomoto, 1993). Because 'H arid 'H shifts
correspoind except for a small isotope effect and broadening,
'H assignment was accomplished based on that of 'H. The
I3C enrichment was calculated from the line interisity of the
C-3 posifion. The 15N-NMRspectra of 2'-deoxymugineic acid
(D20) wlere measured at 30.5 MHz (GN-300 spectrometer)
under the following conditions: pulse width, 10.0 ps; acquisition time, 1.36 s; recycle time, 2.35 s; number of acquisitions,
22,112; data size, 16,384. NH4N03 was used as ,an extemal
reference to calibrate the chemical shift. FAB-MS (JEOLJMSHX 110/110A) spectra for 'H- and "N-labeled ,md nonlabeled 2'-deoxymugineic acid were recorded at 10 kV using a
glycerol matrix. The 'H and 15N enrichment of 2'-deoxymugineic acid were calculated from the mass spectra.
1.o
Refer to Figure 2 for numbering
RESULTS AND DlSCUSSlON
The acquisition of iron by gramineous plants is characterized by secretion of femc-chelating substances, th e mugineic
acids. Most species and cultivars secrete two or more compounds (Ma and Nomoto, 1993). However, wheat seaetes
only one compound, 2'-deoxymugineic acid, which is the
X 10-4 MnCl', 2 X 10-4 CuS04, 4 x 10-4 ZnS04, 1.0 x 10-3
(NH4)6M07024,
1.1 X 10-' FeCI3]. The nutrient solution was
adjusted to pH 5.5 with 0.1 N KOH and replaced every 2 d.
The light intensity was about 40 W/m2, and the light/
dark period and temperatures were 14/10 h and 17/1OoC,
respectively .
After 2 weeks, the plants were subjected to iron deficiency
by transferring them to pots containing the above nutrient
solution but free of iron. Feeding experiments were performed with 80 plants each with nutrient solution minus iron
for 11 to 13 d in 3-L Wagner pots containing the Hoagland
solution. After the secretion of 2'-deoxymugineic acid, the
roots were fed with the following labeled compounds: (a)
100 ~ L M~-[l-'~C]Met
(99.0% I3C, Isotec, Inc., Miamisburg,
(95% "N, Berlin-Adlershof,
OH); (b) 100 p~ D,L-['~N]M~~
Berlin, Germany); (c) 100 p~ ~,~-[2,3,3-~H~-S-methyl-~H~]Met
(98.2% 'H, Merck Sharp & Dohme/Isotopes, Montreal, Canada); (d) 50 PM [2,2,3,3-'H4]ACC (98% 'H, Sigma). The
compounds were dissolved in the nutrient solution. Because
a prior experiment showed that the surface-active agent
Decaglyn 1-L (Nikko Chemicals, Tokyo, Japan) stimulated
Met uptake, 40 pg mL-' pot-' was added during feeding
experiments.
~
lsolation and Purification
Isolation of 2'-deoxymugineic acid was performed as previously reported (Ma and Nomoto, 1993). Briefly, root washings were collected the following day by soaking the roots in
distilled water (3 L x 3) from 3 to 5 h after the onset of the
light period. The root washings were then purified by chromatography (Ma and Nomoto, 1993).
'
~
'
~
l
'
"
'
I
'
'
'
'
l
'
~
'
Chemical shift (ppm)
Figure 1. 'H-NMR spectrum of unlabeled 2'-deoxymugineic acid
(A) and 2H-NMR spectrum of 2'-deoxymugineic acid (E;) biosynthesized froni ~ , ~ - [ 2 , 3 , 3 - ' H ~ - . - m e t h y I - ~ H ~in
] Mwheat.
et
CNpectra were
measuredi at 400 MHz ('H-NMR, D20) and 61.3 MHz ('H-NMR,
H 2 0 ) . Tht? peak at 4.83 ppm represents a signal from either H 2 0 or
D 2 0 . Refer to Figure 2 for numbering.
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Biosynthesis of 2’-Deoxy.mugineic Acid
L-Met-”N
~.[3.3.4.4-~H,]Met
Figure 2. lncorporation of Met molecules labeled with ”C, 2H,and
15Nat different positions during conversion to 2’-deoxymugineic
acid.
key phytosiderophore. Therefore, wheat is a good experimental model to study the biosynthetic pathway from L-Met
to 2’-deoxymugineic acid.
To obtain information about the biosynthetic pathway
between L-Met and 2’-deoxymugineic acid, we studied how
the first precursor, L-Met, is incorporated into 2’-deoxymugineic acid. When [l-13C]Met was fed to the roots, 11.7 mg
of purified 2’-deoxymugineic acid was obtained. The I3CNMR study showed that the C-1, -4‘, and -4‘’ positions of
2‘-deoxymugineic acid were I3C enriched 20.3, 19.8, and
21.8 times, respectively, by comparison of their peak heights
with those of unlabeled 2’-deoxymugineicacid (Table I). This
result suggests that the three carboxyl groups of 2’-deoxymugineic acid were formed from the carboxyl groups of three
Met molecules. This result is in agreement with previous
findings in barley (cv Minorimugi) (Kawai et al., 1988).
When D,L-[”N]M~~
was fed to the roots, a ”N-NMR study
of isolated 2’-deoxymugineicacid (9.9 mg) showed two peaks
at 61.8 and 54.6 ppm, which represented approximately a
10-fold ”N enrichment. These peaks were assigned to the
azetidine ring nitrogen and the secondary amino nitrogen.
This result suggests that two nitrogen atoms in the 2‘-deoxymugineic acid resulted from nitrogen of L-Met.
(98.2%’H) was fed
When ~,~-[2,3,3-’H~-S-methyl-~H~]Met
to the roots, the isolated 2’-deoxymugineic acids (21.6 mg)
were 2H enriched by about 10-fold. The ’H-NMR study
indicated that the ratio of the peak intensities at 2.72, 2.52,
and 2.15 ppm was about 1:1:4 (Fig. 1); in all, six deuterium
atoms were present. According to the ‘H-NMR assignment,
these peaks correspond to deuterium on the C-3, C-2’, and
C-2” positions, respectively. If 2 ’-deoxymugineic acid is
formed from three molecules of Met (Kawai et al., 1988; Ma
and Nomoto, 1993), nine deuterium atoms from [2,3,3-’H3S-methyl-*H3]Metshould be present in the 2’-deoxymugineic
acid. Even if transamination and subsequent reduction at the
C-3” position caused the loss of deuterium at the C-3”
609
position, the deuterium at the C-2 and C-3’ positions would
be retained. However, the peak intensity of the deuterium at
the C-3‘ position was much lower than that of deuterium at
the C-2’ position originating from the same L-Met (Fig. 1).
The signal for the C-2 deuterium atom overlapped with the
D 2 0 signal (Fig. 1). To observe this signal, ‘H-NMR measurement of the same sample was performed at a lower
temperature (10OC). As a result, the D20signal was shifted
to a lower field (data not shown), but the peak for the C-2
deuterium atom was observed to be as small as the one for
the C-3’ deuterium. This indicates that the deuterium atoms
at the C-2 and C-3‘ positions from the C-2 position of Met
are almost completely lost when the Met molecules are converted to 2’-deoxymugineic acid.
In a previous study, when ~,~-[3,3,4,4-’H,]Metwas fed to
the roots, 12 deuterium atoms were observed in the 2’deoxymugineicacid (Ma and Nomoto, 1993). This suggested
that a11 deuterium atoms from C-3 and C-4 positions of three
’H-labeled Met molecules were incorporated during biosynthesis. Based on these results, Figure 2 summarizes the incorporation of each Met molecule labeled with 13C,’H, and 15N
COOH
H3C‘sdNH2
L-Met
COOH
I
COOH
COOH
IrTN)-
6N-i.N..
2s. 3’S-N-(3-amino-3-carboxypropyl)
COOH
azetidine-2-carboxylicacid
COOH
COOH
H3cxS&NH2
COOH
c N 4 N d NH H 2
2s. 3’S, 3”S-N-[N-(3-amino-3-carboxypmpyl)3-amino-3-carboxypropyl]azetidine-2-carbxylic
acid
2’-Deoxymugineic acid
Figure 3. A hypothetical pathway from L-Met to 2’-deoxymugineic
acid.
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Copyright © 1994 American Society of Plant Biologists. All rights reserved.
610
Ma and Nomoto
at different positions during conversion to 2’-deoxymugineic
acid in the roots of wheat. It is clear that except for the
hydrogen at C-2, C-3‘, and C-3” and the terminal hydroxyl,
a11 other atoms in 2’-deoxymugineic acid are formed from
three molecules of Met. These results eliminate aspartic psemialdehyde and 4-amino-2-oxobutanoic acid as intermediates between Met and 2’-deoxymugineic acid, because if
they were intermediates, the deuteriums from the C-4 position of [3,3,4,4-’H4]Met and nitrogen from [15N]Met would
not be observed in the 2‘-deoxymugineic acid biosynthesized
from these labeled Met molecules (Ma’and Nomoto, 1993;
Fig. 1).
The mechanism for loss of deuterium from the C-2 position
of Met during the conversion is still unknown. If each of the
Met precursor forms a Schiff‘s base with pyridoxal phosphate, an enzyme cofactor, before they are converted to 2’deoxymugineic acid, the deuterium from the C-2 position of
Met will be lost. However, an in vitro study indicated that
the step from Met to nicotianamine needs no cofactor (Mori,
1993), suggesting that this mechanism for deuterium loss is
unlikely.
The precursor of ethylene is ACC, and it is also formed
from L-Met (Yang and Hoffman, 1984). The formation of
ACC from L-Met could also similarly result in the loss of the
deuterium at the C-2 position in Met as described above.
Therefore, ACC could be the precursor of 2‘-deoxymugineic
acid. However, feeding [2,2,3,3-*H4]ACCto the wheat roots
did not result in the incorporation of ’H atom into the isolated
2’-deoxymugineic acid (8.2 mg).
Azetidine-2-carboxylic acid is a four-membered cyclic imino acid that is distributed among severa1 members of the
Liliaceae, a few species of Agavaceae and Amaryllidaceae,
and other plant species (Leete et al., 1974). Its biosynthesis
has been intensively investigated, especially in Convalaria
majalis (lily of the valley). In this plant, L-Met was found to
be the precursor for azetidine-2-carboxylic acid (Leete et al.,
1974, 1986). Met labeled with 14Cat C-1 and with 3H at the
C-4 position is incorporated into azetidine-2-carboxylic acid
with complete retention of the 3H relative to 14C(Leete et al.,
1974). Feeding [l-14C,15N]Metresults in the same specific
incorporation of these two isotopes into azetidine-2-carboxylic acid (Leete et al., 1986). The azetidine-2-carboxylic acid
derived from [1-14C,2-3H]Metalmost completely loses the 3H
(95%) from the C-2 position (Leete et ai., 1974), and [l-13C~arboxyl-’~C-’~N]ACC
is not incorporated. These results are
consistent with the present findings (Table I; Fig. 1) and our
previous observation of L-Met conversion to 2‘-deoxymugineic acid (Ma and Nomoto, 1993), thus suggesting that L-Met
is first converted to azetidine-2-carboxylic acid and then to
2’-deoxymugineic acid.
2S,3 ’S-N-(3-amino-3-carboxypropyl)azetidine-2-carboxylic acid and nicotianamine are easily produced chemically
from azetidine-2-carboxylic acid (Kristensen and Larsen,
1974). An in vitro study indicated that nicotianamine serves
Plant Physiol. Vol. 105, 1994
as a precursor of 2’-deoxymugineic acid (Shojilna et al.,
1990). These findings led us to postulate a hypothetical
biosynthetic pathway between L-Met and 2’-deoxymugineic
acid (Fig. 3). Studies on this pathway are now in progress.
ACKNOWLEDCMENTS
We are indebted to Dr. H. Naoki and Ms. T. Matsuda of our
institute antd Mr. J. Matsuda of Shimadzu Corp. for their c:onstructive
advice andi skillful NMR determinations. We are also very grateful
to Mr. T. Fujita for FAB-MS measurements and to Dr. I’. Escoubas
of our instiitute for his correction of the English text.
Received December 20, 1993; accepted February 28, 1994.
Copyright Clearance Center: 0032-0889/94/105/0607/04.
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