Identification of two novel phytosiderophores secreted by perennial grasses

Authors


Author for correspondence: Jian Feng Ma Tel/Fax: +81 86 4341209 Email: maj@rib.okayama-u.ac.jp

Summary

  • • It has been suggested that some perennial grasses secrete phytosiderophores in response to iron (Fe) deficiency, but the compounds have not been identified. Here, we identified and characterized the phytosiderophores secreted by two perennial grasses, Lolium perenne cv. Tove and Poa pratensis cv. Baron.
  • • Root exudates were collected from the roots of Fe-deficient grasses and then purified with various chromatographies. The structure of the purified compounds was determined using both nuclear magnetic resonance and fast atom bombardment mass spectrometry.
  • • Both species secreted phytosiderophores in response to Fe deficiency, and the amount of phytosiderophores secreted increased with the development of Fe deficiency. The type of phytosiderophores secreted differed with plant species; L. perenne cv. Tove secreted 3-epihydroxy-2′-deoxymugineic acid (epiHDMA), 2′-deoxymugineic acid (DMA) and an unknown compound, whereas P. pratensis cv. Baron secreted DMA, avenic acid A (AVA) and an unknown compound.
  • • Purification and subsequent analysis with nuclear magnetic resonance and mass led to identification of the two novel phytosiderophores; 3-hydroxy-2′-deoxymugineic acid (HDMA) from L. perenne, and 2′-hydroxyavenic acid A (HAVA) from P. pratensis. Both novel phytosiderophores have similar chelating activity to known phytosiderophores.

Introduction

Iron acquisition by strategy II plants (gramineous plants) is characterized by the secretion of ferric chelating substances (phytosiderophores) and by a specific uptake system (Takagi, 1976; Takemoto et al., 1978; Römheld & Marschner, 1986; Ma & Nomoto, 1996; Ma, 2005). The biosynthetic pathway for various phytosiderophores has been elucidated (Mori & Nishizawa, 1987; Shojima et al., 1990; Ma & Nomoto, 1993; Ma et al., 1999) and several genes involved in phytosiderohopore biosynthesis have been cloned (Higuchi et al., 1999; Takahashi et al., 1999; Nakanishi et al., 2000; Higuchi et al., 2001; Kobayashi et al., 2001; Kobayashi et al., 2005). The secretion of phytosiderophores shows a distinct diurnal rhythm (Takagi et al., 1984) and polar vesicle transport seems to be involved in this process (Negishi et al., 2002), although the genes responsible for the process have not been cloned. For the uptake of the Fe3+–phytosiderophore complex, a gene yellow stripe 1 (YS1) encoding this transporter was identified in maize (Zea mays) (Curie et al., 2001) and, recently, a gene specific to the transport of the Fe3+–phytosiderophore complex was isolated in barley (Hordeum vulgare) (Murata et al., 2006).

However, knowledge of iron (Fe) acquisition by strategy II plants has been gained from studies using a few annual crop species including wheat (Triticum aestivum), barley, maize and sorghum (Sorghum vulgare), with only a few research papers investigating perennial grasses (Ma & Nomoto, 1996). Gries & Runge (1992) showed that some wild grass species secreted phytosiderophores in response to Fe deficiency. Ma et al. (2003) found that Festuca rubra (cvs Rubina and Barnica) secreted a phytosiderophore in response to Fe deficiency. Recently, Cesco et al. (2006) also reported that intercropping with either F. rubra or Poa pratensis improved Fe-deficiency-induced chlorosis in citrus, and this effect has been attributed to the release of Fe-chelating substances from the grasses. However, in most studies with perennial grasses, the compounds secreted were not identified. In the present study, we examined secretion of phytosiderophores by Lolium perenne cv. Tove and Poa pratensis cv. Baron under Fe deficiency and identified two novel phytosiderophores from the root exudates of these species.

Materials and Methods

Plant material and growth conditions

Lolium perenne L. cv. Tove and Poa pratensis L. cv. Baron were used in the present study. A total of 0.5 g of seeds (seeds of L. perenne cv. Tove and P. pratensis cv. Baron weigh on average 1.9 and 0.4 mg each, respectively) were germinated in the dark at 20°C on a mesh in contact with deionized water in a 3.5-l pot. After germination, seedlings were precultured in 1/5 Hoagland nutrient solution (pH 5.3) in a temperature-controlled growth chamber at 20°C with natural sunlight. The nutrient solution was renewed every 3 d. After 7 d of culture, the seedlings were transferred to 1/5 Hoagland nutrient solution with or without Fe and used for the following experiments. All experiments were performed with three replicates.

Collection and fractionation of root exudates

The root exudates were collected from Fe-deficient plants at different times. The day before collection of root exudates, the roots were washed with deionized water twice, and then placed in 3 l of deionized water. Collection of the root exudates was started before sunrise (c. 06:00 h), and terminated at 14:00 h. The root exudates collected were immediately concentrated using a rotary evaporator (Eyela, Tokyo, Japan) at 40°C. The residuals were re-dissolved in 1 ml of distilled water and then fractionated into cationic, anionic, and neutral fractions according to Ma et al. (2003).

Quantitative determination of phytosiderophores in root exudates

For quantitative analysis, the roots exudates collected were immediately passed through a cation-exchange column (16 mm × 14 cm) filled with Amberlite IR 120B (H+ form; Organo Co., Tokyo, Japan) and eluted with 2 m NH4OH. The eluates were concentrated using a rotary evaporator at 40°C. After the residues had been dissolved in 1 ml of distilled water, the amounts of phytosiderophores were determined by measuring its Fe-solubilizing capacity according to Takagi (1976), with some modifications (Ma et al., 2003).

For comparison of Fe-solubilizing capacity between the two novel phytosiderophores and known phytosiderophores, 80 nmol of each purified phytosiderophore, including 3-hydroxy-2′-deoxymugineic acid (HDMA), 2′-deoxymugineic acid (DMA), 2′-hydroxyavenic acid A (HAVA) and avenic acid A (AVA), was incubated with Fe(OH)3 suspension and the amount of Fe solubilized was determined according to Ma et al. (2003).

High-performance liquid chromatography (HPLC) analysis of root exudates

Root exudates were analyzed with HPLC using a cation exchange column (Shim-Pack, Amino-Li; Shimadzu Co., Kyoto, Japan) according to Kawai et al. (1987). The mobile phase was 0.15 m lithium citrate (pH 2.6), mixed with 0.2 m LiOH at a proportion of 5%. The total flow rate of the mobile phase was 0.4 ml min−1 at 50°C. Fluorescence was detected after reaction with NaClO and o-phthalaldehyde (Shimadzu Co.) at emission 450 nm and excitation 350 nm. Purified phytosiderophores including DMA, mugineic acid (MA), 3-epihydroxymugineic acid (epiHMA), 3-hydroxymugineic acid (HMA), and AVA were obtained from the root exudates of Fe-deficient wheat, barley, oat (Avera sativa) and rye (Secale cereale) (Ma & Nomoto, 1993). Their purity was c. 99%, determined by H+-nuclear magnetic resonance (NMR). The concentration was calculated based on the peak area.

Purification and identification of Fe-chelating substances

The cationic fractions with Fe-chelating activity were purified by combination of ion exchange and gel filtration chromatography (Ma & Nomoto, 1993). The 1H and 13C NMR spectra of the purified compound in D2O were recorded on a AVANCE-750 spectrometer (Bruker BioSpin GMBH, Rheinstetten, Germany) operated at 750.13 MHz for proton resonance at 298 K (Ma et al., 2003). Signal assignment of 1H and 13C was accomplished by two-dimensional NMR correlation spectroscopy such as double quantum filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), 1H {13C}-heteronuclear single quantum coherence (1H{13C}-HSQC), 1H {13C}-heteronuclear multiple bond correlation (1H{13C}-HMBC), and nuclear Overhauser and exchange spectroscopy (NOESY). The mass spectrum of the purified compounds was measured on a JMX-HX/HX110A spectrometer (JEOL, Akishima, Japan) using Fast Atom Bombardment (FAB) negative ionization with a dithiodiethanol matrix (Ma et al., 2003).

Results and Discussion

When plants were subjected to Fe deficiency, secretion of Fe-chelating substances was observed in both L. perenne cv. Tove and P. pratensis cv. Baron. Furthermore, quantitative analysis showed that the amount of Fe-chelating substances increased with the development of Fe deficiency (Fig. 1). No Fe-chelating activity was observed in the exudates from the two plant species supplied with sufficient Fe, suggesting that secretion of Fe-chelating substances from the roots is a response to Fe deficiency. Fractionation of the crude exudates showed that the Fe-chelating activity was found only in the cationic fraction. Analysis of this fraction with HPLC showed that three different peaks were detected in the samples of L. perenne and P. pratensis, respectively (Fig. 2a,b). The retention times of the first and third peaks in L. perenne were consistent with those of 3-epihydroxy-2′-deoxymugineic acid (epiHDMA) (Fig. 2c) and DMA (Fig. 2d), respectively, whereas the retention time of the second peak did not correspond to any of known phytosiderophores tested (Fig. 2a). The unknown compound accounted for 54% of total phytosiderophores secreted, while DMA and epiDMA accounted for 30 and 15%, respectively. In P. pratensis, the second and third peaks had the same retention times as those of DMA (Fig. 2e) and AVA (Fig. 2e), respectively, whereas the first peak represented an unidentified compound (Fig. 2b). The relative proportions of the unknown compound, DMA, and AVA were 56, 22, and 22%, respectively.

Figure 1.

Time-dependent secretion of iron (Fe)-chelating substances by Fe-deficient Lolium perenne cv. Tove (closed diamonds) and Poa pratensis cv. Baron (open squares). DW, dry weight.

Figure 2.

High-performance liquid chromatography (HPLC) spectra of the root exudates from iron (Fe)-deficient (a) Lolium perenne cv. Tove, and (b) Poa pratensis cv. Baron. Standards of (c) 3-epihydroxy-2′-deoxymugineic acid (epiHDMA), (d) 3-epihydroxymugineic acid (epiHMA), 3-hydroxymugineic acid (HMA), and 2′-deoxymugineic acid (DMA), and (e) mugineic acid (MA), DMA, and avenic acid A (AVA) are also shown. DW, dry weight.

To determine the structure of the possible new phytosiderophores, the compounds secreted by L. perenne and P. pratensis were purified by various chromatographies. These compounds were then subjected to Fast Atom Bombardment Mass Spectrometry (FAB-MS) and two-dimensional NMR measurements. The FAB-MS spectrum of the unknown compounds from L. perenne exhibited a deprotonated molecule [M-H] at m/z 319. From this observation, together with the structural information obtained by DQF-COSY, TOCS, NOESY, and 1H{13C} HMBC (Fig. 3a), this compound was identified as HDMA. However, the FAB-MS spectrum of the unknown compound from the root exudates of P. pratensis showed a deprotonated molecule [M-H] at m/z 337. By combination of this result with structural information from NMR analysis (Fig. 3b), this compound was identified as 2′-hydroxyavenic acid A (HAVA). Neither of the phytosiderophores identified in the present study has been reported previously.

Figure 3.

Two-dimensional nuclear magnetic resonance (NMR) spectrum of the unknown compound released from the roots of iron (Fe)-deficient (a) Lolium perenne cv. Tove, and (b) Poa pratensis cv. Baron. The unknown compounds were purified with various chromotographies and then subjected to NMR measurement. The total correlation spectroscopy (TOCSY) spectrum is shown. The three colored lines in the spectrum show three fragmental parts of the spin system connected by the spin-spin coupling.

The two novel phytosiderophores were characterized in terms of Fe-chelating ability. A quantitative test showed that the capacity to solubilize Fe from insoluble Fe(OH)3 was similar between HDMA and DMA, and between HAVA and AVA (Fig. 4). The molar ratio of HDMA to Fe and HAVA to Fe was close to 1, suggesting that both compounds form complexes with Fe in a 1 : 1 fashion.

Figure 4.

Iron (Fe)-chelating capacities of two novel phytosiderophores (PS), 3-hydroxy-2′-deoxymugineic acid (HDMA) and 2′-hydroxyavenic acid A (HAVA). A comparison of the capacity to solubilize Fe from insoluble Fe was performed for HDMA and HAVA and two known phytosiderophores (2′-deoxymugineic acid (DMA) and avenic acid A (AVA)). Error bars represent ± standard deviation (n = 3).

Since the structure of the first phytosiderophore, mugineic acid, was identified from barley root exudates by Takemoto et al. (1978), seven phytosiderophore analogs in total have been isolated and identified from barley, oat, rye, and wheat (Fushiya et al., 1980; Nomoto et al., 1981; Ma et al., 1999). Identification of two novel phytosiderophores in the present study indicates that diverse phytosiderophores are found in gramineous species in nature.

Biosynthetic studies have shown that all phytosiderophores are synthesized from three molecules of l-methionine (l-Met) (Ma & Nomoto, 1992, 1993, 1994b; Mori & Nishizawa, 1987; Shojima et al., 1990), which is supplied by the Met cycle (Ma et al., 1995) (Fig. 5). All phytosiderophores share the same biosynthetic pathway from l-Met to DMA, whereas hydroxylation at different positions of DMA gives rise to different derivatives of phytosiderophore in various plant species (Ma & Nomoto, 1996; Fig. 5). In L. perenne, DMA, epiHDMA and HDMA were secreted (Fig. 2). This suggests that the novel phytosiderophore HDMA is synthesized from DMA by hydroxylation at the C-3 position. Unlike in rye (Ma & Nomoto, 1994a), HMA has not been detected in L. perenne, suggesting that there is no enzyme for further hydroxylation at the C-2′ position in this species (Fig. 5).

Figure 5.

Biosynthetic pathways of various phytosiderophores. l-methionine serves as the precursor for all phytosideriophores, and is produced via the methionine (Met) cycle. All phytosiderophores share the same pathway from Met to 2′-deoxymugineic acid. inline image, proposed pathway for two novel phytosiderophores. KMB, 2-keto-4-methyl-thibutyric acid; MTA, 5′-Methyl-thioadenosine; MTR, 5-Methylthioribose; MTR-p, 5-Methylthioribose-1-phosphate; NA, Nicotianamine; SAM, 5-adenosyl-methionine.

In P. pratensis, secretion of DMA, AVA and HAVA was found (Fig. 2). Avenic acid A, which was first isolated from oat (Fushiya et al., 1980), is biosynthesized from DMA by cleavage of the azetidine ring (Ma & Nomoto, 1992, 1993). Therefore, it is likely that HAVA is synthesized from AVA by hydroxylation at the C-2′ position, which is probably catalyzed by IDS3 (Iron deficiency specific clone no. 3) (Fig. 5). It appears that hydroxylation of phytosiderophores does not change the capacity to solubilize Fe from insoluble Fe because the Fe-solubilizing capacities of HDMA and HAVA were similar to those of DMA and AVA, respectively (Fig. 4). This result is consistent with previous findings by von Wiren et al. (2000), who found that DMA and its hydroxyl derivatives MA and epiHMA solubilized Fe to similar extents. This is because all these phytosiderophores contain the same six functional groups, which are necessary to chelate Fe. It has also been reported that there is no difference in Fe uptake from phytosiderophores with different numbers of hydroxyl groups (Ma et al., 1993).

The identification of two novel phytosiderophores in the present study contributes to better understanding of the mechanisms of Fe acquisition in perennial grasses. It is possible that there are more phytosiderophores in perennial grasses; their structures are yet to be identified.

Acknowledgements

This work was supported in part by grants from Sunbor and the Ohara Foundation for Agriculture. We thank Fangjie Zhao at Rothamsted Research for his critical reading of this manuscript.

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