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.
We isolated two muskmelon (Cucumis melo) cDNA homologs of the Arabidopsis ethylene receptor genes ETR1 and ERS1 and designated them Cm-ETR1 (C. melo ETR1; accession no. AF054806) and Cm-ERS1 (C. melo ERS1; accession no. AF037368), respectively. Northern analysis revealed that the level of Cm-ERS1 mRNA 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 and Cm-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.
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 symbiotic interactions between a legume and (brady) rhizobia result in a unique, nitrogen-fixing plant organ, the nodule. Recent studies have shown that the phytohormone ethylene inhibits nodule formation in some legumes (8,9,16,24,25). Application of 1-aminoocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene, inhibits nodulation in Medicago truncatula (24).Rhizobitoxine [2-amino-4-(2-amino-3-hydropropoxy)-transbut-3-enoic acid] is an ethylene synthesis inhibitor that is produced by the legume symbiont Bradyrhizobium elkanii (15, 17-19, 22, 39). It is thought that production of this compound enhances nodulation of the host legume because of its inhibitory effect on ethylene synthesis. However, some reports have shown that there is not a significant difference in nodule number between plants inoculated with B. elkanii USDA61 and plants inoculated with rhizobitoxine-deficient mutants during nodulation of Glycine max, Glycine soja, Vigna unguiculata, and Macroptilium atropurpureum (26,39). Recently, Duodu et al. observed a significant difference in nodule number between plants inoculated with isogenic variants of USDA61 during nodulation of Vigna radiata (7). Although these findings do not seem to be consistent with the hypothesis that rhizobitoxine has a positive effect on nodulation, the inconsistency can be explained by differences in the ethylene sensitivity of nodulation among leguminous species; nodulation of G. max is generally not sensitive to ethylene (10, 31, 38), while nodulation of V. radiata is sensitive (7). The inconsistency could also result from differences in the abilities of the strains used in the experiments to produce rhizobitoxine; strain USDA61 is a weak producer of rhizobitoxine (39).In addition to G. max, the leguminous plant M. atropurpureum is a nodulating host for B. elkanii and Bradyrhizobium japonicum (12,15). Although the effect of ethylene on nodulation has been studied in many leguminous host plants so far, the effect of ethylene in M. atropurpureum is not known. B. elkanii was found to be more competitive than B. japonicum for nodulation of M. atropurpureum in a multistrain environment when a field soil was inoculated with a mixture of several strains isolated from the field soil (21). In general, B. elkanii accumulates rhizobitoxine in cultures and in nodules, while B. japonicum does not (5,15,18,19). These results led us to investigate the role of rhizobitoxine production on the nodulation and competitiveness of B. elkanii on M. atropurpureum by using a B. elkanii strain that produces...
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 largedeletion mutant of B. elkanii, USDA94⌬rtx::⍀1, 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 USDA94⌬rtx::⍀1. 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.Rhizobitoxine [2-amino-4-(2-amino-3-hydropropoxy)-transbut-3-enoic acid] is synthesized by the legume symbiont Bradyrhizobium elkanii (37) and the plant pathogen Burkholderia andropogonis (29). Because it induces foliar chlorosis of soybeans, rhizobitoxine has been regarded as a plant toxin (18,36,57). In terms of biochemical functions, rhizobitoxine inhibits -cystathionase in the methionine biosynthesis pathway (39, 57) and 1-aminocyclopropane-1-carboxylate (ACC) synthase in the ethylene biosynthesis pathway (59).Recently, a beneficial role for rhizobitoxine in Rhizobiumlegume symbiosis has been revealed. Using a rhizobitoxine mutant, Yuhashi et al. (60) found that rhizobitoxine production by B. elkanii enhances nodulation and competitiveness in the legume Macroptilium atropurpureum (siratro), probably via the inhibition of endogenous ethylene production in the host plant. Duodu et al. (7) reported that rhizobitoxine mutants formed fewer mature nodules on Vigna radiata (mung bean) than the wild-type strain. In addition, application of ethylene inhibitors to the rhizobitoxine mutants partly restored the nodulation phenotype. Therefore, rhizobitoxine is a nodulation enhancer rather than a phytotoxin for siratro and mung bean, although it is unlikely that rhizobitoxine exerts this positive effect in nodulation of soybean cultivars (28,43,60).The biosynthetic pathway for rhizobitoxine ...
Summary• Ethylene evolution from plants inhibits Agrobacterium-mediated genetic transformation, but the mechanism is little understood. In this study, the possible role of ethylene in Agrobacterium-mediated genetic transformation was clarified.• It was tested whether or not plant ethylene sensitivity affected genetic transformation; the sensitivity might regulate bacterial growth during co-cultivation and vir gene expression in Agrobacterium tumefaciens. For these experiments, melon (Cucumis melo) was used, in which ethylene sensitivity was controlled by chemicals, and Arabidopsis ethylene-insensitive mutants.• Agrobacterium-mediated genetic transformation was inhibited in ethylene-sensing melon, whereas, in Arabidopsis ethylene-insensitive mutant, it was enhanced. However, the ethylene sensitivity did not affect bacterial growth. vir gene expression was inhibited by application of plant exudate from ethylene-sensitive plants. The inhibitory effect of the ethylene sensitivity on genetic transformation relieved the activation of vir gene expression in A. tumefaciens with vir gene inducer molecule (acetosyringone, AS) or A. tumefaciens mutant strain which has constitutive vir gene expression.• These results indicate that ethylene evolution from a plant inoculated with A. tumefaciens inhibited vir gene expression in A. tumefaciens through the ethylene signal transduction in the plant, and, as a result, Agrobacterium-mediated genetic transformation was inhibited.
The effect of ethylene on gene transfer mediated by an Agrobacterium tumefaciens harbouring a binary vector with the β‐glucuronidase (uid A) 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 μM), and inhibited by the addition of aminoethoxy‐vinylglycin (AVG, 10 μM). 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 uid A 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.
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.Agrobacterium-mediated gene transfer is widely used for plant molecular genetics and its applications (14). In particular, efficient systems of genetic transformation are required for plant functional genomics and molecular breeding to improve traits (20,21). Recent studies showed that ethylene is one of the factors that inhibits Agrobacterium-mediated gene transfer (1, 3, 5). Therefore, if Agrobacterium tumefaciens has the ability to decrease the ethylene level in the host plant, the frequency of gene transfer will increase. To suppress ethylene evolution in plant cells during cocultivation, we introduced the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene from Pseudomonas sp. strain ACP (7, 18) into A. tumefaciens. ACC deaminase cleaves ACC (the immediate ethylene precursor) into ␣-ketobutyrate and ammonia, and as a result, the ethylene level is decreased (4, 12, 16).The ACC deaminase gene was amplified and cloned into pBBR1MCS-5 (10), a broad-host-range plasmid, to generate a lacZ::acdS translational fusion (Fig. 1A). The resulting plasmid was designated pBBRacdS and was introduced into A. tumefaciens C58 (17) or C58C1Rif R (2) by electroporation (19). The binary vector pIG121-Hm, involved in T-DNA transfer (6), was also harbored in A. tumefaciens C58C1Rif R (pBBRacdS, pIG121-Hm). The ACC deaminase activity in A. tumefaciens C58C1Rif R (pBBRacdS, pIG121-Hm) was assayed according to the method of Honma and Shimomura (7). The amount of ␣-ketobutyrate in the reaction buffer was estimated from a standard curve based on a dilution of 10 to 400 M (detected at 340 nm). The controls for this experiment were C58C1Rif R (pBBRMCS-5, pIG121-Hm) and samples in reaction buffer without the substrate 200 M ACC. The accumulation of ␣-ketobutyrate was observed only in the lysate from A. tumefaciens C58C1Rif R (pBBRacdS, pIG121-Hm) in the presence of the substrate (Fig. 1B). Therefore, we succeeded in conferring ACC deaminase activity on A. tumefaciens.Surface-sterilized melon (Cucumis melo L. var. cantaloupensis cv. Vedrantais) seeds were sown on a half-strength preparation of Murashige and Skoog's medium (MS) (13) and incubated at 25°C with 16 h of light per day for 5 days. Cotyledons from the germinated seedlings were transversely sectioned by hand into five pieces, and among these five, three internal pieces were inoculated. The segments were soaked in an A. tumefaciens cell suspension of 10 7 cells ml Ϫ1 for 20 min and then placed on cocultivation medium (MS containing 1.0 mg of 6-benzylamino-purine liter Ϫ1 , 2% glucose, and 0.4% Gelrite [Wako, Tokyo, Japan], pH 5.5) in a gas vial with 16 h of light per day. Thirty melon cotyledon seg...
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 4°C 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 4°C, 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.
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