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Biological nitrogen fixation (BNF) by plants and its bacterial associations represent an important natural system for capturing atmospheric dinitrogen (N2) and processing it into a reactive form of nitrogen through enzymatic reduction. The study of BNF in non-leguminous plants has been difficult compared to nodule-localized BNF in leguminous plants because of the diverse sites of N2 fixation in non-leguminous plants. Identification of the involved N2-fixing bacteria has also been difficult because the major nitrogen fixers were often lost during isolation attempts. The past 20 years of molecular analyses has led to the identification of N2 fixation sites and active nitrogen fixers in tissues and the rhizosphere of non-leguminous plants. Here, we examined BNF hotspots in six reported non-leguminous plants. Novel rhizobia and methanotrophs were found to be abundantly present in the free-living state at sites where carbon and energy sources were predominantly available. In the carbon-rich apoplasts of plant tissues, rhizobia such as Bradyrhizobium spp. microaerobically fix N2. In paddy rice fields, methane molecules generated under anoxia are oxidized by xylem aerenchyma-transported oxygen with the simultaneous fixation of N2 by methane-oxidizing methanotrophs. We discuss the effective functions of the rhizobia and methanotrophs in non-legumes for the acquisition of fixed nitrogen in addition to research perspectives.

Bacterial ACC deaminase is one of the key tools to ameliorate plant stress by lowering ethylene level in plants. The effects of ACC deaminase-producing bacteria on the volatile profiles in plants have not been examined to date. To address this, we performed metabolic profiling of volatiles in carrots following inoculation of the bacteria producing ACC deaminase. We isolated ACC deaminase-producing bacteria from the inner part of the fruits and vegetables grown on organic farms by culturing on ACC-containing media, and screened them with PCR for the acdS gene, mungbean growth assay, and in vitro ACC deaminase activity. The isolated endophytes were evaluated for their ability to alter volatile profiles in carrots. Eleven bacterial strains possessing the activity to cleave ACC were selected among the 60 isolates grown on the medium containing ACC as a sole N source. Three of them that belonged to Pseudomonas could reduce the levels of (E)-2-hexenal and the other green leaf volatiles (GLVs) and terpenoids in the carrot leaves following inoculation of the seeds. The isolated endophytes with ACC deaminase activity could alter the composition of volatiles in plants, probably through lowering ethylene level in the plant.

Sweet potato (Ipomoea batatas) is known for its ability to grow under nitrogen-limited conditions. To clarify the possible contribution of biological nitrogen fixation, we tried to isolate and identify diazotrophic bacteria from sweet potatoes. By using cultivation technique, we isolated putative endophytes, which possess nifH genes, from surface-sterilized sweet potatoes. Their nitrogen-fixing abilities were demonstrated by the acetylene reduction assay in a semi-solid malate medium and sweet potato extracts. We also examined the colonization of an isolated strain (AT1) in sweet potatoes and their influence on growth and nitrogen fixation in plants as assessed by an acetylene reduction assay and 15N-isotope dilution technique. The isolates were identified as strains of Bradyrhizobium sp. AT1, Paenibacillus sp. AS2 and Pseudomonas sp. T16 based on their 16S rRNA gene sequences. They showed acetylene reduction activity (ARA) in the semi-solid malate medium. Among them, B. sp. AT1 showed ARA in sweet potato extracts under micro-aerobic conditions whereas both P. sp. AS2 and P. sp. T16 showed no ARA. The inoculation of B. sp. AT1 to the sweet potatoes resulted in increases in the fresh weights and detection of ARA in the inoculated plants. Moreover, the reduction of 15N atom % was observed in the inoculated plants compared to uninoculated controls. B. sp. AT1 actively expresses nitrogenase activity in sweet potatoes and may contribute to the nitrogen nutrition of host plants.

In leguminous plants, infection with rhizobia leads to the formation of root nodules that fix atmospheric nitrogen and supply it as ammonium to the host plant cells. The formation of nitrogen-fixing nodules is nutritionally benefi- cial, but excessive nodule production appears to be detrimental to the host legumes because of the resultant over-consumption of photosynthetic products. Therefore, the number of root nodules in leguminous plants is tightly regulated by shoot-root signaling known as a feedback or autoregulation of nodulation. Recently, we found that foliar application of polyamines and an effective inhibitor of polyamine biosynthesis regulates the number of nodules in soybean plants. In this review, we discuss the possible function of polyamines as a systemic regulator of nodule formation.

In legumes, the number of root nodules is controlled by a mechanism called autoregulation. Recently, we found that the foliar brassinosteroid (BR), a plant growth-regulating hormone, systemically regulates the nodule number in soybean plants. In the present study we report that such down-regulation of root nodule formation by a BR may occur through a change of the polyamine contents, with the experimental evidence as follows. The foliar contents of both spermidine (Spd) and spermine (Spm) in the super-nodulating soybean mutant, En6500, were always lower than those in its parent line, Enrei. This lower Spd and Spm content accompanied a striking accumulation of putrescine (Put) in the former plant. This finding indicates that Spd and Spm biosynthesis from their precursor Put is repressed in En6500. The foliar treatments with Spd or Spm of En6500 led to a reduction of both nodule number and root growth. On the other hand, foliar treatment with MDL74038, a specific inhibitor of Spd biosynthesis, apparently increased the root nodule number in Enrei. Foliar application of brassinolide (BL) of En6500 increased the leaf Spd level and reduced the nodule number. These results suggested that BL-induced Spd synthesis in shoots might suppress the root nodule formation.

Neuroactive aromatic amines acting on the central nervous system are widespread in the plant kingdom. We have previously found β-phenethylamine (β-PHA), one of the aromatic alkaloids, in root nodules of various annual legume crops. The present study was undertaken to determine the site of β-PHA accumulation within root nodules of the adzuki bean Vigna angularis. High concentrations of β-PHA were always detected in the alkaloid fraction of adzuki bean root nodules. Related aromatic amines such as tyramine, dopamine, and other β-PHA derivatives, which are found in various medicinal plants, were not detected in adzuki bean root nodules. The amounts of β-PHA in root nodules varied not only with the growth stage of the host plant, but also with nodule age; β-PHA levels increased with nodule development, but declined with nodule senescence. Adzuki bean nodules, after crushing with a grinding medium, were separated into bacteroids and a nodule cytosol fraction. A large amount of β-PHA was detected in the bacteroids, while a very small amount was prsent in the nodule cytosol fraction derived from plant cells. The bacteroids in the mature nodules contained considerably higher amounts of β-PHA than did those in immature or senescent nodules. The formation of β-PHA in root-nodule bacteria was then tested using eight strains of Rhizobiaceae (Rhizobium, Bradyrhizobium and Sinorhizobium), including a strain isolated from root nodules of field-grown adzuki bean plant. None of the cultured cells produced β-PHA in liquid media in the presence or absence of phenylalanine, a putative precursor of β-PHA. Nitrogen-fixing bacteroids within nodules are the cells uniquely differentiated from root-nodule bacteria. The present results suggest that β-PHA is formed associated with the differentiation of vegetative bradyrhizobia into nitrogen-fixing bacteroids with the plant host cells.

Leguminous plants form nitrogen-fixing root nodules and the number of nodules is controlled by a self-regulating mechanism called autoregulation. However, signaling substances involved in nodule regulation have not been identified. In the present study, we used brassinolide, a most effective molecular species of plant hormone brassinosteroids, and brassinazole, an effective inhibitor of brassinosteroid biosynthesis to determine whether brassinolide played a role in systemic regulation of noduel formation in wild type soybean and its super-nodulating mutant. Foliar application or direct injection of brassinolide into the root base inhibited nodule formation and root development in the super-nodulating mutant (En6500), but not in the parent line (cv. Enrei). The internodes in the plants subjected to foliar application were significantly longer than those in the untreated plants. In contrast, the application of brassinazole on mature leaves or into the culture media resulted in the increase of the nodule num...

The changes in the cAMP and cGMP contents in root nodules were investigated using pea (Pisum sativum L. cv. Izusen) and soybeans (Glycine max L. (Merr.) cv. Enrei and its supernodulating mutant En6500). The concentration of cAMP increased in the nodules from pea and soybean cv. Enrei with nodule development, but not in the nodules from soybean En6500. The concentrations of cGMP remained low in pea and soybean cv. Enrei, while the levels of cGMP in the roots and nodules from soybean En6500 showed a temporal increase after rhizobium infection. The external application of dibutyryl-cAMP (db-cAMP) significantly decreased nodule production in these leguminous plants, indicating the possible involvement of cAMP in the regulation of nodule formation. On the other hand, dibutyryl-cGMP (db-cGMP) did not significantly affect the nodule fresh weight of these legumes. The data further suggest the existence of a difference of the functional role of cGMP from that of cAMP in the regulation of root nodule forma...

The aromatic amine, β-phenethylamine, was identified in various field-grown leguminous plants by analyses with HPLC, GC, GC-MS and 1H-NMR. High concentration of β-phenethylamine was generally detected only in mature root nodules, but not in other plant organs such as root, stem, leaf, pod and grain. Occurrence was specific to the root nodules formed by Bradyrhizobium infection. Ten of eleven legume crops including soybean [Glycine max (L.) Merr.], pigeon pea [Cajanus cajan (L.) Millsp.], adzuki bean (Vigna angularis), mung bean [V. radiata (L.) Wilczek] and cowpea (V. unguiculata) contained this aromatic amine, but groundnut (Arachis hypogaea L.) also nodulated by Bradyrhizobium sp. did not. Root nodules collected from garden pea (Pisum sativum L.), broad bean (Vicia fava L.), kidney bean (Phaseolus vulgaris L.) and various other herbaceous legumes nodulated by Rhizobium sp., Mesorhizobium sp., Sinorhizobium sp. or Azorhizobium caulinodans, and root-nodulated, woody non-legumes, nodulated by Frankia spp., contained little β-phenethylamine.

Cyclic adenosine 3′,5′-monophosphate (cAMP) and cyclic guanosine 3′,5′-monophosphate (cGMP) contents of cultured cells of Frankia strains originally isolated from nodules of Alnus sieboldiana, Myrica rubra and Elaeagnus macrophylla were measured by enzyme immunoassays (EIA). Frankia cells, cultured for 59–121 d, had cAMP contents ranging from 2.9 to 76.1 pmol mg −1 protein and cGMP contents ranging from 0.9 to 5.2 pmol mg −1 protein. Following Frankia culture, the media contained extremely large quantities of cAMP and significant levels of cGMP. The nature of accumulation and secretion of cyclic nucleotides by slow-growing Frankia cells was comparable to that by a fast-growing actinomycete Streptomyces lividans TK24, suggesting that secretion of cAMP by Frankia cells may occur through the cell membrane but not by cell lysis. cAMP and cGMP contents in the symbiotic nodules, leaves and roots of actinorrhizal plants and leaves of non-actinorrhizal trees were also measured. The nodules of actinorrhizal woody plants (A. sieboldiana, E. macrophylla, E. umbellata, E. pungens and M. rubra) had cAMP contents ranging from 4 to 258 pmol g −1 f. wt and cGMP contents ranging from 1.1 to 5.2 pmol mg −1 protein. Most leaves and some roots of actinorrhizal plants and all the leaves of non-actinorrhizal woody plants examined contained small but significant amounts of cAMP and cGMP. This is the first report of significant contents of cAMP and cGMP in cultured Frankia cells and Frankia- infected nodules. Possible roles of cyclic nucleotides as symbiotic signals are discussed.

Cyclic adenosine 3′,5′-monophosphate (cAMP) content of various cultured rhizobia strains and tissues of legumes and non-leguminous plants was measured by enzyme immunoassays. Most rhizobia, cultured for 44 to 165 h, contained cAMP ranging from 0.6 to 5 pmol mg−1protein except forAzorhizobium caulinodansORS571. The culture media also contained varying amounts of cAMP depending on the strain of rhizobia.Azorhizobiumcells and their media contained no detectable cAMP. Nodules from most legumes and non-legumes had cAMP contents ranging from 2–70 pmol g−1f.wt. However, nodules fromSesbania rostrata,Crotalaria spectabilisandParasponia andersoniishowed undetectable cAMP levels, and those fromGlycine maxandVigna angularisoccasionally showed levels below the detection limit. The leaves of non-legumes mostly had cAMP levels below detection limit (approx. 1.0 pmol g−1 f.wt), while the leaves of a few legumes occasionally had detectable cAMP. The possible role of cAMP as a symbiotic signal is discussed.