AOBPreview originally published online on May 24, 2007
Annals of Botany 2007 100(1):55-59; doi:10.1093/aob/mcm087
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Rhizobitoxine-induced Chlorosis Occurs in Coincidence with Methionine Deficiency in Soybeans
Graduate School of Life Sciences, Tohoku University, Katahira, Aoba-ku, Sendai 980-8577, Japan
* For correspondence. E-mail: kiwamu{at}ige.tohoku.ac.jp
Received: 14 January 2007 Returned for revision: 12 February 2007 Accepted: 20 March 2007 Published electronically: 24 May 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Background and Aims: Rhizobitoxine, produced by the legume symbiont Bradyrhizobium elkanii, inhibits cystathionine-ß-lyase (EC 4·4·1·8) in methionine biosynthesis and 1-aminocyclopropane-1-carboxylate synthase (ACC) in ethylene biosynthesis. Rhizobitoxine production by B. elkanii enhances nodulation of host legumes via the inhibition of ethylene synthesis, but causes foliar chlorosis in susceptible soybeans, though how it does so remains to be investigated. The aim of this study was to examine the physiological basis of rhizobitoxine-induced chlorosis in soybeans.
Methods: Wild-type B. elkanii and a rhizobitoxine-deficient mutant were inoculated in Glycine max ‘Lee’. Thirty days after inoculation, the upper parts of soybean shoots were analysed for amino acid contents. Chlorotic soybeans inoculated with wild-type B. elkanii were treated with methionine and ACC to assess the effects of the chemicals on the chlorosis.
Key Results: Chlorotic upper shoots of soybeans inoculated with wild-type B. elkanii had a lower methionine content and higher accumulation of the methionine precursors than those with the rhizobitoxine-deficient mutant. In addition, the foliar chlorosis was alleviated by the application of methionine.
Conclusions: Rhizobitoxine-induced chlorosis occurs in coincidence with methionine deficiency as a result of cystathione-ß-lyase inhibition during methionine biosynthesis.
Key words: Bradyrhizobium elkanii, chlorosis, methionine, nodulation, rhizobitoxine, Glycine max, soybean
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Rhizobitoxine [2–amino–4–(2–amino–3–hydropropoxy)–trans–but–3–enoic acid] is produced by the legume symbiont Bradyrhizobium elkanii (Owens et al., 1972) and the plant pathogen Burkholderia andropogonis (Mitchell et al., 1986). Rhizobitoxine inhibits cystathionine–ß–lyase (EC 4·4·1·8) in methionine biosynthesis (Owens et al., 1968; Giovanelli et al., 1972) and 1-aminocyclopropane-1-carboxylate (ACC) synthase in ethylene biosynthesis (Yasuta et al., 1999). Several genes responsible for rhizobitoxine production are known in B. elkanii (Ruan and Peters, 1992; Yasuta et al., 2001).
Rhizobitoxine production by B. elkanii enhances nodulation of the host legume species Vigna radiata (mung bean) (Duodu et al., 1999), Macroptilium atropurpureum (siratro) (Yuhashi et al., 2000; Okazaki et al., 2003) and Amphicarpaea edgeworthii (Parker and Peters, 2001). Inoculation with rhizobitoxine-producing B. elkanii caused lower ethylene evolution in host legumes, whereas the loss of rhizobitoxine production resulted in the restoration of ethylene evolution in the host (Yuhashi et al., 2000) and in fewer nodules and lower nodulation competitiveness of the bacteria (Duodu et al., 1999; Yuhashi et al., 2000; Parker and Peters, 2001; Okazaki et al., 2003; Sugawara et al., 2006). Ethylene suppresses nodulation in most legume species, including M. atropurpureum (Okazaki et al., 2004a) and Lotus japonicus (Nukui et al., 2000, 2004). Rhizobitoxine production by B. elkanii enhances ethylene-sensitive nodulation in the host plants as a result of inhibition of ACC synthase and blocking of ethylene biosynthesis of the hosts.
Nodulation by B. elkanii causes foliar chlorosis in soybean (Glycine max) (Erdman et al., 1956; Owens and Wright, 1965; Teaney and Fuhrmann, 1992). Plants show yellow leaves mainly in newly developing leaves, lower chlorophyll content, growth inhibition (Eaglesham and Hassouna, 1982; Fuhrmann, 1990; Bruce and Fuhrmann, 1993) and higher accumulation of rhizobitoxine in leaves, stems and nodules (Minamisawa and Kume, 1987). Rhizobitoxine production by B. elkanii is responsible for the induction of foliar chlorosis, because the loss of rhizobitoxine production by B. elkanii abolishes the chlorosis induction (Ruan and Peters, 1992; Okazaki et al., 2004b). Okazaki et al. (2004b) reported that the rtxC gene mutant of B. elkanii USDA94, designated 
rtxC, produced dihydrorhizobitoxine but no rhizobitoxine. Dihydrorhizobitoxine, the oxidative form of rhizobitoxine, was accumulated in the upper shoot of G. max inoculated with rtxC but revealed no chlorosis on the plant. The wild-type strain of B. elkanii USDA94 resulted in the accumulation of rhizobitoxine in the upper shoot and caused severe chlorosis on G. max. However, the mechanism of chlorosis induction remains to be solved.
The induction of foliar chlorosis should involve methionine or ACC in the host plants, because rhizobitoxine inhibits the biosynthesis of both (Yasuta et al., 1999). Here, the accumulation of rhizobitoxine-related amino acids in host shoots and the effect of methionine and ACC application on foliar chlorosis induction are assessed.
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MATERIALS AND METHODS |
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Bacterial strains, plasmids and growth conditions
Wild-type B. elkanii USDA94 and the rtxC mutant
rtxC (Okazaki et al., 2004b) were grown aerobically at 30 °C for 7 d in HM medium (Cole and Elkan, 1973) supplemented with 0·1 % arabinose and 0·025 % yeast extract (and kanamycin at 150 mg L–1 for culturing the mutant). Bacterial cells were collected by centrifugation at 5000 g for 10 min at room temperature and washed twice with sterile water. The bacterial cell suspension was adjusted to 1 x 107 cells mL–1 in sterile water just before inoculation (Miyamoto et al., 2004).
Soybean cultivation
Seeds of Glycine max ‘Lee’ were surface-sterilized with 0·5 % hydrogen peroxide for 1 min and washed ten times with sterile distilled water. The seeds were sown in sterile vermiculite with a nitrogen-free plant nutrient solution (Akao and Kouchi, 1989) in sterile Leonard jar assemblies composed of two 300-mL plant boxes (Ye et al., 2005; Saito and Minamisawa, 2006). The seeds were inoculated with bacterial cell suspension at 1 x 107 cells per seed. The plants were cultivated at 25 °C under a light–dark cycle of 16-h light and 8-h dark in a plant growth cabinet (LH300; NK Systems Co. Ltd, Osaka, Japan) that provided 65 µmol photons m–2 s–1 of photosynthetically active radiation (You et al., 2006).
Chemical application
Methionine, ACC, aspartate, glutamate and potassium nitrate (KNO3) were dissolved in plant nutrient solution at 500 µM each. These solutions were applied to the lower reservoir of the Leonard jar assemblies, and changed every 2 d from 16 d after inoculation.
Amino acid content in soybean
Thirty days after sowing, the upper part of the shoots (from the shoot tip to the third trifoliate leaf position) were harvested and weighed. The materials were homogenized in hot 80 % methanol and extracted at 80 °C for 1 h. The extract was centrifuged at 5000 g for 15 min, and the supernatant was collected. The pellet was re-extracted twice with hot methanol. The combined supernatant was dried in vacuo and re-dissolved in distilled water (3 mL of distilled water per gram fresh weight of initial plant materials). The samples were derivatized with phenylisothiocyanate (PITC) and analysed by liquid chromatography and mass spectrometry (LC/MS) to quantify major amino acids according to Yasuta et al. (2001) as follows. A 500-µL aliquot of the samples was mixed with 10 nmol of aminoethoxyvinylglycine (a structural analogue of rhizobitoxine), used as an internal standard, before PITC derivatization. PITC derivatization was carried out according to the method of Yamaya and Matsumoto (1988). A 50-µL aliquot of the sample solution was evaporated in vacuo in a 1·5 mL tube, and the pellet was dissolved in 20 µL of ethanol–triethylamine–water (2:1:2, v/v/v). After evaporation, the pellet was dissolved in 10 µL of ethanol-triethylamine-water-PITC (7:1:1:1, v/v), incubated for 20 min at room temperature, and then evaporated to dryness. Each pellet of PITC derivative was dissolved in 100 µL of deionized water and passed through a 0·2-µm cellulose nitrate filter prior to LC/MS analysis. A JMS-LCmate (JEOL, Tokyo, Japan) equipped with an electrospray ionization system and high-performance liquid chromatograph (HP-1100; Hewlett Packard, Waldbronn, Germany) was used for analysis of PITC-labelled amino acids under the following conditions: column, Inertsil ODS-2 (1·5 x 150 mm; GL Sciences Inc., Tokyo, Japan); column temperature, 40 °C; flow rate, 0·1 ml min–1; mobile phase, a linear gradient from 30 % solvent B (100 % MeCN) in solvent A (0·1 % HCOOH) to 100 % solvent B for 15 min. The concentrations of each amino acid were calculated according to the ratio between the peak area of PITC-aminoethoxyvinylglycine (m/z 431) and the peak area of the PITC derivative of each amino acid. PITC-derivatives of measured amino acids (m/z in parentheses) were methionine (285), aspartate (269), homoserine (255), serine (241), leucine (267), isoleucine (267), valine (253), alanine (225), glycine (211) and glutamate (283).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The rtxC gene is involved in the final step of rhizobitoxine biosynthesis, desaturation of dihydrorhizobitoxine, to produce biologically active rhizobitoxine in B. elkanii (Okazaki et al., 2004b). Therefore, by comparing the wild type and the rtxC mutant of B. elkanii USDA94 it is possible to determine the biological effects of rhizobitoxine in host plants. The soybeans inoculated with the wild-type strain showed foliar chlorosis in the upper shoots and growth inhibition (Fig. 1A), typical host responses in the B. elkanii–soybean nodulation interaction (Teaney and Fuhrmann, 1992). On the other hand, plants inoculated with the rtxC mutant (
rtxC) did not show these negative phenotypes (Fig. 1D). The shoot fresh weight of soybeans inoculated with the wild-type strain was 3·4 ± 0·4 g per plant, whereas that of soybeans inoculated with rtxC was at 5·0 ± 0·4 g plant– 1.
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Amino acid analysis showed differences in amino acid accumulation in soybean shoots between inoculations with the wild-type and
rtxC (Table 1). The methionine content of plants inoculated with the wild-type strain was significantly lower, and the aspartate content was significantly higher, than that of plants inoculated with rtxC.
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Because aspartate and homoserine are generally metabolized in methionine biosynthesis as precursors of cystathionine, the substrate of cystathione-ß-lyase (Yasuta et al., 2001) (Fig. 2), the ratios of aspartate and homoserine contents to methionine content were calculated. The ratio of homoserine to methionine was increased in the plants inoculated with the wild-type strain more than in the plants inoculated with
rtxC. This difference indicates a higher accumulation of homoserine, suggesting the inhibition of cystathionine-ß-lyase in methionine biosynthesis by rhizobitoxine (Fig. 2). A similar increase was observed in the ratio of aspartate to methionine, although the aspartate level depends on the synthesis of methionine and other amino acids.
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When 500 µM methionine was applied to soybeans from 16 d after inoculation with the wild-type strain (before the occurrence of rhizobitoxine-induced chlorosis), it alleviated the foliar chlorosis and plant growth inhibition (Fig. 1B). The application of 10 µM or 2 mM methionine showed either no discernible effect or inhibition of shoot growth (data not shown). The application of ACC led to a slight inhibition of shoot growth but did not alleviate the chlorosis (Fig. 1C). These results indicate that the chlorosis was due not to ACC deficiency but to methionine deficiency. The application of aspartate, glutamate and KNO3 did not alter the chlorosis (data not shown).
These results show that the rhizobitoxine-induced foliar chlorosis was due to insufficient methionine biosynthesis in soybean shoots, which was supported by application of methionine (Fig. 1). The shoots showed lower content of methionine and higher accumulation of aspartate and homoserine (Table 1). These observations illustrated that rhizobitoxine-induced foliar chlorosis is the result of methionine deficiency due to inhibition of cystathione-ß-lyase. In higher plants, most cystathionine-ß-lyase is localized in chloroplasts (Wallsgrove et al., 1983; Droux et al., 1995, Ravanel et al., 1998). Methionine is required as a precursor of S-adenosylmethionine for chlorophyll biosynthesis (Bollivar et al., 1994). Soybean leaves with rhizobitoxine-induced foliar chlorosis had a lower chlorophyll content (Teaney and Fuhrmann, 1992). The inhibition by rhizobitoxine of cystathionine-ß-lyase would affect chloroplast function owing to insufficient chlorophyll biosynthesis in soybean shoots deficient in methionine. Methionine depletion and subsequent amino acid imbalance may have caused impaired protein synthesis in the plant cells. Indeed, inoculation of rhizobitoxine-producing B. elkanii resulted in significant reduction in total leaf protein content of G. max (Teaney and Fuhrmann, 1992). The limited protein synthesis may well have obstructed the synthesis and maintenance of photosynthesis-related proteins, resulting in the development of chlorosis.
Soybean cultivars differ in their susceptibility to rhizobitoxine-induced chlorosis (Erdman et al., 1957; Owens and Wright, 1965). Since the foliar chlorosis is caused by a methionine deficiency due to inhibition of cystathionine-ß-lyase, the basis of the rhizobitoxine resistance or susceptibility of soybeans should involve the overall sensitivity of cystathionine-ß-lyase to rhizobitoxine. However, the rhizobitoxine inhibition constant of cystathionine-ß-lyase partially purified from four soybean cultivars was not clearly correlated with the resistance or susceptibility of the cultivars to rhizobitoxine (Xiong and Fuhrmann, 1996). The different methionine demands and tolerance to methionine deficiency among soybean cultivars might also account for the susceptibility to chlorosis development.
Because rhizobitoxine-induced foliar chlorosis in B. elkanii–soybean symbiosis reduces soybean growth and yield (Vasilas and Fuhrmann, 1993), the present results will contribute to the diagnosis of chlorosis symptoms induced by rhizobitoxine and to the breeding of rhizobitoxine-resistant cultivars.
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ACKNOWLEDGEMENTS |
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This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan (nos. 17658034 and 15380002) and by a Grant-in-Aid for Scientific Research on Priority Areas ‘Comparative Genomics’ to K.M. We are grateful to H. Ezura (Tsukuba University, Japan) for his continuing interest and encouragement.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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