Effect of Light Quality on Metabolomic, Ionomic, and Transcriptomic Profiles in Tomato Fruit.

Light quality affects plant growth and the functional component accumulation of fruits. However, there is little knowledge of the effects of light quality based on multiomics profiles. This study combined transcriptomic, ionomic, and metabolomic analyses to elucidate the effects of light quality on metabolism and gene expression in tomato fruit. Micro-Tom plants were grown under blue or red light-emitting diode light for 16 h daily after anthesis. White fluorescent light was used as a reference. The metabolite and element concentrations and the expression of genes markedly changed in response to blue and red light. Based on the metabolomic analysis, amino acid metabolism and secondary metabolite biosynthesis were active in blue light treatment. According to transcriptomic analysis, differentially expressed genes in blue and red light treatments were enriched in the pathways of secondary metabolite biosynthesis, carbon fixation, and glycine, serine, and threonine metabolism, supporting the results of the metabolomic analysis. Ionomic analysis indicated that the element levels in fruits were more susceptible to changes in light quality than in leaves. The concentration of some ions containing Fe in fruits increased under red light compared to under blue light. The altered expression level of genes encoding metal ion-binding proteins, metal tolerance proteins, and metal transporters in response to blue and red light in the transcriptomic analysis contributes to changes in the ionomic profiles of tomato fruit.

Characterization of the FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 Homolog SlFKF1 in Tomato as a Model for Plants with Fleshy Fruit.

FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) is a blue-light receptor whose function is related to flowering promotion under long-day conditions in Arabidopsis thaliana. However, information about the physiological role of FKF1 in day-neutral plants and even the physiological role other than photoperiodic flowering is lacking. Thus, the FKF1 homolog SlFKF1 was investigated in tomato, a day-neutral plant and a useful model for plants with fleshy fruit. It was confirmed that SlFKF1 belongs to the FKF1 group by phylogenetic tree analysis. The high sequence identity with A. thaliana FKF1, the conserved amino acids essential for function, and the similarity in the diurnal change in expression suggested that SlFKF1 may have similar functions to A. thaliana FKF1. CONSTANS (CO) is a transcription factor regulated by FKF1 and is responsible for the transcription of genes downstream of CO. cis-Regulatory elements targeted by CO were found in the promoter region of SINGLE FLOWER TRUSS (SFT) and RIN, which are involved in the regulation of flowering and fruit ripening, respectively. The blue-light effects on SlFKF1 expression, flowering, and fruit lycopene concentration have been observed in this study and previous studies. It was confirmed in RNA interference lines that the low expression of SlFKF1 is associated with late flowering with increased leaflets and low lycopene concentrations. This study sheds light on the various physiological roles of FKF1 in plants.

Characterization of FLOWERING LOCUS C Homologs in Apple as a Model for Fruit Trees.

To elucidate the molecular mechanism of juvenility and annual flowering of fruit trees, FLOWERING LOCUS C (FLC), an integrator of flowering signals, was investigated in apple as a model. We performed sequence and expression analyses and transgenic experiments related to juvenility with annual flowering to characterize the apple FLC homologs MdFLC. The phylogenetic tree analysis, which included other MADS-box genes, showed that both MdFLC1 and MdFLC3 belong to the same FLC group. MdFLC1c from one of the MdFLC1 splice variants and MdFLC3 contain the four conserved motives of an MIKC-type MADS protein. The mRNA of variants MdFLC1a and MdFLC1b contain intron sequences, and their deduced amino acid sequences lack K- and C-domains. The expression levels of MdFLC1a, MdFLC1b, and MdFLC1c decreased during the flowering induction period in a seasonal expression pattern in the adult trees, whereas the expression level of MdFLC3 did not decrease during that period. This suggests that MdFLC1 is involved in flowering induction in the annual growth cycle of adult trees. In apple seedlings, because phase change can be observed in individuals, seedlings can be used for analysis of expression during phase transition. The expression levels of MdFLC1b, MdFLC1c, and MdFLC3 were high during the juvenile phase and low during the transitional and adult phases. Because the expression pattern of MdFLC3 suggests that it plays a specific role in juvenility, MdFLC3 was subjected to functional analysis by transformation of Arabidopsis. The results revealed the function of MdFLC3 as a floral repressor. In addition, MdFT had CArG box-like sequences, putative targets for the suppression of flowering by MdFLC binding, in the introns and promoter regions. These results indicate that apple homologs of FLC, which might play a role upstream of the flowering signals, could be involved in juvenility as well as in annual flowering. Apples with sufficient genome-related information are useful as a model for studying phenomena unique to woody plants such as juvenility and annual flowering.

From miracle fruit to transgenic tomato: mass production of the taste-modifying protein miraculin in transgenic plants.

The utility of plants as biofactories has progressed in recent years. Some recombinant plant-derived pharmaceutical products have already reached the marketplace. However, with the exception of drugs and vaccines, a strong effort has not yet been made to bring recombinant products to market, as cost-effectiveness is critically important for commercialization. Sweet-tasting proteins and taste-modifying proteins have a great deal of potential in industry as substitutes for sugars and as artificial sweeteners. The taste-modifying protein, miraculin, functions to change the perception of a sour taste to a sweet one. This taste-modifying function can potentially be used not only as a low-calorie sweetener but also as a new seasoning that could be the basis of a new dietary lifestyle. However, miraculin is far from inexpensive, and its potential as a marketable product has not yet been fully developed. For the last several years, biotechnological production of this taste-modifying protein has progressed extensively. In this review, the characteristics of miraculin and recent advances in its production using transgenic plants are summarized, focusing on such topics as the suitability of plant species as expression hosts, the cultivation method for transgenic plants, the method of purifying miraculin and future advances required to achieve industrial use.

Uniform accumulation of recombinant miraculin protein in transgenic tomato fruit using a fruit-ripening-specific E8 promoter.

The E8 promoter, a tomato fruit-ripening-specific promoter, and the CaMV 35S promoter, a constitutive promoter, were used to express the miraculin gene encoding the taste-modifying protein in tomato. The accumulation of miraculin protein and mRNA was compared among transgenic tomatoes expressing the miraculin gene driven by these promoters. Recombinant miraculin protein predominantly accumulated in transgenic tomato lines using the E8 promoter (E8-MIR) only at the red fruit stage. The accumulations were almost uniform among all fruit tissues. When the 35S promoter (35S-MIR) was used, miraculin accumulation in the exocarp was much higher than in other tissues, indicating that the miraculin accumulation pattern can be regulated by using different types of promoters. We also discuss the potential of the E8-MIR lines for practical use.

High-level accumulation of recombinant miraculin protein in transgenic tomatoes expressing a synthetic miraculin gene with optimized codon usage terminated by the native miraculin terminator.

In our previous study, a transgenic tomato line that expressed the MIR gene under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator (tNOS) produced the taste-modifying protein miraculin (MIR). However, the concentration of MIR in the tomatoes was lower than that in the MIR gene's native miracle fruit. To increase MIR production, the native MIR terminator (tMIR) was used and a synthetic gene encoding MIR protein (sMIR) was designed to optimize its codon usage for tomato. Four different combinations of these genes and terminators (MIR-tNOS, MIR-tMIR, sMIR-tNOS and sMIR-tMIR) were constructed and used for transformation. The average MIR concentrations in MIR-tNOS, MIR-tMIR, sMIR-tNOS and sMIR-tMIR fruits were 131, 197, 128 and 287 µg/g fresh weight, respectively. The MIR concentrations using tMIR were higher than those using tNOS. The highest MIR accumulation was detected in sMIR-tMIR fruits. On the other hand, the MIR concentration was largely unaffected by sMIR-tNOS. The expression levels of both MIR and sMIR mRNAs terminated by tMIR tended to be higher than those terminated by tNOS. Read-through mRNA transcripts terminated by tNOS were much longer than those terminated by tMIR. These results suggest that tMIR enhances mRNA expression and permits the multiplier effect of optimized codon usage.

Flavonoids in the flowers of Primula ×polyantha Mill. and Primula primulina (Spreng.) H. Hara (Primulaceae).

Two undescribed anthocyanins and two undescribed flavonols were isolated from the flowers of Primula ×polyantha Mill., along with five known anthocyanins and four known flavonols. The two undescribed anthocyanins and the two undescribed flavonols were determined to be hirsutidin 3-O-ß-galactopyranoside-5-O-ß-glucopyranoside, 7-O-methyl-petunidin 3-O-ß-galactopyranoside-5-O-ß-glucopyranoside, quercetin 3-O-ß-[(6""-acetylglucopyranosyl)-(1 â†’ 2)-ß-glucopyranosyl-(1 â†’ 6)-ß-glucopyranoside], and kaempferol 3-O-ß-[(6""-acetylglucopyranosyl)-(1 â†’ 2)-ß-glucopyranosyl-(1 â†’ 6)-ß-glucopyranoside] using chemical and spectroscopic methods. They were also found in the flowers of the Himalayan wild species, Primula primulina (Spreng.) H. Hara except for quercetin 3-O-ß-[(6""-acetylglucopyranosyl)-(1 â†’ 2)-ß-glucopyranosyl-(1 â†’ 6)-ß-glucopyranoside]. The flower color variations of P. ×polyantha cultivars, reflected by the hue values (b*/a*) of the colors, were due to the glycosidic patterns in the anthocyanins and their concentrations in the petals. Moreover, in the P. ×polyantha cultivars with violet-blue flowers, both the intermolecular copigmentation occurs between hirsutidin 3-O-ß-galactopyranoside-5-O-ß-glucopyranoside and another flavonol, quercetin 3-O-ß-glucopyranosyl-(1 â†’ 2)-ß-glucopyranosyl-(1 â†’ 6)-ß-glucopyranoside. Moreover, the flower color variation was affected by the pH value.

Characterization of PcLEA14, a Group 5 Late Embryogenesis Abundant Protein Gene from Pear (Pyrus communis).

Fruit trees need to overcome harsh winter climates to ensure perennially; therefore, they are strongly influenced by environmental stress. In the present study, we focused on the pear homolog PcLEA14 belonging to the unique 5C late embryogenesis abundant (LEA) protein group for which information is limited on fruit trees. PcLEA14 was confirmed to belong to this protein group using phylogenetic tree analysis, and its expression was induced by low-temperature stress. The seasonal fluctuation in its expression was considered to be related to its role in enduring overwinter temperatures, which is particularly important in perennially. Moreover, the function of PcLEA14 in low-temperature stress tolerance was revealed in transgenic Arabidopsis. Subsequently, the pear homolog of dehydration-responsive element-binding protein/C-repeat binding factor1 (DREB1), which is an important transcription factor in low-temperature stress tolerance and is uncharacterized in pear, was analyzed after bioinformatics analysis revealed the presence of DREB cis-regulatory elements in PcLEA14 and the dormancy-related gene, both of which are also expressed during low temperatures. Among the five PcDREBs, PcDREB1A and PcDREB1C exhibited similar expression patterns to PcLEA14 whereas the other PcDREBs were not expressed in winter, suggesting their different physiological roles. Our findings suggest that the low-temperature tolerance mechanism in overwintering trees is associated with group 5C LEA proteins and DREB1.

Characterization and Expression Analysis of the Ca2+/Cation Antiporter Gene Family in Tomatoes.

The Ca2+/cation antiporter (CaCA) superfamily plays an important role in the regulation of the essential element Ca2+ and cation concentrations. Characterization and expression analyses of CaCA superfamily genes were performed in the tomato (Solanum lycopersicum) as a representative of dicotyledonous plants and fruit crops. Sixteen CaCA candidate genes were found and identified as tomato CaCA, SlCaCA, by a domain search. In a phylogenetic analysis of the SlCaCA superfamily, the 16 genes were classified into SlCAX, SlNCL, SlCCX, and SlMHX families. Among them, Solyc12g011070, belonging to the SlCAX family, had four splice variants, three of which were predicted to be nonfunctional because of a lack of important motifs. EF-hand domains were only found in SlNCL, in addition to consensus Na_Ca_ex domains, and the region containing EF-hand domains was characteristically long in some members of SlNCL. Furthermore, four genes of the SlCCX family were found to be intronless. As for intracellular localization, one SlCCX member was predicted to be localized to the plasma membrane, while other SlCCXs, SlCAXs, and SlMHXs were predicted to be localized to the vacuolar membrane. The expression patterns of SlCaCAs in various organs, including during several developmental stages of fruit, were classified into four groups. Genes involved in each of the SlCAX, SlNCL, and SlCCX gene families were categorized into three or four groups according to expression patterns, suggesting role sharing within each family. The main member in each subfamily and the members with characteristic fruit expression patterns included genes whose expression was regulated by sugar or auxin and that were highly expressed in a line having metabolite-rich fruit.

Acylated cyanidin 3-sophoroside-5-glucoside in purple-violet flowers of Moricandia arvensis (Brassicaceae).

A new acylated anthocyanin was isolated as a major pigment, along with a known anthocyanin (Moricandia arvensis anthocyanin 1: MAA-1), from a strain of Moricandia arvensis (Code No. MOR-ARV-3) with purple-violet flowers, and identified as cyanidin 3-O-[2-O-(2-O-(4-O-(6-O-(4-O-(ß-glucopyranosyl)-trans-caffeoyl)-ß-glucopyranosyl)-trans-sinapoyl)-ß-glucopyranoside]-5-O-[6-O-(malonyl)-ß-glucopyranoside].

Triacylated peonidin 3-sophoroside-5-glucosides from the purple flowers of Moricandia ramburii Webb.

Triacylated peonidin 3-sophoroside-5-glucosides were isolated from the purple flowers of Moricandia ramburii Webb. (Family: Brassicaceae), and determined to be peonidin 3-O-[2-O-(2-O-(trans-feruloyl)-glucosyl)-6-O-(trans-p-coumaroyl)-glucoside]-5-O-[6-O-(malonyl)-glucoside] (1), peonidin 3-O-[2-O-(2-O-(trans-feruloyl)-glucosyl)-6-O-(cis-p-coumaroyl)-glucoside]-5-O-[6-O-(malonyl)-glucoside] (2) and peonidin 3-O-[2-O-(2-O-(trans-sinapoyl)-glucosyl)-6-O-(trans-p-coumaroyl)-glucoside]-5-O-[6-O-(malonyl)-glucoside] (3), respectively, by chemical and spectroscopic methods. In addition, one known acylated cyanidin glycoside, cyanidin 3-O-[2-O-(2-O-(trans-feruloyl)-glucosyl)-6-O-(trans-p-coumaroyl)-glucoside]-5-O-[6-O-(malonyl)-glucoside] (4), was also identified in the flowers. Peonidin glycosides have not been reported hitherto in floral tissues in to Brassicaceae.

Covalent anthocyanin-flavonol complexes from the violet-blue flowers of Allium 'Blue Perfume'.

Three covalent anthocyanin-flavonol complexes (pigments 1-3) were extracted from the violet-blue flower of Allium 'Blue Perfume' with 5% acetic acid-MeOH solution, in which pigment 1 was the dominant pigment. These three pigments are based on delphinidin 3-glucoside as their deacylanthocyanin and were acylated with malonyl kaempferol 3-sophoroside-7-glucosiduronic acid or malonyl-kaempferol 3-p-coumaroyl-tetraglycoside-7-glucosiduronic acid in addition to acylation with acetic acid. By spectroscopic and chemical methods, the structures of these three pigments 1-3 were determined to be: pigment 1, (6(I)-O-(delphinidin 3-O-(3(I)-O-(acetyl)-ß-glucopyranoside(I))))(2(VI)-O-(kaempferol 3-O-(2(II)-O-(3(III)-O-(ß-glucopyranosyl(V))-ß-glucopyranosyl(III))-4(II)-O-(trans-p-coumaroyl)-6(II)-O-(ß-glucopyranosyl(IV))-ß-glucopyranoside(II))-7-O-(ß-glucosiduronic acid(VI)))) malonate; pigment 2, (6(I)-O-(delphinidin 3-O-(3(I)-O-(acetyl)-ß-glucopyranoside(I))))(2(VI)-O-(kaempferol 3-O-(2(II)-O-ß-glucopyranosyl(III))-ß-glucopyranoside(II))-7-O-(ß-glucosiduronic acid(VI)))); and pigment 3, (6(I)-O-(delphinidin 3-O-(3(I)-O-(acetyl)-ß-glucopyranoside(I))))(2(VI)-O-(kaempferol 3-O-(2(II)-O-(3(III)-O-(ß-glucopyranosyl(V))-ß-glucopyranosyl(III))-4(II)-O-(cis-p-coumaroyl)-6(II)-O-(ß-glucopyranosyl(IV))-ß-glucopyranoside(II))-7-O-(ß-glucosiduronic acid(VI)))) malonate. The structure of pigment 2 was analogous to that of a covalent anthocyanin-flavonol complex isolated from Allium schoenoprasum where delphinidin was observed in place of cyanidin. The three covalent anthocyanin-flavonol complexes (pigment 1-3) had a stable violet-blue color with three characteristic absorption maxima at 540, 547 and 618nm in pH 5-6 buffer solution. From circular dichroism measurement of pigment 1 in the pH 6.0 buffer solution, cotton effects were observed at 533 (+), 604 (-) and 638 (-) nm. Based on these results, these covalent anthocyanin-flavonol complexes were presumed to maintain a stable intramolecular association between delphinidin and kaempferol units closely related to that observed between anthocyanin and hydroxycinnamic acid residues in polyacylated anthocyanins. Additionally, an acylated kaempferol glycoside (pigment 4) was isolated from the same flower extract, and its structure was determined to be kaempferol 3-O-sophoroside-7-O-(3-O-(malonyl)-ß-glucopyranosiduronic acid).

The HSP terminator of Arabidopsis thaliana induces a high level of miraculin accumulation in transgenic tomatoes.

High-level accumulation of the target recombinant protein is a significant issue in heterologous protein expression using transgenic plants. Miraculin, a taste-modifying protein, was accumulated in transgenic tomatoes using an expression cassette in which the miraculin gene was expressed by the cauliflower mosaic virus (CaMV) 35S promoter and the heat shock protein (HSP) terminator (MIR-HSP). The HSP terminator was derived from heat shock protein 18.2 in Arabidopsis thaliana . Using this HSP-containing cassette, the miraculin concentration in T0 transgenic tomato lines was 1.4-13.9% of the total soluble protein (TSP), and that in the T1 transgenic tomato line homozygous for the miraculin gene reached 17.1% of the TSP. The accumulation level of the target protein was comparable to levels observed with chloroplast transformation. The high-level accumulation of miraculin in T0 transgenic tomato lines achieved by the HSP terminator was maintained in the successive T1 generation, demonstrating the genetic stability of this accumulation system.

A trial of production of the plant-derived high-value protein in a plant factory: photosynthetic photon fluxes affect the accumulation of recombinant miraculin in transgenic tomato fruits.

One of the ultimate goals of plant science is to test a hypothesis obtained by basic science and to apply it to agriculture and industry. A plant factory is one of the ideal systems for this trial. Environmental factors affect both plant yield and the accumulation of recombinant proteins for industrial applications within transgenic plants. However, there have been few reports studying plant productivity for recombinant protein in closed cultivation systems called plant factories. To investigate the effects of photosynthetic photon flux (PPF) on tomato fruit yield and the accumulation of recombinant miraculin, a taste-modifying glycoprotein, in transgenic tomato fruits, plants were cultivated at various PPFs from 100 to 400 (µmol m(-2) s(-)1) in a plant factory. Miraculin production per unit of energy used was highest at PPF100, although miraculin production per unit area was highest at PPF300. The commercial productivity of recombinant miraculin in transgenic tomato fruits largely depended on light conditions in the plant factory. Our trial will be useful to consider the trade-offs between the profits from production of high-value materials in plants and the costs of electricity.

The development of chronic kidney disease in Japanese patients with non-alcoholic fatty liver disease.

Objective Chronic kidney disease (CKD) is present in patients with nonalcoholic fatty liver disease (NAFLD). The aim of this retrospective study was to assess the cumulative development incidence and predictive factors for new onset of CKD in Japanese patients with NAFLD. Methods A total of 5,561 NAFLD patients without CKD were enrolled. CKD was defined as either an estimated glomerular filtration rate of <60 mL/min/1.73 m(2) or dipstick proteinuria (≥+1). A blood sample and a urine sample were taken for routine analyses during follow-up. The mean observation period was 5.5 years. The primary goal is the new development of CKD. Independent factors associated with new development of CKD were analyzed by using the Kaplan-Meyer method and the Cox proportional hazards model. Results Of 5.561 NAFLD patients, 263 patients developed CKD. The cumulative development rate of CKD was 3.1% at the 5th year and 12.2% at the 10th year. Multivariate Cox proportional hazards analysis showed that CKD development in patients with NAFLD occurred when patient had low level of GFR of 60-75 mL/min/1.73 m(2) [hazard ratio:2.75; 95% confidence interval (CI)=1.93-3.94; p<0.001], age of ≥50 years (hazard ratio: 2.67; 95% CI=2.06-3.46; p<0.001), diabetes (hazard ratio: 1.92; 95% CI=1.45-2.54; p<0.001), hypertension (hazard ratio: 1.69; 95% CI=1.25-2.29; p<0.001), and elevated serum gamma-glutamyltransferase of ≥109 IU/L (hazard ratio: 1.35; 95% CI=1.02-1.78; p=0.038). Conclusion Our retrospective study indicates that the annual incidence of CKD in Japanese patients with NAFLD is about 1.2%. Five factors of low eGFR level, aging, type 2 diabetes, hypertension, and elevated gamma-glutamyltransferase, increases the risk of the development of CKD.

Involvement of nitric oxide in the inhibition of nitrogenase activity by nitrate in Lotus root nodules.

Nitrogenase activity, as acetylene-reduction activity (ARA), in Lotus root nodules was clearly inhibited 27h after the addition of nitrate. Nitric oxide (NO) production was detected at that time in nitrate-supplied root nodules using the NO-reactive fluorescent probe diaminofluorescein-2 diacetate. The involvement of NO production in the inhibition of nitrogenase activity by nitrate was investigated using the NO donor sodium nitroprusside (SNP) and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO). SNP inhibited ARA at 1mM, and c-PTIO suppressed the inhibition of ARA by nitrate. These results suggest that NO is involved in the inhibition of nitrogenase activity by nitrate in Lotus root nodules.

Spatial and developmental profiling of miraculin accumulation in transgenic tomato fruits expressing the miraculin gene constitutively.

We previously developed a transgenic tomato that expresses the miraculin gene using a constitutive promoter. In this study, we profiled the developmental and spatial accumulation of the miraculin protein and mRNA in transgenic tomato fruits. Miraculin mRNA expression was almost constant up to orange stage, and then the expression increased at red stage. The miraculin protein accumulated gradually during fruit development and reached its highest level at the overripe stage. At the red stage of fruit, miraculin protein was accumulated at the highest level in the exocarp, and similar in other fruit tissues: mesocarp, dissepiment, upper placenta, lower placenta and jelly. Moreover, the pattern of miraculin accumulation in fruit tissues was the same regardless of genetic background and position at which the miraculin gene was inserted in the genome. We also discuss suitable tomato types expressing miraculin for their commercial use.

Molecular breeding of tomato lines for mass production of miraculin in a plant factory.

A transgenic tomato line (56B, "Moneymaker") that expresses the miraculin gene driven by the CaMV 35S promoter was crossed with a dwarf tomato ("Micro-Tom") for the molecular breeding of cultivars that are suitable for miraculin production in a closed cultivation system. Plant size, miraculin accumulation, and self-pruning growth were used as selection indicators for F2 plants. Two lines were chosen for further analysis, bred to the F6 or F7 generation and cultivated in a closed cultivation system. In 56B and the two crossed lines, the concentrations of miraculin in the pericarp were 140, 367, and 343 microg/g FW, respectively. We also estimated that 26.2, 73.6, and 45.9 kg FW/m2 of tomatoes and 2.2, 16.6, and 9.8 mg/m2 of miraculin in the pericarp, respectively, could be harvested per year. These two crossed lines will be useful for the mass production of miraculin, especially in a closed cultivation system.

Nitrate-independent expression of plant nitrate reductase in Lotus japonicus root nodules.

Nitrate-independent nitrate reductase (NR) activity is generally found in legume root nodules. Therefore, the effects of nitrate on plant NR activity and mRNA were investigated in the root nodules of Lotus japonicus (L. japonicus). Both NR activity and mRNA levels in roots and root nodules were up-regulated by the addition of nitrate. In the absence of nitrate, NR activity and mRNA were detected in root nodules but not in roots. Southern blotting analysis indicates that NR is encoded by a single gene in L. japonicus. No nitrate was detected in the root nodules or roots of plants grown in the absence of nitrate, while its accumulation was observed in plants supplied with exogenous nitrate. These results indicate that inducible-type NR can be expressed in root nodules in the absence of nitrate. The activation state of the nitrate-independent activity of NR was as high as that of NR activity induced by nitrate. NR mRNA expressed independently of nitrate in root nodules without nitrate was localized in the infected regions of the root nodules. Thus, the expression could be related to the specific structure and environment of root nodules.