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†Background and Aims Rhizobitoxine, produced by the legume symbiont Bradyrhizobium elkanii, inhibits cystathionine-b-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 rhizobitoxineinduced 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-b-lyase inhibition during methionine biosynthesis.

Soybean bradyrhizobia, Bradyrhizobium japonicum and Bradyrhizobium elkanii differ in various traits such as DNA fingerprint, rhizobitoxine production, indole-3-acetic acid production and uptake of hydrogenase. In this communication, we investigated whether the differences between both species extend to host preference in multistrain environments. Nodule occupancy of B.japonicum and B. elkanii significantly depended on host plants. B. japonicum and B. elkanii preferentially nodulated Glycine max and M. atropurpureum, respectively. Both bacterial species were shown to nodulate G. soja with similar efficiency. There was a significant divergence of DNA sequences in and around nodulation genes between the B. japonicum and B. elkanii field isolates. However, flavonoid and lipo-chitin nodulation signals were not involved in the difference in host preference of B. japonicum and B. elkanii. The ecological implications of host preference are discussed.

Regression analysis of results from a mathematical competition model showed that rhizobitoxine production by Bradyrhizobium elkanii USDA94 gave this strain a nodulation competitiveness about 10 times greater than that of a non-rhizobitoxine-producing mutant strain on Macroptilium atropurpureum (Siratro). Rhizobitoxine enhancement of competitive nodulation occurred at a late stage in the time-course of nodulation. All other known rhizobial factors that affect nodulation competitiveness act in the rhizosphere and during the initial interaction with legumes. This unique late action of rhizobitoxine could prove advantageous in inoculant production, because inoculum often fails to nodulate in the latter stages of nodulation kinetics.

We investigated the horizontal transfer of nodulation (nod) genes to a Bradyrhizobium elkanii strain, lacking common nod genes as a recipient, in soils and microcosms using selection systems of antibiotic resistance and legume nodulation. We observed the horizontal transfer of nod genes at 4C in Nakazawa soil where peculiar strains (HRS strains) of B. japonicum harboring high copy numbers of insertion sequences dominated. In microcosms containing HRS strains as donors, we detected a similar horizontal transfer from B. japonicum HRS strain NK5 to the B. elkanii recipient more efficiently at 4C, which was verified by examining hybridization, nodulation and Nod factor production. These traits were, however, gradually lost during successive cultures. Plasmid analysis indicated that this event was not due to the simple transfer of plasmid carrying common nod genes. These results suggest the potential for horizontal transfer of nod genes among bradyrhizobia and other bacterial populations in soil environments.

We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii . The nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxA showed that several open reading frames (ORFs) were located downstream of rtxA . A large-deletion mutant of B. elkanii , USDA94Δ rtx ::Ω1, which lacks rtxA , ORF1 ( rtxC ), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains rtxA and these ORFs, complemented rhizobitoxine production in USDA94Δ rtx ::Ω1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxA and rtxC are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions of rtxA indicated that rtxA is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis. An insertional mutant of rtxC produced serinol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, the rtxC product was highly homologous to the fatty acid desaturase of Pseudomonas syringae and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase. This result suggested that rtxC encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production. In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia.

Inhibitors of ethylene synthesis or its physiological function enhanced nodulation in Lotus japonicus and Macroptilium atropurpureum. In contrast, the application of 1-aminocyclopropane-1-carboxylic acid, a precursor of ethylene biosynthesis, reduced the nodule number in these legumes. These results suggest that an ethylene-mediated signaling pathway is involved in the nodulation process even in the determinate nodulators.

Application of 1-aminoocyclopropane-1-carboxylic acid, an ethylene precursor, decreased nodulation of Macroptilium atropurpureum by Bradyrhizobium elkanii. B. elkanii produces rhizobitoxine, an ethylene synthesis inhibitor. Elimination of rhizobitoxine production in B. elkanii increased ethylene evolution and decreased nodulation and competitiveness on M. atropurpureum . These results suggest that rhizobitoxine enhances nodulation and competitiveness of B. elkanii on M. atropurpureum .

The effect of ethylene on gene transfer mediated by an Agrobacterium tumefaciens harbouring a binary vector with the beta-glucuronidase (uidA) gene was investigated in melon, Cucumis melo L. Explants excised from melon cotyledons produced ethylene, the production of which was increased by the addition of 1-aminocyclopropane-1-carboxylic acid (ACC, 20 or 200 micromolar), and inhibited by the addition of aminoethoxy-vinylglycin (AVG, 10 micromolar). Agrobacterium inoculation of explants increased ethylene production, while application of AVG during inoculation reduced it. After 4 days of co-cultivation with Agrobacterium, gene transfer in the explants was assayed by transient uidA expression. Application of ACC to the co-cultivation medium reduced Agrobacterium-mediated gene transfer to explants and that of AVG increased it. These results suggest that ethylene affects the A. tumefaciens-mediated gene transfer to the explants excised from melon cotyledons, and the efficiency of Agrobacterium-mediated gene transfer can be improved by inhibiting ethylene production from the explants.

We isolated two muskmelon (Cucumis melo) cDNA homologs of the Arabidopsis ethylene receptor genes ETR1 andERS1 and designated them Cm-ETR1(C.meloETR1; accession no. AF054806) andCm-ERS1 (C.meloERS1; accession no. AF037368), respectively. Northern analysis revealed that the level of Cm-ERS1mRNA in the pericarp increased in parallel with the increase in fruit size and then markedly decreased at the end of enlargement. In fully enlarged fruit the level of Cm-ERS1 mRNA was low in all tissues, whereas that of Cm-ETR1 mRNA was very high in the seeds and placenta. During ripening Cm-ERS1 mRNA increased slightly in the pericarp of fruit before the marked increase of Cm-ETR1 mRNA paralleled climacteric ethylene production. These results indicate that both Cm-ETR1 andCm-ERS1 play specific roles not only in ripening but also in the early development of melon fruit and that they have distinct roles in particular fruit tissues at particular developmental stages.

Ethylene is a gaseous plant hormone which gives cues to various developmental processes and stress responses in plants. Plants regulate their ethylene sensitivity for programmed development and responses to environmental stresses. After the successful isolation of the Arabidopsis ethylene receptor gene ETR1, ethylene receptor genes have been found in various plant species. Characterization of ethylene receptor genes provides clues to understanding how plants regulate their ethylene sensitivity. This knowledge also gives us potential methods for engineering the ethylene sensitivity of plants. Genetically-engineered plants with reduced ethylene sensitivity will be potential resources for improving crop performance. Moreover, those transgenic plants will provide further information to elucidate the mechanism of how plants regulate their ethylene sensitivity. The recent progress in genetic and protein analyses of ethylene receptors is summarized in this review. The possible strategies for altering the ethylene sensitivity of plants using the ethylene receptor genes are also discussed.

Agrobacterium -mediated gene transfer is widely used for plant molecular genetics, and efficient techniques are required. Recent studies show that ethylene inhibits the gene transfer. To suppress ethylene evolution, we introduced 1-aminocyclopropane-1-carboxylate (ACC) deaminase into Agrobacterium tumefaciens . The ACC deaminase enhanced A. tumefaciens -mediated gene transfer into plants.

Attempts to improve the symbiotic nitrogen fixation with effective (Brady) rhizobium strains do not always succeed under field conditions due to the lower nodulation competitiveness of the introduced strains than that of the indigenous rhizobia (Triplett and Sadowsky 1992). An introduced strain needs to be marked for monitoring its nodule occupancy under competitive nodulation conditions.

Macroptilium atropurpureum is a model legume with a broad symbiont range for nodulation. We have achieved the first in vitro plant regeneration of this species using cv. Siratro. Hypocotyl explants excised from dark-grown seedlings generated slimy, friable calli after three weeks’ culture on B5 medium containing 1‐2 mg:l kinetin and 0.05 mg:l a-naphthaleneacetic acid. This was followed by the generation of green organogenic callus with shoot buds by subculturing the explants to hormone-free B5 medium 20 days after the start of culture. The green organogenic calli with shoot buds were maintained as organogenic callus by subculturing on the same medium, and shoots were elongated on hormone-free B5 medium. Elongated shoots were rooted on half-strength B5 medium. Most regenerated plants were morphologically normal, diploid and fertile, although tetraploid plants appeared at a low frequency (8%). © 2000 Elsevier Science Ireland Ltd. All rights reserved.