©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Biosynthesis of Phytosiderophores, Mugineic Acids, Associated with Methionine Cycling (*)

Jian Feng Ma (§) , Tetsuro Shinada , Chitose Matsuda , Kyosuke Nomoto

From the (1)Suntory Institute for Bioorganic Research, Wakayamadai 1-1-1, Shimamoto-cho, Mishima-gun, Osaka 618, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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-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.


INTRODUCTION

Mugineic acids (MAs),()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).

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.


EXPERIMENTAL PROCEDURES

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.

D-5-[5-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 CHCl. 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).

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. 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 HO (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).

A mixture of the acetonide (6 g, 27.2 mmol) and Amberite IR-120B (H form) (20 g) in HO (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 (CHCl-MeOH, 9:1) gave a colorless oil as a 1:2 anomeric mixture (3.2 g, 71%). H NMR (400 MHz, DO) 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% DO/HO) ppm: 2.65 (1H, br s), 2.80 (1H, br s). High resolution mass spectrometry (FAB) calculated for CHOSD (M + Na)m/z 205.0479, found 205.0477.

2-[1-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 NHOH 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 HO as a solvent.

NMR Study

C NMR measurements of isolated DMA (in DO) 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 DO) 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 HO 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.


RESULTS AND DISCUSSION

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.


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.

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 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.

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 CHS group of Met is conserved for continued regeneration of Met.

To examine whether this Met recycling pathway is operative during DMA biosynthesis in wheat roots, incorporation experiments using ribose labeled with 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 DO). 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, DO) and 61.3 MHz (H NMR, deuterium-depleted water). The peak at 4.8 ppm represents a signal for either HO or DO.



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 DO). ★ 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.

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.


FOOTNOTES

*
This research was supported in part by the Ministry of Education, Science and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Research Institute for Bioresources, Okayama University, Chuo 2-20-1, Kurashiki 710, Japan.

The abbreviations used are: MA, mugineic acid; DMA, 2`-deoxymugineic acid; MTR, 5-methylthioribose; KMB, 2-keto-4-methylthiobutyric acid; AdoMet, S-adenosylmethionine.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.