From the
The biosynthesis of 2`-deoxymugineic acid, a key
phytosiderophore, was examined in association with the putative
methionine recycling pathway in the roots of wheat using labeling
experiments and structural analysis. Feeding with D-[1-
Mugineic acids (MAs),
The biosynthesis of MAs
is induced by iron deficiency stress in the roots (Takagi, 1976); the
more severe the iron deficiency, the greater the amount of mugineic
acids synthesized (Takagi et al., 1984). The biosynthesis of
MAs proceeds throughout the day, and the biosynthesized MAs are then
accumulated in the roots until their secretion to the rhizosphere the
following morning. The amount of biosynthesized MAs reaches as high as
1-2% of root dry weight. However, the concentration of L-Met, the precursor, is extremely low in the roots. As such
low levels of Met can maintain the observed high rate of MAs synthesis,
we hypothesized that Met may be recycled during MAs biosynthesis in a
manner similar to that occurring in ethylene production in ripening
apples and tomatoes (Yang and Hoffman, 1984). Several label experiments
were conducted here to verify this hypothesis.
D-5-[5-
To a solution of tosylate (10
g, 28 mmol) in N,N-dimethylformamide (30 ml), sodium
thiomethoxide (2.3 g, 33 mmol) was added at 0 °C under
N
A mixture
of the acetonide (6 g, 27.2 mmol) and Amberite IR-120B (H
2-[1-
2`-Deoxymugineic acid is the first phytosiderophore to be
biosynthesized and serves as a precursor for other MAs (Fig. 1)
(Ma and Nomoto, 1993, 1994a). Furthermore, as significant differences
in solubilization and uptake of iron were not observed among different
MAs (Ma et al., 1993), DMA is suggested as a key
phytosiderophore. Since the aim of the present study was to determine
how L-Met is derived during MAs biosynthesis, wheat, which
secretes only DMA, was used as a model plant.
Generally, the level of Met is low in higher plants, being about
1/100 those of other amino acids such as Asp and Glu. The pathway
leading to formation of L-Met in higher plants is Asp
Methionine also serves as a precursor of
ethylene (Yang and Hoffman, 1984), and it has been established that Met
is recycled during ethylene production as shown in Fig. 1. The
first step of the recycling pathway is the activation of Met by ATP to
give AdoMet. S-Adenosylmethionine is fragmented to give
5`-methylthioadenosine and 1-aminocyclopropane-1-carboxylic acid, which
is then converted to ethylene. 5`-Methylthioadenosine is hydrolyzed to
MTR and adenine, and phosphorylation of MTR by MTR kinase yields
5-methylthioribose-1-phosphate. Loss of the phosphate group of
5-methylthioribose-1-phosphate, concurrent with the rearrangement of
the ribose carbon atoms, leads to the synthesis of KMB. In the final
step of the sequence, KMB is converted to Met via transamination. The
overall result of this cycle is that the ribose moiety of ATP (carbons
2`, 3`, 4`, and 5`) furnishes the 4-carbon moiety of Met, and the
CH
To examine whether this Met recycling pathway is operative
during DMA biosynthesis in wheat roots, incorporation experiments using
ribose labeled with
L-Methionine was also found to be
activated into AdoMet first by ATP before leading to DMA in vitro (Shojima et al., 1990). In the case of ethylene
biosynthesis, AdoMet is fragmented to 1-aminocyclopropane-1-carboxylic
acid and 5`-methylthioadenosine, and the latter enters the Met
recycling pathway. 1-Aminocyclopropane-1-carboxylic acid is not
incorporated into DMA, and azetidine-2-carboxylic acid has been
suggested to be an intermediate (Ma and Nomoto, 1994b). This suggests
that AdoMet splits into azetidine-2-carboxylic acid and
5`-methylthioadenosine during DMA biosynthesis (Fig. 1).
The
process of iron acquisition by some gramineous plants includes MAs
biosynthesis inside the roots, diurnal rhythmic secretion of MAs to the
rhizosphere, effective solubilization of insoluble iron in soils by
chelation, and specific uptake of MAs-iron complexes. To date, only
some gramineous plants have been found to have the ability to
biosynthesize MAs. The strategy of iron acquisition by secreting MAs
adopted by gramineous plants has advantages over others (such as those
in dicotyledonous and nongramineous monocotyledonous plants) for
adaptation to calcareous soils from an ecological point of view
(Römheld and Marschner, 1987). Therefore, this strategy has been
targeted for breeding crops resistant to iron deficiency, and some
attempts have been made to isolate genes encoding MAs biosynthesis. The
present results represent important information for plant breeders that
genes responsible for both MAs biosynthesis and Met cycling should be
considered.
C]ribose did not result in
C enrichment of 2`-deoxymugineic acid, while D-[2-
C]ribose resulted in
C enrichment at the C-4", -1, -4` positions, and D-[5-
C]ribose did in C-1`, -4, and -1"
positions of 2`-deoxymugineic acid, respectively. Furthermore, two
isotope-labeled intermediates of the methionine recycling pathway,
5-[5-
H
]methylthioribose and
2-[1-
C]keto-4-methylthiobutyric acid, were
synthesized, and their incorporation into 2`-deoxymugineic acids was
investigated. Six deuterium atoms at the C-4, -1`, and -1" positions of
2`-deoxymugineic acid were observed after feeding with
5-[5-
H
]methylthioribose. Feeding with
2-[1-
C]keto-4-methylthiobutyric acid yielded
2`-deoxymugineic acid enriched with
C at the C-4`, -1, and
-4" positions. These results demonstrated for the first time that the
biosynthesis of 2`-deoxymugineic acid is associated with the methionine
recycling pathway. This association system functions to recycle
methionine required for continued synthesis of mugineic acids in the
roots of gramineous plants.
(
)a group of ferric
iron chelating substances which have been isolated from root exudates
of some gramineous plants such as barley and wheat, have been
recognized as phytosiderophores (see e.g. Nomoto et
al.(1987)). Hitherto six such compounds have been isolated and
identified from different gramineous plants. Although the chemical
structures and numbers of biosynthesized MAs differ among plant species
and even among cultivars within a species, all contain the same six
functional groups which are coordinated to ferric iron. As these
compounds play an important role in acquiring insoluble iron from soil
for optimal growth of gramineous plants in addition to their unique
structures, many efforts have been made to elucidate the biosynthetic
pathways of MAs. In vivo and in vitro biosynthetic
studies have consistently indicated that L-Met serves as a
precursor for all MAs (Mori and Nishizawa, 1987; Kawai et al.,
1988; Shojima et al., 1990; Ma and Nomoto, 1992, 1993, 1994a).
A series of feeding studies demonstrated that all MAs share the same
pathway from L-Met to 2`-deoxymugineic acid (DMA) although the
subsequent steps differ and are dependent on the plant species and
cultivar (Ma and Nomoto, 1992, 1993, 1994a).
Chemicals
D-[1-C]Ribose, D-[2-
C]ribose, and D-[5-
C]ribose (99.0%
C)
were purchased from ICON, Mt. Marion, NY. L-[1-
C]Met (99.0%
C) was
from Isotec, Inc., Miamisburg, OH. L-Amino acid oxidase (type
VI) and catalase were from Sigma.
H
]Methylthioribose
(MTR) was prepared with D-ribose as the starting material. The
latter (20 g) was converted to
1-o-[5-
H
]methyl-D-ribose
acetonide (6.1 g) in four steps according to De Voss et al. (1994). Introduction of a methylthio group and hydrolysis were
conducted as follows. To a solution of the acetonide prepared (6.1 g,
30 mmol) in pyridine (20 ml), p-toluenesulfonyl chloride (6 g,
31.5 mmol) was added at room temperature under N
. The
mixture was stirred for 16 h and poured into ice (100 g), then
acidified with 1 M HCl at 0 °C and extracted twice with
CH
Cl
. The combined extracts were washed with
saturated NaHCO
and saturated NaCl, dried over
MgSO
, filtered, and evaporated under reduced pressure. The
crude tosylate (10 g, 93%) was used for the next conversion without any
purification.
H NMR (400 MHz, CDCl
) ppm: 7.80
(2H, d, J = 8.4 Hz), 7.36 (2H, d, J =
8.4 Hz), 4.93 (1H, s), 4.60 (1H, d, J = 5.6 Hz), 4.53
(1H, d, J = 5.6 Hz), 4.30 (1H, s), 3.24 (3H, s), 2.46
(3H, s), 1.45 (3H, s), 1.23 (3H, s).
. The mixture was stirred at 0 °C for 30 min, then
allowed to warm to room temperature. After further stirring for 2.5 h,
the mixture was diluted with H
O (100 ml) and extracted
twice with hexane. The combined organic extracts were dried over
MgSO
, filtered, and evaporated under reduced pressure. The
corresponding D-[5-
H
]MTR
acetonide was obtained as a pale yellow oil (6.1 g, 99%), and was used
for the next conversion without any purification.
H NMR
(400 MHz, CDCl
) ppm: 4.98 (1H, s), 4.72 (1H, d, J = 6 Hz), 4.61 (1H, d, J = 6 Hz), 4.27 (1H,
s), 3.5 (3H, s), 2.15 (3H, s), 1.49 (3H, s), 1.33 (3H, s).
form) (20 g) in H
O (80 ml) was stirred at 80 °C
for 8 h, then filtered through Celite to remove the ion exchange resin.
The filtrate was concentrated under reduced pressure. Purification of
the crude mixture by silica gel column chromatography
(CH
Cl
-MeOH, 9:1) gave a colorless oil as a 1:2
anomeric mixture (3.2 g, 71%).
H NMR (400 MHz,
D
O) ppm: the major isomer; 5.23 (1H, d, J =
2 Hz), 4.21 (1H, dd, J = 5.2, 6.4 Hz), 4.07 (1H, d, J = 6.4 Hz), 4.02 (1H, dd, J = 5.2, 2
Hz), 2.17 (3H, s) and the minor isomer; 5.38 (1H, d, J = 4 Hz), 4.23 (1H, d, J = 5.6 Hz), 4.16
(1H, dd, J = 5.2, 4.0 Hz), 4.06 (1H, m), 2.16 (3H, s).
H NMR (61.25 MHz, 10% D
O/H
O) ppm:
2.65 (1H, br s), 2.80 (1H, br s). High resolution mass spectrometry
(FAB
) calculated for
C
H
O
SD
(M +
Na)
m/z 205.0479, found 205.0477.
C]Keto-4-methylthiobutyric acid (KMB)
sodium salt was enzymatically prepared from L-[1-
C]Met with L-amino acid
oxidase (Meister, 1952). Catalase solution (containing 100 units) and L-amino acid oxidase (20 units) were added successively to a
suspension of 0.5 g L-[1-
C]Met in 30 ml
of water. The pH was adjusted to 7.2 by the addition of 1 M
sodium hydroxide and the solution was incubated for 3 h in a water bath
at 37 °C. After the oxidation reaction was completed, the mixture
was dialyzed with agitation against three changes of distilled water (1
liter each time) at 4 °C for 12 h. The combined dialysates were
evaporated in vacuo at 40 °C to about 20 ml. The mixture
was then passed through a cation exchange resin (Dowex 50, hydrogen
form) to remove unconverted Met. The eluate was adjusted to pH 4.5 with
1 M NaOH, and the solution was concentrated in vacuo at 40 °C. Finally, 120 mg of white powder was obtained by
freeze-drying. The
H and
C NMR data were
consistent with unlabeled KMB purchased from Ardrich except that C-1 of
the synthesized product was
C-enriched (
96%).
Plant Growth
Wheat plants (Triticum
aestivum, L. cv Nourin 61) were cultured hydroponically as
previously reported (Ma and Nomoto, 1993). Briefly, about 80 selected
seedlings (7 days old) were transplanted to continuously aerated
nutrient solution in 3-liter plastic pots in an environmental chamber.
Hoagland solution (0.3 strength for macronutrients and full strength
for micronutrients) was used for culture. The nutrient solution was
adjusted to pH 5.5 with 0.1 M KOH and replaced every 2 days.
Light intensity was adjusted to 40 W m, and the
light/dark period and temperatures were set at 14/10 h and 17/10
°C, respectively. After 11 days, the plants were subjected to iron
deficiency.
Feeding Experiments
Feeding experiments were
performed with 80 plants exposed to iron deficiency for 10-12
days as described above in 3-liter Wagner pots containing Hoagland
solution. As the secretion of MAs is characterized by diurnal rhythm
(Takagi et al., 1984), all feeding experiments were conducted
after secretion. The roots were fed with the following labeled
compounds: 1) 300 µMD-[1-C]ribose; 2) 300 µMD-[2-
C]ribose; 3) 300 µMD-[5-
C]ribose; 4) 250 µMD-[2-
C]ribose and 250 µMD-[5-
C]ribose; 5) 500 µM [5-
H
]MTR; and 6) 200 µM [1-
C]KMB.
Isolation and Purification
Root washings were
collected the following day by soaking the roots in distilled water (3
liters, three times) from 3 to 5 h after the onset of the light period.
The root washings were then charged on Amberlite IR-120
(H), and the cationic fractions of 2 N NH
OH were eluted. Subsequent chromatography on Dowex
50W-X8 was performed, and gradient eluates with ammonia-formate buffer
(pH 2.5-3.1) provided a concentrated active fraction. Chelating
activities were determined by the o-phenanthroline method
(Takagi, 1976). Concentrated DMA fractions were finally subjected to
gel filtration through Sephadex G-10 with H
O as a solvent.
NMR Study
C NMR measurements of
isolated DMA (in D
O) were obtained with a 75.5-MHz
spectrometer (GE-300). 3-(trimethylsilyl)propionic acid sodium
salt-d
was used as an external reference for
calibration of the chemical shift, and assignments for
C
were accomplished by
C-
H COSY,
H-detected multiple-bond heteronuclear multiple-quantum
coherence and
H-detected heteronuclear multiple-quantum
coherence experiments. Calculation of
C enrichment was
based on the line intensity of the C-3 position.
H NMR
spectra of isolated DMA (in D
O) were recorded on a 400-MHz
spectrometer (JNM-EX400), and assignments of
H were
accomplished by
H-
H COSY. The
H NMR
spectra (in deuterium-depleted water) were measured at 61.3 MHz
(JNM-EX400 spectrometer). The natural abundance of
H in
H
O was used as a chemical shift reference for the
H NMR experiment. As
H chemical shifts
correspond to
H shifts, the assignment was accomplished
after analysis of the corresponding proton spectra.
Figure 1:
Biosynthesis of mugineic acids in
relation to methionine cycling. MTA, 5`-methylthioadenosine; MTR-P, 5-methylthioribose-1-phosphate; Ade, adenine; A-2-C, azetidine-2-carboxylic acid.
2`-Deoxymugineic acid
is biosynthesized from three molecules of L-Met (Kawai et
al., 1988; Ma and Nomoto, 1993, 1994b). A series of label studies
demonstrated that the 4-C moieties (C-1, -2, -3, and -4) of three Met
contribute to the skeleton of DMA while the methylthio group does not
(Ma and Nomoto, 1994b). The amount of secreted DMA reached about 1.5
µmol plant day
on the 12th
day after commencement of iron deficiency, and usually more DMA was
biosynthesized than was secreted since some DMA is retained within the
roots. Despite this active biosynthesis of DMA in the wheat roots, the
level of its first precursor, L-Met, was negligible.
Hse
o-phosphohomoserine
homocysteine
Met
(Giovanelli et al., 1974); however, there is no evidence to
show that this pathway is stimulated during DMA biosynthesis. Also, the
fate of the methylthio group derived from L-Met when it is
converted to DMA is not known. Furthermore, Met biosynthesized by the
above pathway is usually used for protein synthesis, and its activated
form, S-adenosylmethionine (AdoMet), is used for methylation
and polyamine biosynthesis. If most biosynthesized Met is additionally
consumed for DMA biosynthesis, the basal metabolic pathways will not
function. Therefore, it is reasonable to consider that Met is
biosynthesized by some other pathway to maintain the active
biosynthesis of DMA.
S group of Met is conserved for continued regeneration of
Met.
C at different positions were carried
out in wheat roots under iron deficient conditions. After feeding with
C-labeled ribose, ATP labeled with
C will be
formed. If Met used for DMA biosynthesis is regenerated by the
recycling pathway (Fig. 1), carbon 1 of the ribose moiety would
not be incorporated into DMA because it is degraded into
HCO
during the cycle, while carbons 2, 3,
4, and 5 of the ribose moiety would incorporated into specific
positions of DMA. When 300 µMD-[1-
C]ribose was fed to the wheat
roots, 18.5 mg of purified DMA were obtained. The
C NMR
study showed that there was no
C enrichment at any carbon
of DMA (Fig. 2B). When roots were fed D-[2-
C]ribose under the same condition,
the C-4", -1, and -4` positions of isolated DMA (18.2 mg) were
C-enriched 1.86-, 1.71-, and 1.84-fold, respectively, by
comparison of their peak heights with those of unlabeled DMA (Fig. 2, A and C). Feeding with D-[5-
C]ribose also resulted in
C enrichment of DMA (44.4 mg) by 1.81-, 1.68-, and
2.07-fold at the C-1`, -4, and -1" positions, respectively (Fig. 2D). A dual label experiment using equal
concentration of D-[2-
C]ribose and D-[5-
C]ribose showed that
C was equally incorporated into the specific positions of
DMA (47.6 mg) (Fig. 2E). Although the pool of ribose is
very large in the plants, the selective incorporation of
C-labeled ribose to DMA suggests that Met is recycled
during DMA biosynthesis as speculated (Fig. 1).
Figure 2:
C NMR spectra of unlabeled
2`-deoxymugineic acid (A) and 2`-deoxymugineic acid derived
from D-[1-
C]ribose (B), D-[2-
C]ribose (C), D-[5-
C]ribose (D), and the
equal mixture of D-[2-
C]ribose and D-[5-
C]ribose (E) in wheat
roots. Spectra were measured with a 75.5-MHz spectrometer (in
D
O). Refer to Fig. 1 for numbering. ★ represents the
positions enriched by
C.
To further
confirm this speculation, two isotope-labeled intermediates of the Met
cycling pathway, [5-H
]MTR and
[1-
C]KMB, were synthesized.
5-[5-
H
]Methylthioribose was
synthesized according to the scheme shown in Fig. 3. If the Met
cycling pathway is associated with DMA biosynthesis, the label (
H) of [5-
H
]MTR would be
incorporated into C-4, -1`, and -1" positions of DMA. In addition, in a
previous study, when Met labeled with
H at the C-4 position
was fed to the wheat roots, 6 deuterium atoms from 3 molecules of
H-labeled Met were completely incorporated into DMA (Ma and
Nomoto, 1993). Therefore, 6 deuterium atoms would be similarly observed
when fed with [5-
H
]MTR if the above
speculation is correct. When [5-
H
]MTR
was fed to the wheat roots,
H signal of the isolated DMA
(21.8 mg) was observed on the
H NMR spectrum (Fig. 4). Relative peak intensities at 4.05, 3.95, 3.35, and 3.21
ppm were, respectively, approximated to 1:1:2:2, and a total of 6
deuterium atoms were observed. Based on the
H NMR
assignment, these peaks corresponded to the deuterium atoms at the C-4,
C-1`, and C-1" positions, respectively. As can be seen in Fig. 4,
both the numbers and positions of
H incorporated are
completely consistent with the above speculation, demonstrating that
the Met recycling pathway is operative during DMA biosynthesis.
Figure 3:
Reaction scheme for the synthesis of
5-[5-H
]methylthioribose.
Figure 4:
H NMR spectrum of unlabeled
2`-deoxymugineic acid (A) and
H NMR spectrum of
2`-deoxymugineic acid (B) derived from
5-[5-
H
]methylthioribose in wheat
roots. Spectra were measured at 400-MHz (
H NMR,
D
O) and 61.3 MHz (
H NMR, deuterium-depleted
water). The peak at 4.8 ppm represents a signal for either
H
O or D
O.
2-[1-C]Keto-4-methylthiobutyric acid was
enzymatically synthesized from [1-
C]Met. If the
Met recycling pathway is related to DMA biosynthesis,
[1-
C]KMB would be converted into
[1-
C]Met by transamination, resulting in
C enrichment of three carboxyl groups in DMA. When roots
were fed [1-
C]KMB, 22.8 mg of DMA were isolated.
The results of
C NMR analysis showed that the C-4", -1,
and -4` positions were
C-enriched by 3.66-, 3.64-, and
3.94-fold, respectively (Fig. 5). This result further confirmed
the relationship between the Met recycling pathway and DMA
biosynthesis.
Figure 5:
C NMR spectra of unlabeled
2`-deoxymugineic acid (A) and 2`-deoxymugineic acid (B)
derived from 2-[1-
C]keto-4-methylthiobutyric
acid in wheat roots. Spectra were measured with a 75.5-MHz spectrometer
(in D
O). ★ represents positions enriched by
C.
The above findings demonstrate for the first time that
DMA biosynthesis is associated with the Met recycling pathway in wheat
roots (Fig. 1). Our results also suggest that the Met recycling
pathway is not limited to ripening apples and tomatoes but is also
active in wheat roots. This pathway allows continuous generation of ATP
resulting in the continuous synthesis of DMA in wheat roots even where
the Met pool is small, enabling DMA to be synthesized without depleting
the cellular Met pools.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.