Biotechnology Advances 24 (2006) 382 – 388

www.elsevier.com/locate/biotechadv

Research review paper

Rhizobitoxine modulates plant–microbe interactions
by ethylene inhibition
Masayuki Sugawara a , Shin Okazaki a , Noriyuki Nukui a , Hiroshi Ezura b ,
Hisayuki Mitsui a , Kiwamu Minamisawa a,⁎
a
Graduate School of Life Sciences, Tohoku University, Katahira, Aoba-ku, Sendai 980-8577, Japan
b
Gene Research Center, Tsukuba University, Ten-nodai, Tsukuba 305-8572, Japan
Available online 3 March 2006

Abstract

Bradyrhizobium elkanii produces rhizobitoxine, an enol-ether amino acid, which has been regarded as a phytotoxin because it
causes chlorosis in soybeans. However, recent studies have revealed that rhizobitoxine plays a positive role in establishing symbiosis
between B. elkanii and host legumes: rhizobitoxine enhances the nodulation process by inhibiting ACC (1-aminocyclopropane-
1-carboxylate) synthase in the ethylene biosynthesis of host roots. B. elkanii rtxA and rtxC genes are required for rhizobitoxine
production. In particular, rtxC gene is involved in the desaturation of dihydrorhizobitoxine into rhizobitoxine. A legume with a
mutated ethylene receptor gene produced markedly higher numbers of rhizobial infection threads and nodule primordia. Thus,
endogenous ethylene in legume roots negatively regulates the formation of nodule primordia, which is overcome by rhiozbitoxine.
Although a plant pathogen Burkholderia andropogonis has been known to produce rhizobitoxine, the genome sequence of
Xanthomonas oryzae showed the existence of a putative rhizobitoxine transposon in the genome. The cumulative evidence suggests
that rhizobitoxine-producing bacteria modulate plant–microbe interactions via ethylene in the rhizosphere and phyllosphere
environments. In addition, rhizobitoxine-producing capability might be utilized as tools in agriculture and biotechnology.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Bradyrhizobium elkanii; Ethylene; Legume; Rhizobia; Rhizobitoxine

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
2. Effect of ethylene on nodulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
3. Biochemical functions of rhizobitoxine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
4. Positive role of rhizobitoxine production in symbiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
5. Rhizobitoxine biosynthesis genes and pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
6. Rhizobitoxine-producing pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
7. Nodulation enhancement via ethylene perception . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
8. Alternative strategy for decreasing ethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
9. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386

⁎ Corresponding author. Tel./fax: +81 22 217 5684.

E-mail address: [email protected] (K. Minamisawa).

0734-9750/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2006.01.004
 M. Sugawara et al. / Biotechnology Advances 24 (2006) 382–388 383

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387

1. Introduction Inhibition of ethylene synthesis and responses has been

shown to enhance nodule formation in Medicago
In the symbiotic nitrogen-fixation process, bacteria of truncatula (Oldroyd et al., 2001), Medicago sativa
the family Rhizobiaceae convert atmospheric dinitrogen (Nukui et al., 2000; Peters and Crist-Estes, 1989), Lotus
(N2) to ammonia (NH3), which can be effectively utilized japonicus (Bras et al., 2000; Nukui et al., 2000), P.
by host legume plants. The family Rhizobiaceae cur- sativum (Guinel and Sloetjes, 2000; Lorteau et al.,
rently includes six genera: Rhizobium, Sinorhizobium, 2001), and Macroptilium atropurpureum (Nukui et al.,
Mesorhizobium, Allorhizobium, Azorhizobium, and 2000). In addition, a hypernodulating mutant of M.
Bradyrhizobium, which are collectively referred to as truncatula has ethylene-insensitive phenotypes (Pen-
rhizobia. They have the ability to infect the roots of metsa and Cook, 1997). Unlike in most legume species,
legumes and to produce nodules. The differentiated however, ethylene may not play a significant role in the
forms of rhizobia in the nodule, called bacteroids, fix nodulation of modern soybean cultivars (Hunter, 1993;
atmospheric nitrogen into ammonia and export the fixed Schmidt et al., 1999). Ethylene evolution in legume
nitrogen to the host plant (Long, 1989). Symbiotic ni- roots increases after application of rhizobial cells
trogen fixation is of great importance not only in the (Ligero et al., 1986; Suganuma et al., 1995), Nod
production of leguminous crops but also in the global factor (van Spronsen et al., 1995), nitrate (Caba et al.,
nitrogen cycle. 1998; Ligero et al., 1987), and illumination (Lee and
Nitrogen-fixing nodules are formed as a consequence LaRue, 1992a), suggesting that these environmental
of a series of interactions between rhizobia and legu- factors control nodulation through their effects on the
minous host plants (Fisher and Long, 1992). The sym- levels of ethylene.
biotic partners show a high degree of mutual specificity,
mediated by the exchange of chemical signals (Spaink, 3. Biochemical functions of rhizobitoxine
1996, 2000). The plant signals, flavonoids excreted by
the roots, activate the expression of nodulation genes in In 1956, Erdman et al. (1956) first reported that
rhizobia, resulting in the production of rhizobial lipo- certain strains of rhizobia induce chlorosis in new leaves
chitooligosaccharide signals called Nod factors. Al- of soybeans. Owens and colleagues (Owens and Wright,
though legumes form root nodules mainly in response to 1965a,b) purified the toxic compound from nodule
Nod factors, it has been thought that the plant perception extracts and culture media of Rhizobium japonicum
of endogenous signals, particularly plant hormones, is (now Bradyrhizobium elkanii) and demonstrated that the
also important for the establishment of proper symbiotic compound is phytotoxic to soybeans (Johnson et al.,
interactions between rhizobia and legumes (Caetano- 1959; Owens, 1973). The toxic compound was later
Anolles and Gresshoff, 1991). called rhizobitoxine. Among soybean bradyrhizobia, all
strains of B. elkanii (formerly DNA homology group II)
2. Effect of ethylene on nodulation produce rhizobitoxine, whereas those of B. japonicum
(formerly DNA homology group I) do not produce
Ethylene is produced and sensed in response to a rhizobitoxine at all (Hollis et al., 1981; Minamisawa,
wide variety of environmental and developmental cues, 1989, 1990; Minamisawa and Mitsui, 2000). Rhizobi-
including germination, flowering, drought, pathogen toxine has therefore been regarded as a phytotoxin that
attack, and nodulation (Abeles et al., 1992; Spaink, causes chlorosis symptoms in soybeans.
1997). In particular, ethylene inhibits infection of rhi- The structure of rhizobitoxine was solved in 1972
zobia and nodulation of most legumes, as shown by (Owens et al., 1972b). Rhizobitoxine is an enol-ether
many studies (Okazaki et al., 2004a). Exogenously amino acid (2-amino-4-[2-amino-3-hydroxypropoxy]-
applied ethylene or ethylene precursor inhibits nodula- trans-3-butenoic acid) with a molecular weight of 190
tion in many legumes. In Pisum sativum, exogenous (Fig. 1). The first biochemical function identified for
ethylene application inhibits elongation of infection rhizobitoxine was that of an inhibitor of β-cystathionase
threads into the inner cortex (Lee and LaRue, 1992b). in the methionine biosynthesis pathway (cystathionine
In P. sativum, endogenous ethylene exerts positions on β-lyase, E.C.4.4.1.8) (Fig. 1). β-Cystathionase catalyzes
nodule meristem morphogenesis (Heidstra et al., 1997). the conversion of cystathionine to homocysteine, pyruvate,
384 M. Sugawara et al. / Biotechnology Advances 24 (2006) 382–388

and ammonium. As an analog of cystathionine, rhizobi- atropurpureum (siratro) and enhances nodule formation
toxine irreversibly inhibits β-cystathionase in bacteria by using B. elkanii USDA94 and its mutant lacking
(Owens et al., 1968) and plants (Giovanelli et al., 1972). rhizobitoxine production (Yuhashi et al., 2000). In
Yasuta et al. (1999) found that rhizobitoxine also particular, the nodulation competitiveness of B. elkanii
strongly inhibits 1-aminocyclopropane-1-carboxylate USDA94 was significantly enhanced by rhizobitoxine
(ACC) synthase (E.C.4.4.1.14) in the ethylene biosyn- production. Duodu et al. also reported a positive role of
thesis pathway (Fig. 1), which can explain the early rhizobitoxine in the symbiosis between B. elkanii
observation of rhizobitoxine inhibition of ethylene USDA61 and Vigna radiata (mungbean) (Duodu et al.,
evolution in apple tissues (Owens et al., 1971). ACC 1999). Rhizobitoxine-minus mutants of USDA61 formed
synthase is the rate-limiting enzyme in ethylene bio- dramatically fewer mature nodules than the wild-type
synthesis in plants, catalyzing the conversion of S- strain. Interestingly, the rhizobitoxine-minus mutants
adenosylmethionine to ACC. A closely related ethoxy induced many immature nodules, indicating that the initial
analog of rhizobitoxine, aminoethoxyvinylglycine stages of root hair infection were not impaired but that the
(AVG), also inhibits ACC synthase (Devine and development to mature nodules was obstructed. Therefore,
Breithaut, 1980; Yasuta et al., 1999). The oxidized ethylene might affect abortion of the developing nodules in
form of rhizobitoxine (dihydrorhizobitoxine) is approx- the association between USDA61 and mungbean, and
imately 99% less potent than rhizobitoxine as an inhi- rhizobitoxine production by the bacterium would over-
bitor of ACC synthase, so the double bond between C3 come this effect by inhibiting ethylene biosynthesis.
and C4 may be critical for the inhibition of ACC syn- Another study on rhizobitoxine and nodulation was
thase (Yasuta et al., 1999). carried out by Parker et al. using Amphicarpaea (Parker
and Peters, 2001). Nodulation experiments using B.
4. Positive role of rhizobitoxine production in elkanii USDA61 and its rhizobitoxine-minus mutants
symbiosis revealed that efficient nodulation in A. edgeworthii but
not in A. bracteata is highly dependent on rhizobitoxine
Rhizobitoxine has been regarded as a phytotoxin ever production.
since the discovery that it induces chlorosis to soybeans.
However, recent studies have shown a positive role of 5. Rhizobitoxine biosynthesis genes and pathway
rhizobitoxine in the symbiosis between B. elkanii strains
and their host legumes. Yuhashi et al. reported that The genes involved in rhizobitoxine biosynthesis
rhizobitoxine production in B. elkanii USDA94 reduces have been identified in two B. elkanii strains, USDA61
ethylene evolution from the associated roots of M. (Ruan and Peters, 1992) and USDA94 (Yasuta et al.,

Aspartate Methionine
biosynthetic
pathway
Homoserine

S Rhizobitoxine
Cystathionine
β-cystathionase

Homocysteine
+
3 S
3 S
Ado
Methionine SAM ACC synthase Ethylene
ACC
Ethylene biosynthetic pathway

Fig. 1. Rhizobitoxine inhibition in methonine and ethylene biosynthetic pathway. SAM, S-adenosylmethionine; ACC, 1-aminocyclopropane-1-
carboxylate.
 M. Sugawara et al. / Biotechnology Advances 24 (2006) 382–388 385

2001). Ruan et al. isolated Tn5-induced rhizobitoxine- dihydrorhizobitoxine by an rtxC-encoded protein is

null mutants of B. elkanii USDA61 and identified the essential for the bacterium to elicit these phenotypes in
rtxA gene, which is responsible for rhizobitoxine bio- planta.
synthesis in culture and in planta (Ruan and Peters,
1992). Yasuta et al. (2001) cloned and sequenced the 6. Rhizobitoxine-producing pathogens
genetic locus involved in rhizobitoxine biosynthesis
from B. elkanii USDA94, which produces more rhizobi- A plant pathogen, Burkholderia andropogonis, is also
toxine than USDA61 in culture. B. elkanii USDA94 known to be a rhizobitoxine producer. The pathogen
possesses several open reading frames (ORFs) down- causes chlorotic symptoms in corn and sorghum, pre-
stream of the rtxA gene. A large deletion mutant of B. sumably as a result of rhizobitoxine production in planta
elkanii, USDA94Δrtx::Ω1, which lacks rtxA, rtxC, and (Mitchell and Frey, 1988). Recently, rtx genes found in
the downstream ORFs, does not produce rhizobitoxine, the genome of Xanthomonas oryzae pathovar oryzae
dihydrorhizobitoxine, or serinol. The latter two com- KACC10331 (Lee et al., 2005), a bacterium that causes
pounds have been found to be co-produced with rhizobi- bacterial blight in rice (Oryza sativa L.), although the rtx
toxine and are regarded as intermediates (Minamisawa genes were misannotated (Lee et al., 2005). Amino acid
and Watanabe, 1986; Owens et al., 1972a; Yasuta et al., sequences of rtxA and rtxC genes are highly homologous
2001). Complementation analyses with cosmids con- with those of B. elkanii (Fig. 2). The rtx gene clusters in
taining an insertional mutagenesis within the putative X. oryzae and B. elkanii, contained additional two genes
rtx operon suggested that (i) at least rtxA and rtxC are that are relevant to glutamine (ORF2; Glutamine amido-
necessary for rhizobitoxine production in B. elkanii, transferase, ORF4; Glutamine synthetase). These genes
(ii) the newly discovered rtxC gene, which is located probably supply glutamine-related substrates for rhizo-
immediately downstream of the rtxA gene, is involved in bitoxine biosynthesis (Sugawara et al., unpublished re-
desaturation of dihydrorhizobitoxine into rhizobitoxine, sults). From the comparison, we expect rhizobitoxine
and (iii) other ORFs downstream of rtxC affect the production in X. oryzae KACC10331. Interestingly, the
levels of production of rhizobitoxine and its intermedi- rtx gene cluster in X. oryzae pv. oryzae KACC10331
ates by unknown functions. resides between two insertion sequences (ISs), and
Large amounts of dihydrorhizobitoxine are generally formed a composite transposon structure for rhizobi-
co-produced with rhizobitoxine (Minamisawa and toxine biosynthesis (Fig. 2). Therefore, such rhizobi-
Kume, 1987; Owens et al., 1972a). However, no con- toxine transpson may transfer to other plant-associated
clusive data have been reported on the biological effects bacteria in the environments.
of dihydrorhizobitoxine in planta. Okazaki et al. (2004b)
demonstrated that dihydrorhizobitoxine production does 7. Nodulation enhancement via ethylene perception
not affect the plant phenotypes of B. elkanii USDA94 in
terms of nodulation competitiveness and chlorosis Rhizobitoxine is secreted outside rhizobial cells and
induction. They thus concluded that desaturation of delivered to the plant. In fact, rhizobitoxine has been

Bradyrhizobium elkanii USDA94

rtxA rtxC ORF2 ORF3.1 ORF3.2 ORF4

N-domain C-domain

Identity (%) 55.2 72.6 64.5 48.8 30.3

ISXo5 IS1113

1 kb
Xanthomonas oryzae pv. oryzae KACC10331

Fig. 2. Comparison of rtx genes in Bradyrhizobium elkanii USDA94 and Xanthomonas oryzae pv. oryzae KACC10331. Sequence comparison were
carried out using sequence of rtx genes in B. elkanii USDA94 (accession no. AB062279) and X. oryzae pv. oryzae KACC10331 (accession no.
AE013598). Arrows show rtx genes and predicted open reading frame (ORFs). Numbers in the middle space indicate percent identity based on the
amino acids sequences. ISXo5 and IS113 are insertion sequences with transposase gene (Lee et al., 2005).
386 M. Sugawara et al. / Biotechnology Advances 24 (2006) 382–388

detected in leaves as well as in nodules of soybeans genome of the fast-growing M. loti MAFF303099
inoculated with B. elkanii (Minamisawa and Kume, presented in a database (Kaneko et al., 2000), and
1987). Moreover, ethylene evolution is blocked in le- identified ACC deaminase gene as a candidate (Uchiumi
gume root systems inoculated with B. elkanii (Yuhashi et al., 2004). Indeed, ACC deaminase was verified to
et al., 2000). Therefore, the explanation of how rhizobi- enhance nodulation and competitiveness to the host
toxine decreases endogenous levels of ethylene in le- legume by using a disruption mutant of ACC deaminase
gume roots appears straightforward. gene (mlr5932) (Uchiumi et al., 2004). Recently, genes
The next question is how a decreased ethylene level encoding ACC deaminase have been also found in other
enhances nodulation. Recently, several models have rhizobia, such as Rhizobium leguminosarum and B.
been proposed depicting the relationships between japonicum (Kaneko et al., 2002; Ma et al., 2003a,b).
signal transduction, ethylene sensing, and the develop- ACC deaminase from R. leguminosarum bv. viciae has
ment of nodulation (Gresshoff et al., 2003; Guinel and been confirmed to enhance nodulation of P. sativum as
Geil, 2002; Stearns and Glick, 2003). One promising well (Ma et al., 2003a). These recent findings on ACC
approach will be to construct transgenic legumes with deaminase suggest that rhizobia can adopt more than
altered ethylene sensitivities. The expression of ethylene one strategy to reduce the amount of ethylene syn-
receptors that cannot bind ethylene confers reduced thesized by their host legumes and thus enhance nodule
ethylene sensitivity to heterologous plants in a geneti- formation. Indeed, since many processes in nodule for-
cally dominant manner (Bleecker, 1999). In petunia and mation are blocked by ethylene, it seems reasonable
tomato, transformation with a mutant Arabidopsis that, in order to enable efficient nodulation, rhizobia
ethylene receptor gene (etr1-1) conferred reduced would decrease ethylene levels by producing both ACC
sensitivity to ethylene (Wilkinson et al., 1997). deaminase and rhizobitoxine.
To address the mechanism by which ethylene inhibits
nodulation, Nukui et al. (2004) transformed L. japonicus 9. Concluding remarks
B-129 ‘Gifu’ (Handberg and Stougaard, 1992) with a
mutated ethylene receptor gene Cm-ERS1/H70A: A point The cumulative evidence reveals general strategies
mutation was introduced into the melon ethylene receptor by which rhizobia produce an inhibitor and an enzyme
Cm-ERS1 (Sato-Nara et al., 1999) by changing the 70th to decrease ethylene levels in host roots and thereby
amino acid, histidine, to alanine, which abolished its enhance nodulation. Plant pathogens, such as B.
ethylene-binding ability. The resultant transgenic L. andropogonis and X. oryzae, probably produce rhizo-
japonicus plants showed reduced ethylene sensitivity. bitoxine to inhibit ethylene biosynthesis and reduce
When inoculated with M. loti, the transgenic L. defense reactions by the host plants. The existence of
japonicus produced markedly higher numbers of in- putative rhizobitoxine transposon in X. oryzae genome
fection threads and nodule primordia on their roots than prompts us to speculate that more plant-associated bac-
did either wild-type or azygous plants without the trans- teria produce rhizobitoxine and modulate plant–microbe
gene. In addition, the amount of transcripts of NIN, a interactions in the rhizosphere and phyllosphere
gene governing formation of infection threads (Schauser environments.
et al., 1999), increased in the inoculated transgenic plants If some rhizobial strains lack the ability to decrease
as compared with the wild-type plants. These results ethylene levels in host legumes, the introduction of
imply that endogenous ethylene in L. japonicus roots genes for rhizobitoxine biosynthesis and ACC deami-
inhibits the formation of nodule primordia, as well as nase into these rhizobia ought to enhance their sym-
other infection processes (Nukui et al., 2004). biotic interactions with host legumes. This idea may also
be extended to other plant-associated bacteria, such as
8. Alternative strategy for decreasing ethylene Rhizobium radiobacter (formerly Agrobacterium tume-
faciens), which has been used for plant transformation.
The ability to synthesize rhizobitoxine is confined to Indeed, application of AVG, an analog of rhizobitoxine,
the slow-growing B. elkanii among rhizobia so far. increased the efficiency of Agrobacterium-mediated
Thus, Yasuta et al. (1999) addressed the question as to gene transfer to explants of melons (Ezura et al.,
whether fast-growing rhizobia possess another inhibi- 2000). The ethylene-decreasing strategies of rhizobia
tory factor for ethylene biosynthesis of host plants, are interesting and suggestive for the further under-
because it could enhance nodulation (Nukui et al., standing of plant–microbe interactions and should be a
2000). They sought potential enzymes and compounds promising tool for overcoming the problem of rhizobial
for reducing ethylene biosynthesis from the entire competition and for making further progress toward
 M. Sugawara et al. / Biotechnology Advances 24 (2006) 382–388 387

developing more environmentally friendly biotechnol- Handberg K, Stougaard J. Lotus japonicus, an autogamous, diploid
ogy and agriculture. legume species for classical and molcular genetics. Plant J
1992;2:487–96.
Heidstra R, Yang WC, Yalcin Y, Peck S, Emons AM, van Kammen A,
Acknowledgements et al. Ethylene provides positional information on cortical cell
division but is not involved in Nod factor-induced root hair tip
This work was supported in part by grants to K. M. growth in Rhizobium–legume interaction. Development
1997;124:1781–7.
and H. E. from the Ministry of Education, Science,
Hollis TA, Kloos WE, Elkan GE. DNA : DNA hybridization studies of
Sports, and Culture of Japan (nos. 11556012 and Rhizobium japonicum and related Rhizobiaceae. J Gen Microbiol
14360037) and by a Research Fellowship of the Japan 1981;123:215–22.
Society for the Promotion of Young Scientists to N. N. Hunter WJ. Ethylene production by root nodules and effect of ethylene
We thank PROBRAIN (Japan) for supporting the on nodulation in Glycine max. Appl Environ Microbiol
research of K. M. We are grateful to K. Yuhashi (Tsukuba 1993;59:1947–50.
Johnson HW, Means UM, Clark FE. Responses of seedlings to extracts
University) for their collaboration and helpful discussions of soybean nodules bearing selected strains of Rhizobium
for ethylene perception and competitive nodulation. japonicum. Nature 1959;183:308–9.
Kaneko T, Nakamura Y, Sato S, Asamizu E, Kato T, Sasamoto S, et al.
Complete genome structure of the nitrogen-fixing symbiotic
References bacterium Mesorhizobium loti. DNA Res 2000;7:331–8.
Kaneko T, Nakamura Y, Sato S, Minamisawa K, Uchiumi T, Sasamoto
Abeles FB, Morgan PW, Saltveit Jr ME. Ethylene in plant biology. 2nd S, et al. Complete genomic sequence of nitrogen-fixing symbiotic
ed. New York: Academic Press, 1992. bacterium Bradyrhizobium japonicum USDA110. DNA Res
Bleecker A. Ethylene perception and signaling: an evolutionary 2002;31:189–97.
perspective. Trends Plant Sci 1999;4:269–74. Lee KH, LaRue TA. Ethylene as a possible mediator of light- and
Bras CP, Jorda MA, Wijfjes AHM, Harteveld M, Stuurman N, nitrate-induced inhibition of nodulation of Pisum sativum L. cv
Thomas-Oates JE, et al. A Lotus japonicus nodulation system Sparkle. Plant Physiol 1992a;100:1334–8.
based on heterologous expression of the fucosyl transferase NodZ Lee KH, LaRue TA. Exogenous ethylene inhibits nodulation of Pisum
and the acetyl transferase NolL in Rhizobium leguminosarum. Mol sativum L. cv Sparkle. Plant Physiol 1992b;100:11759–63.
Plant-Microbe Interact 2000;13:475–9. Lee BM, Park YJ, Park DS, Kang HW, Kim JG, Song ES, et al. The
Caba JM, Recalde L, Ligero F. Nitrate-induced ethylene biosynthesis genome sequence of Xanthomonas oryzae pathovar oryzae
and the control of nodulation in alfalfa. Plant Cell Environ KACC10331, the bacterial blight pathogen of rice. Nucleic
1998;21:87–93. Acids Res 2005;33:577–86.
Caetano-Anolles G, Gresshoff M. Plant genetic control of nodulation. Ligero F, Lluch C, Olivares J. Evolution of ethylene from roots of
Annu Rev Microbiol 1991;45:345–82. Medicago sativa plants inoculated with Rhizobium meliloti. J Plant
Devine TE, Breithaut BH. Effect of the ethoxy analog of rhizobitoxine Physiol 1986;125:361–5.
on nodulation of soybeans. Crop Sci 1980;20:819–21. Ligero F, Lluch C, Olivares J. ,Evolution of ethylene from roots and
Duodu S, Bhuvaneswari TV, Stokkermans TJW, Peters NK. A positive nodulation rate of alfalfa (Medicago sativa L.) plants inoculated
role for rhizobitoxine in Rhizobium-legume symbiosis. Mol Plant- with Rhizobium meliloti as affected by the presence of nitrate.
Microbe Interact 1999;12:1082–9. J Plant Physiol 1987;129:461–7.
Erdman LW, Johnson HW, Clark F. A bacterial-induced chlorosis in Long SR. Rhizobium–legume nodulation: life together in the
the Lee soybean. Plant Dis Rep 1956;40:646. underground. Cell 1989;56:203–14.
Ezura H, Yuhashi K, Yasuta T, Minamisawa K. Effect of ethylene on Lorteau MA, Ferguson BJ, Guinel FC. Effects of cytokinin on ethylene
Agrobacterium tumefaciens-mediated gene transfer to melon. Plant production and nodulation in pea (Pisum sativum) cv. Sparkle.
Breeding 2000;119:75–9. Physiol Plant 2001;112:421–8.
Fisher RF, Long SR. Rhizobium-plant signal exchange. Nature Ma W, Guinel FC, Glick BR. Rhizobium leguminosarum biovar viciae
1992;357:655–60. 1-aminocyclopropane-carboxylate deaminase promotes nodulation
Giovanelli J, Owens LD, Mudd SH. β-cystathionase: in vivo of pea plants. Appl Environ Microbiol 2003a;69:4396–402.
inactivation by rhizobitoxine and role of the enzyme in methionine Ma W, Sebestianova SB, Sebestian J, Burd GI, Guinel FC, Glick BR.
biosynthesis in corn seedlings. Plant Physiol 1972;51:492–503. Prevalence of 1-aminocyclopropane-carboxylate deaminase in
Gresshoff PM, Rose RJ, Singh M, Rolfe BG. Symbiosis signals. Rhizobium spp. Antonie van Leeuwenhoek 2003b;83:285–91.
Todays' LifeScience May/June, 2003; http://cirl.rsbs.anu.edu.au/ Minamisawa K. Comparison of extracellular polysaccharide compo-
News/tls0503Gresshoff HR.pdf. sition, rhizobitoxine production, and hydrogenase phenotype
Guinel FC, Geil RD. A model for the development of the rhizobial and among various strains of Bradyrhizobium japonicum. Plant Cell
arbuscular mycorrhizal symbioses in legumes and its use to Physiol 1989;30:877–84.
understand the roles of ethylene in the establishment of these two Minamisawa K. Division of rhizobitoxine-producing and hydrogen-
symbioses. Can J Bot 2002;80:695–720. uptake positive strains of Bradyrhizobium japonicum by nifDKE
Guinel FC, Sloetjes LL. Ethylene is involved in the nodulation sequence divergence. Plant Cell Physiol 1990;31:81–9.
phenotype of Pisum sativum R50 (sym 16), a pleiotropic mutant Minamisawa K, Kume N. Determination of rhizobitoxine and
that nodulates poorly and has pale green leaves. J Exp Bot dihydrorhizobitoxine in soybean plants by amino acid analyzer.
2000;51:885–94. Soil Sci Plant Nutr 1987;33:645–9.
388 M. Sugawara et al. / Biotechnology Advances 24 (2006) 382–388

Minamisawa K, Mitsui H. Genetic ecology of soybean bradyrhizobia. Penmetsa RV, Cook DR. A legume ethylene-insensitive mutant
In: Bollag J-M, Stotzky G, editors. Soil biochemistry. New York, hyperinfected by its rhizobial symbiont. Science 1997;275:527–30.
N.Y.: Marcel Dekker; 2000. p. 349–77. Peters NK, Crist-Estes DK. Nodule formation is stimulated by the
Minamisawa K, Watanabe H. Serinol (2-amino-1,3-propanediol) and ethylene inhibitor aminoethoxyvinylglycine. Plant Physiol
3-amino-1,2-propanediol in soybean nodules. Plant Cell Physiol 1989;91:690–3.
1986;27:1109–16. Ruan X, Peters NK. Isolation and characterization of rhizobitoxine
Mitchell RE, Frey EJ. Rhizobitoxine and hydroxythreonine production mutants of Bradyrhizobium japonicum. J Bacteriol 1992;174:
by Pseudomonas andropogonis strains, and the implications to 3467–3473.
plant disease. Physiol Mol Plant Pathol 1988;32:335–41. Sato-Nara K, Yuhashi K, Higashi K, Hosoya K, Kubota M, Ezura H.
Nukui N, Ezura H, Yuhashi K, Yasuta T, Minamisawa K. Effect of Stage- and tissue-specific expression of ethylene receptor homolog
ethylene precursor and inhibitors for ethylene biosynthesis and genes during fruit development in muskmelon. Plant Physiol
perception on nodulation in Lotus japonicus and Macroptilium 1999;120:321–30.
atropurpureum. Plant Cell Physiol 2000;41:893–987. Schauser L, Roussis A, Stiller J, Stougaard J. A plant regulator
Nukui N, Ezura H, Minamisawa K. Transgenic Lotus japonicus with controlling development of symbiotic root nodules. Nature
an ethylene receptor gene Cm-ERS1/H70A enhances formation of 1999;402:191–5.
infection threads and nodule primordia. Plant Cell Physiol Schmidt JS, Harper JE, Hoffman TK, Bent AF. Regulation of soybean
2004;45:427–35. nodulation independent of ethylene signaling. Plant Physiol
Okazaki S, Nukui N, Sugawara M, Minamisawa K. Rhizobial 1999;119:951–9.
strategies to enhance symbiotic interaction: rhizobitoxine and Spaink HP. Regulation of plant morphogenesis by lipo-chitin
1-aminocyclopropane-1-carboxylate deaminase. Microbes Environ oligosaccharides. Crit Rev Plant Sci 1996;15:559–82.
2004a;19:99-111. Spaink HP. Ethylene as a regulator of Rhizobium infection. Trends
Okazaki S, Sugawara M, Minamisawa K. Bradyrhizobium elkanii rtxC Plant Sci 1997;2:203–4.
gene is required for expression of symbiotic phenotypes in the final Spaink HP. Root nodulation and infection factors produced by
step of rhizobitoxine biosynthesis. Appl Environ Microbiol rhizobial bacteria. Annu Rev Microbiol 2000;54:257–88.
2004b;70:535–41. Stearns JC, Glick BR. Transgenic plants with altered ethylene
Oldroyd GE, Engstrom EM, Long SR. Ethylene inhibits the Nod factor biosynthesis or perception. Biotechnol Adv 2003;21:193–210.
signal transduction pathway of Medicago truncatula. Plant Cell Suganuma N, Yamauchi H, Yamamoto K. Enhanced production of
2001;13:1835–49. ethylene by soybean roots after inoculation with Bradyrhizobium
Owens LD. Herbicidal potential of rhizobitoxine. Weed Sci elkanii. Plant Sci 1995;111:163–8.
1973;21:63–6. Uchiumi T, Oowada T, Itakura M, Mitsui H, Nukui N, Dawadi P, et al.
Owens LD, Wright DA. Rhizobial-induced chlorosis in soybeans: Expression islands clustered on symbiosis island of Mesorhizo-
isolation, production in nodules, and varietal specificity of the bium loti genome. J Bacteriol 2004;186:2439–48.
toxin. Plant Physiol 1965a;40:927–30. van Spronsen PC, van Brussel AA, Kijne JW. Nod factors produced by
Owens LD, Wright DA. Production of the soybean-chlorosis toxin by Rhizobium leguminosarum biovar viciae induce ethylene-related
Rhizobium japonicum in pure culture. Plant Physiol changes in root cortical cells of Vicia sativa ssp. nigra. Eur J Cell
1965b;40:931–3. Biol 1995;68:463–9.
Owens LD, Guggenheim S, Hilton JL. Rhizobium-synthesized Wilkinson JQ, Lanahan MB, Clark DG, Bleecker AB, Chang C,
phytotoxin: an inhibitor of β-cystathionase in Salmonella Meyerowitz EM, et al. A dominant mutant receptor from
typhimurium. Biochim Biophys Acta 1968;158:219–25. Arabidopsis confers ethylene insensitivity in heterologous plants.
Owens LD, Lieberman M, Kunishi A. Inhibition of ethylene Nat Biotechnol 1997;15:444–7.
production by rhizobitoxine. Plant Physiol 1971;48:1–4. Yasuta T, Satoh S, Minamisawa K. New assay for rhizobitoxine based
Owens LD, Thompson JF, Fennessy PV. Dihydrorhizobitoxine, a new on inhibition of 1-aminocyclopropane-1-carboxylate synthase.
ether amino-acid from Rhizobium japonicum. J Chem Soc Chem Appl Environ Microbiol 1999;65:849–52.
Commun 1972a;1972:715. Yasuta T, Okazaki S, Mitsui H, Yuhashi K, Ezura H, Minamisawa K.
Owens LD, Thompson JF, Pitcher RG, Williams T. Structure DNA sequence and mutational analysis of rhizobitoxine biosyn-
of rhizobitoxine, an antimetabolic enol-ether amino acid from thesis gene in Bradyrhizobium elkanii. Appl Environ Microbiol
Rhizobium japonicum. J Chem Soc Chem Commun 2001;67:4999–5009.
1972b;1972:714. Yuhashi K, Ichikawa N, Ezura H, Akao S, Minakawa Y, Nukui N, et al.
Parker MA, Peters NK. Rhizobitoxine production and symbiotic Rhizobitoxine production by Bradyrhizobium elkanii enhances
compatibility of Bradyrhizobium from Asian and North American nodulation and competitiveness on Macroptilium atropurpureum.
lineages of Amphicarpaea. Can J Microbiol 2001;47:1–6. Appl Environ Microbiol 2000;66:2658–63.