Graminaceous cereal crops as well as all other green plants require iron (Fe), as Fe is a component of heme and [Fe-S] clusters in the photosystems (PSI, PSII) containing green pigments (chlorophylls). The interveinal yellowing (Fe chlorosis) of growing leaves is caused by insufficient Fe uptake and internal delivery. In the 1970s, Sei-ichi Takagi discovered Fe3+-chelating mugineic acid family phytosiderophores (MAs), which are released from graminaceous roots to directly absorb Fe-MAs complexes. However, the mechanisms underlying the Fe delivery from root cells to terminal interveinal mesophyll cells need intensive investigation. This review first overviews the roles of metal-chelating compounds, i.e., MAs and nicotianamine (NA), and the Fe-chelate transporters involved in primary Fe partitioning; then, the delivery of Fe to the growing leaves via phloem and into the developing chloroplasts/thylakoids via symplastic diffusion and membrane transport is discussed. Fe-MAs are absorbed into the root epidermis cells by YSL transporters and transformed to Fe-NA for symplastic radial movement between root parenchyma cells and into the xylem. If heavy metal ions such as Co2+ and Cu2+ are simultaneously present, competition may occur at their complex formation with NA prior to the radial movement that causes leaf chlorosis because of heavy metal-induced restriction of Fe availability. In xylem saps, large fractions of Fe form Fe-MAs, as in barley plants (Hordeum vulgaris), instead of Fe-citrate, which is predominant in rice plants (Oryza sativa). The former complexes are transferred to the phloem at the stem nodes for the growing leaves, while Fe-citrate is partitioned to the mature leaves by transpiration. Although the major routes of Fe supply to the growing leaves by phloem transport have been confirmed, the mechanisms underlying the synthesis of phloem Fe-compounds, delivery through the phloem system, unloading at the growing sink leaves and Fe trafficking and utilization in the chloroplasts/thylakoids still require intensive investigation. Thus, insufficient root uptake of Fe-MAs and insufficient phloem delivery of Fe to the growing leaves and finally to the chloroplasts may cause the reduced chlorophyll-Fe-protein assembly (chlorosis). However, the schemes of iron phloem delivery presented in this review must be confirmed in future studies.

Although the nitrate assimilation into amino acids in photosynthetic leaf tissues is active under the light, the studies during 1950s and 1970s in the dark nitrate assimilation provided fragmental and variable activities, and the mechanism of reductant supply to nitrate assimilation in darkness remained unclear. 15N tracing experiments unraveled the assimilatory mechanism of nitrogen from nitrate into amino acids in the light and in darkness by the reactions of nitrate and nitrite reductases, glutamine synthetase, glutamate synthase, aspartate aminotransferase, and asparagine synthetase. Nitrogen assimilation in illuminated leaves and non-photosynthetic roots occurs either in the redundant way or in the specific manner regarding the isoforms of nitrogen assimilatory enzymes in their cellular compartments. The electron supplying systems necessary to the enzymatic reactions share in part a similar electron donor system at the expense of carbohydrates in both leaves and roots, but also distinct reducing systems regarding the reactions of Fd-nitrite reductase and Fd-glutamate synthase in the photosynthetic and non-photosynthetic organs.

Roots of the higher plants can assimilate inorganic nitrogen by an enzymatic reduction of the most oxidized form (+6) nitrate to the reduced form (-2) glutamate. For such reactions, the substrates (originated from photosynthates) must be imported to supply energy through the reductant-generating systems within the root cells. Intensive studies over last 70 years (reviewed here) revealed the precise mechanisms of nitrate-to-glutamate transformation in roots with elaborate searches of N-15-tracing, enzymes involved, the reductant-supplying system, and nitrate signaling. In the 1970s, the tracing of N-15-labeled nitrate and ammonia in the roots demonstrated the sequential reduction and assimilation of nitrate to nitrite, ammonia, glutamine amide, and then glutamate. These reactions involve nitrate reductase (NADH-NR, EC 1.7.1.1) in the cytosol, nitrite reductase (ferredoxin [Fd]-NiR, EC 1.7.7.1), glutamine synthetase (GS2, EC 6.3.1.2), and glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in the plastids. NADH for NR is generated by glycolysis in the cytosol, and NADPH for Fd-NIR and Fd-GOGAT are produced by the oxidative pentose phosphate pathway (OPPP). Electrons from NADPH are conveyed to reduce NIR and Fd-GOGAT through Fd-NADP(+) reductase (FNR, EC 1.6.7.1) specifically in the roots. Physiological and molecular analyses showed the parallel inductions of NR, NIR, GS2, Fd-GOGAT, OPPP enzymes, FNR, and Fd in response to a short-term nitrate supply. Recent studies proposed a molecular mechanism of nitrate-induction of these genes and proteins. Roots can also assimilate the reduced form of inorganic ammonia by the combination of cytosolic GS1 and plastidic NADH-GOGAT.

Roots of the higher plants can assimilate inorganic nitrogen by an enzymatic reduction of the most oxidized form (+6) nitrate to the reduced form (-2) glutamate. For such reactions, the substrates (originated from photosynthates) must be imported to supply energy through the reductant-generating systems within the root cells. Intensive studies over last 70 years (reviewed here) revealed the precise mechanisms of nitrate-to-glutamate transformation in roots with elaborate searches of N-15-tracing, enzymes involved, the reductant-supplying system, and nitrate signaling. In the 1970s, the tracing of N-15-labeled nitrate and ammonia in the roots demonstrated the sequential reduction and assimilation of nitrate to nitrite, ammonia, glutamine amide, and then glutamate. These reactions involve nitrate reductase (NADH-NR, EC 1.7.1.1) in the cytosol, nitrite reductase (ferredoxin [Fd]-NiR, EC 1.7.7.1), glutamine synthetase (GS2, EC 6.3.1.2), and glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in the plastids. NADH for NR is generated by glycolysis in the cytosol, and NADPH for Fd-NIR and Fd-GOGAT are produced by the oxidative pentose phosphate pathway (OPPP). Electrons from NADPH are conveyed to reduce NIR and Fd-GOGAT through Fd-NADP(+) reductase (FNR, EC 1.6.7.1) specifically in the roots. Physiological and molecular analyses showed the parallel inductions of NR, NIR, GS2, Fd-GOGAT, OPPP enzymes, FNR, and Fd in response to a short-term nitrate supply. Recent studies proposed a molecular mechanism of nitrate-induction of these genes and proteins. Roots can also assimilate the reduced form of inorganic ammonia by the combination of cytosolic GS1 and plastidic NADH-GOGAT.

Biological nitrogen fixation (BNF) by plants and its bacterial associations represent an important natural system for capturing atmospheric dinitrogen (N-2) 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 N-2 fixation in non-leguminous plants. Identification of the involved N-2-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 N-2 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 N-2. In paddy rice fields, methane molecules generated under anoxia are oxidized by xylem aerenchyma-transported oxygen with the simultaneous fixation of N-2 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.

Despite a general view that asparagine synthetase generates asparagine as an amino acid for long-distance transport of nitrogen to sink organs, its role in nitrogen metabolic pathways in floral organs during seed nitrogen filling has remained undefined. We demonstrate that the onset of pollination in Arabidopsis induces selected genes for asparagine metabolism, namely ASN1 (At3g47340), GLN2 (At5g35630), GLU1 (At5g04140), AapAT2 (At5g19950), ASPGA1 (At5g08100) and ASPGB1 (At3g16150), particularly at the ovule stage (stage 0), accompanied by enhanced asparagine synthetase protein, asparagine and total amino acids. Immunolocalization confined asparagine synthetase to the vascular cells of the silique cell wall and septum, but also to the outer and inner seed integuments, demonstrating the post-phloem transport of asparagine in these cells to developing embryos. In the asn1 mutant, aberrant embryo cell divisions in upper suspensor cell layers from globular to heart stages assign a role for nitrogen in differentiating embryos within the ovary. Induction of asparagine metabolic genes by light/dark and nitrate supports fine shifts of nitrogen metabolic pathways. In transgenic Arabidopsis expressing promoter(CaMV35S)::ASN1 fusion, marked metabolomics changes at stage 0, including a several-fold increase in free asparagine, are correlated to enhanced seed nitrogen. However, specific promoter(Napin2S)::ASN1 expression during seed formation and a six-fold increase in asparagine toward the desiccation stage result in wild-type seed nitrogen, underlining that delayed accumulation of asparagine impairs the timing of its use by releasing amide and amino nitrogen. Transcript and metabolite profiles in floral organs match the carbon and nitrogen partitioning to generate energy via the tricarboxylic acid cycle, GABA shunt and phosphorylated serine synthetic pathway.

During 1981–2000, my research focused on the precise determination of the natural abundances of the 13C/12C and 15N/14N ratios (δ13C, δ15N) in many samples of plant tissues and metabolites. That research benefited from the previous work performed by pioneers in this field. My studies during that period provided the followings; (1) the identification of significant differences in the δ13C values in plant tissues and phloem sap affected by biotic (N acquisition, plant species and cultivars) and abiotic (drought) factors, (2) the discrimination of 15N/14N during the uptake, metabolism, and translocation of nitrate, ammonium and biologically-fixed N, and (3) the application of δ13C and δ15N tracing techniques for the quantification of plant residue dynamics (i.e., accumulation and degradation) in soils, plant N uptake from different sources (soils, chemical fertilizers, organic manures) and field biological N2 fixation by legumes including trees and non-legumes such as sugarcane and sweet potato, respectively. Here I review my publications and findings which gave insight for a precise understanding of whole plant C/N metabolisms.

N2 fixation systems in the nonleguminous crops and bacteria associations have been intensively studied over the last 50 years. Their structure and regulation have been investigated to explore the enhancement of N acquisition in these ecosystems leading to crop-growth with minimum chemical fertilizers. Several lines of important evidence have been accumulated indicating that the magnitudes of associative (nonsymbiotic) N2 fixation in sugarcane (Saccharum spp.), sweet potato (Ipomoea batatas L.), and paddy rice (Oryza sativa L.) are agronomically significant. In these three crops, unique bacterial N2-fixation systems may function in addition to the low-level activity (due to the competition in carbon/energy use) of the commonly occurring rhizosphere-associated system by free-living bacteria such as Beijerinckia, Azotobacter, and Klebsiella. Active expressions of the dinitrogenase reductase-encoded gene (nifH) phylogenetically similar to those of Bradyrhizobium spp. and Azorhizobium sp. were abundantly found in the N2-fixing sugarcane stems, sweet potato stems, and storage tubers. These rhizobia micro-aerobically fix N2 in the carbon compounds-rich apoplasts. Gluconacetobacter diazotrophicus and Herbaspirillum spp. were previously isolated from inside the sugarcane stems, as the candidates of endophytic N2 fixers. However, the current molecular and physiological investigations suggest that their major role is production of phytohormonal substances. In paddy rice fields, methane is produced from organic compounds in anoxia and oxidized by contacting with oxygen gas. An active N2-fixation by methane-oxidizing methanotrophs such as Methylosinus sp. takes place in the root tissues (aerenchyma) and also in the surface soil. This methanotrophic N2-fixation supports the sustainability of soil fertility although the N2-fixation and soil fertility are affected by chemical fertilizers. Finally, we discuss the ecological implications of the newly identified rhizobia and methanotroph systems in the N nutrition in nonlegumes and N reservation in field environments.

The aim of this study was to investigate the role of ASN3-encoded asparagine synthetase (AS, EC 6.3.5.4) during vegetative growth, seed development and germination of Arabidopsis thaliana. Phenotypic analysis of knockout (asn3-1) and knockdown (asn3-2) T-DNA insertion mutants for the ASN3 gene (At5g10240) demonstrated wild-type contents of asparagine synthetase protein, chlorophyll and ammonium in green leaves at 35 days after sowing. In situ hybridization localized ASN3 mRNA to phloem companion cells of vasculature. Young siliques of the asn3-1 knockout line showed a decrease in asparagine but an increase in glutamate. The seeds of asn3-1 and asn3-2 displayed a wild-type nitrogen status expressed as total nitrogen content, indicating that the repression of ASN3 expression had only a limited effect on mature seeds. An analysis of amino acid labeling of seeds imbibed with (15N) ammonium for 24 h revealed that asn3-1 seeds contained 20% less total asparagine while 15N-labeled asparagine ((2-15N)asparagine, (4-15N)asparagine and (2,4-15N)asparagine) increased by 12% compared to wild-type seeds. The data indicate a fine regulation of asparagine synthesis and hydrolysis in Arabidopsis seeds.

A single germinated rice (Oryza sativa L) seed can produce 350 grains with the sequential development of 15 leaves on the main stem and 7 ‒ 10 leaves on 4 productive tillers (forming 5 panicles in total), using nitrogen (N) taken up from the environment over a 150-day growing season. Nitrogen travels from uptake sites to the grain through growing organ-directed cycling among sequentially developed organs. Over the past 40 years, the dynamic system for N allocation during vegetative growth and grain filling has been elucidated through studies on N and 15N transport as well as enzymes and transporters involved. In this review, we synthesize the information obtained in these studies along the following main points: (1) During vegetative growth before grain-filling, about half of the total N in the growing organs, including young leaves, tillers, root tips and differentiating panicles is supplied via phloem from mature source organs such as leaves and roots, after turnover and remobilization of proteins, whereas the other half is newly taken up and supplied via xylem, with an efficient xylem-to-phloem transfer at stem nodes. Thus, the growth of new organs depends equally on both N sources. (2) A large fraction (as much as 80%) of the grain N is derived largely from mature organs such as leaves and stems by degradation, including the autophagy pathway of chloroplast proteins (e.g., Rubisco). (3) Mobilized proteinogenic amino acids, including arginine, lysine, proline and valine, are derived mainly from protein degradation, with amino acid transporters playing a role in transferring these amino acids across cell membranes of source and sink organs, and enabling their efficient reutilization in the latter. On the other hand, amino acids such as glutamine, glutamic acid, γ-amino butyric acid, aspartic acid, and alanine are produced by assimilation of newly taken up N by roots and transported via xylem and phloem. The formation of 350 filled grains over 50 days during the reproductive stage is ascribed mainly to degradation and remobilization of the reserves, previously accumulated over 100 days in the sequentially-developed vegetative organs.

Zinc (Zn) and iron (Fe) are essential but are sometimes deficient in humans, while cadmium (Cd) is toxic if it accumulates in the liver and kidneys at high levels. All three are contained in the grains of rice, a staple cereal. Zn and Fe concentrations in rice grains harvested under different levels of soil/hydroponic metals are known to change only within a small range, while Cd concentrations show greater changes. To clarify the mechanisms underlying such different metal contents, we synthesized information on the routes of metal transport and accumulation in rice plants by examining metal speciation, metal transporters, and the xylem-to-phloem transport system. At grain-filling, Zn and Cd ascending in xylem sap are transferred to the phloem by the xylem-to-phloem transport system operating at stem nodes. Grain Fe is largely derived from the leaves by remobilization. Zn and Fe concentrations in phloem-sap and grains are regulated within a small range, while Cd concentrations vary depending on xylem supply. Transgenic techniques to increase concentrations of the metal chelators (nicotianamine, 2′-deoxymugineic acid) are useful in increasing grain Zn and Fe concentrations. The elimination of OsNRAMP5 Cd-uptake transporter and the enhancement of root cell vacuolar Cd sequestration reduce uptake and root-to-shoot transport, respectively, resulting in a reduction of grain Cd accumulation.

Soil fertility has been an important factor in sustainable plant production in native and agricultural fields in temperate climates such as that in Japan. Soil fertility is assessed based on the availability of nutrients, in particular inorganic nitrogen (N) and phosphorus (P), from soil-accumulated organic matter (SOM) via microbial immobilization and mineralization. However, the pool sizes of SOM in humid tropics such as those in Thailand are small and they are turned over rapidly; under such circumstances, the tropical soil fertility would soon be depleted. To meet the urgent requirement of plant nutrients for high plant productivity, we define a direct supply of plant nutrients (i.e., residue fertility) from raw plant and microbial residues. The residue fertility may be driven by the activities of soil fauna (e.g., earthworms, collembolans, termites) and micro-organisms (e.g., saprophytic fungi, protozoa, bacteria), and the released nutrients may be collected and absorbed directly by plant roots including root hairs, and via arbuscular mycorrhizal phyphae. Here, we propose the Ecosystem Fertility paradigm: the Ecosystem Fertility may consist of various ecological nutrient availabilities including both residue fertility and soil fertility. The structure and function of Ecosystem Fertility driven by the above-mentioned biodiversity in different ecosystems may supply not only inorganic N and P but also various forms of nutrients. However, the underlying mechanisms of the Ecosystem Fertility remain to be determined. For the quantification of the various activities and routes involved, the use of molecular and ecosystem approaches may be highly valuable.

It is known that plants contain ferredoxin (Fd)-dependent nitrite reductase (NiR) and glutamate synthase (GOGAT). The Fd-NiR reaction produces ammonia from nitrite, and the activity is usually measured by nitrite disappearance. The Fd-GOGAT reaction forms two glutamates of different origin, from glutamine and 2-oxoglutarate, and the activity is measured by the oxidation of reductant (NADPH) or by formation of total glutamate. Here, a quantitative probe of the products and efficiency of the process was conducted using (15)N tracing techniques on these reactions in vitro. We quantified the reduction of (15)N-labeled [Formula: see text] to [Formula: see text] and the formation of [(15)N]glutamate and [(14)N]glutamate from [5-(15)N-amide]glutamine plus 2-oxoglutarate by NiR and GOGAT, respectively, with the reductant-Fd-NADP(+) oxidoreductase (FNR)-Fd system as the sequential electron donors. The supply of dithionite or NADPH to recombinant cyanobacterial NiR led to electron donation system-dependent formation of [(15)N]ammonium from [(15)N]nitrite. Addition of 20 mM NaCl and 20 mM Na-ascorbate accelerated nitrite reduction under high concentrations of NADPH. A sufficient supply of NADPH to recombinant Zea mays Fd-GOGAT generated complete GOGAT activity (transferring the [5-(15)N]amide of glutamine to 2-oxoglutarate to form [(15)N]glutamate), whereas a shortage of NADPH resulted in glutaminase activity only, which removed the amide from glutamine and released ammonia and [(14)N]glutamate. We conclude that although the recombinant Fd-GOGAT enzyme has two forms of glutamate synthesis, the first by glutaminase (ammonia release by glutamine amidotransferase) and the second by glutamate synthase (coupling of the ammonia and exogenously applied 2-oxoglutarate), the first works without NADPH, while the second is strictly dependent on NADPH availability.

We examined the concentrations of metals (Cd, Zn, Cu, Fe and Mn) and potential metal-binding compounds [nicotianamine (NA), thiol compounds and citrate] in xylem and phloem saps from 4-week-old castor bean plants (Ricinus communis) treated with 0 (control), 0.1, 1.0, and 10 μM Cd for 3 weeks. Treatment with 0.1 and 1 μM Cd produced no visible damage, while 10 μM Cd retarded growth. Cadmium concentrations in both saps were higher than those in the culture solution at 0.1 μM, similar at 1.0 μM and lower at 10 μM. Cd at 10 μM reduced Cu and Fe concentrations in both saps. NA concentrations measured by capillary electrophoresis-mass spectrometry (MS) in xylem sap (20 μM) were higher than the Cu concentrations, and those in phloem sap (150 μM) were higher than those of Zn, Fe and Cu combined. Reduced glutathione concentrations differed in xylem and phloem saps (1-2 and 30-150 μM, respectively), but oxidized glutathione concentrations were similar. Phloem sap phytochelatin 2 concentration increased from 0.8 μM in controls to 8 μM in 10 μM Cd. Free citrate was 2-4 μM in xylem sap and 70-100 μM in phloem sap. Total bound forms of Cd in phloem and xylem saps from 1 μM Cd-treated plants were 54 and 8%, respectively. Treatment of phloem sap with proteinaseK reduced high-molecular compounds while increasing fractions of low-molecular Cd-thiol complexes. Zinc-NA, Fe-NA and Cu-NA were identified in the phloem sap fraction of control plants by electrospray ionization time-of-flight MS, and the xylem sap contained Cu-NA.

The major component of fused potassium silicate (FPS) fertilizers, produced from steel-making slag, was studied. FPS compounds have received considerable attention as slow-release potassium fertilizers beneficial for crops. This major component was found to be a single phase compound, K2Ca2Si2O7, which had not been previously identified. In order to confirm the presence of the newly identified compound, we synthesized a potassium calcium silicate mixture, K2O-2CaO-2SiO(2), by fusing a mixture of K2CO3, CaCO3, and SiO2. The X-ray diffraction patterns of the synthesized K2O-2CaO-2SiO(2) were largely consistent with those of FPS fertilizer, and Energy Dispersive X-ray Spectroscopy indicated that this compound was a single phase with a K:Ca:Si molar ratio of 1:1:1. It is concluded that the major component of FPS fertilizer is a compound of K2O-2CaO-2SiO(2), newly identified as K2Ca2Si2O7. FPS fertilizers exhibit the characteristic, controlled by their K2Ca2Si2O7 content, of slowly releasing potassium into water and soil.

Graminaceous plants can take up iron-phytosiderophore complexes, whereas non-graminaceous plants absorb ferrous ions after the reduction of ferric compounds at the root cell membranes. The iron (Fe) in the roots may be transported to the aerial plant parts through the xylem. We compared the chemical forms in xylem sap collected from the cut stems of three graminaceous plants (rice [Oryza sativa L.], maize [Zea mays L.], barley [Hordenum vulgare L.]) and three non-graminaceous plants (tomato [Lycopersicon esculentum Mill.], soybean [Glycine max Merr.], castor bean [Ricinus communis L.]) grown in composite soils for the concentrations of iron and iron-chelating compounds (nicotianamine, phytosiderophores, citrate). We also fractionated the xylem saps by size-exclusion chromatography to gain insight into the chemical forms of iron. The Fe concentrations in the xylem sap ranged from 9 to 40 mu M. Nicotianamine was found in the xylem sap from all the plants examined, with higher concentrations in the non-graminaceous plants. In contrast, phytosiderophores (2'-deoxymugineic acid and mugineic acid) were predominantly detected in the graminaceous plants. The concentrations of free citrate varied greatly (from 4 to 2200 mu M) among the six plant species. The xylem sap iron in non-graminaceous plants may form two types of Fe-citrate, whereas in graminaceous plants, the bound Fe forms may be largely two types of Fe-citrate with various Fe-phytosiderophores.

Zinc (Zn) is an essential nutrient for human beings, and most Zn intake occurs through vegetables or cereals such as rice (Oryza sativa L.) grains. Recently, we detected Zn as well as cadmium, which may be partitioned to rice grains, in the phloem saps from the uppermost internodes of rice and in the xylem saps from the cut stems at early grain-filling. To quantify Zn transport to the grains via the phloem and xylem, a mathematical model previously developed for cadmium transport to rice grains was applied. We examined the translocation of zinc into the grains of rice plants at early grain-filling by feeding zinc-65 (Zn-65) via a root-bathing medium, through culm cuts above and below the flag-leaf nodes, and through the flag leaves. The estimate made using the mathematical model and experimental data for three types of Zn-65 transport suggests that the grain Zn may be accumulated predominantly via the phloem through two means of transport, phloem transport of stored Zn from the leaves and, more importantly, xylem-to-phloem transfer at the nodes from Zn as it is being absorbed. The Zn transport via the phloem to the grains is more selective than that of cadmium, a non-nutrient element, as also evidenced by the greater transport of cadmium to the glumes via the xylem.

日本の生態系には，1950～1970年代の大気圏内核実験，1986年4～5月のチェルノブイリ原発事故，そして2011年3月の福島原発事故による放射性物質が降下し広域の複雑な汚染が生じた．降下核種のうち長寿命の放射性セシウム (*Cs) は森林，農耕地，河川や湖沼，海の植物，動物，土，水に不均一に拡散している．この生態系での放射性セシウムの動態の理解は，ヒトの生命と，安全や安心できる食生活と活動のための環境の復興に大切である．

To estimate potential nitrogen-fixing endophytes in sugarcane and sweet potatoes, we used a cultivation-independent approach. The expression (RNA) of dinitrogenase reductase gene (nifH) was investigated by reverse transcription-polymerase chain reaction as well as DNA searches. Gene fragments corresponding to nifH were amplified from mRNA and DNA obtained from stems of pot-grown sugarcane and stems and tubers of field-grown sweet potatoes in Japan. Sequence analysis revealed that large numbers of the clones were homologous to the nifH sequences of rhizobia. Moreover, the expression of nifH genes in the stems of sugarcane was correlated with the nitrogen fixation assessed by the 15N-dilution method. These results suggest that the detection of expressed nifH genes from the host plant tissues enabled an efficient estimate of nitrogen fixation.