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DOI： 10.1007/s13562-019-00487-2

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It has been reported that supply of nitrate to culture solution rapidly and reversibly inhibits nodule growth and nitrogen fixation activity of soybean. In this study, the effects of ammonium, urea, or glutamine on nodule growth and nitrogen fixation activity are compared with that for nitrate. Soybean plants were cultivated with a nitrogen-free nutrient solution, then 1 mM-N of nitrate, ammonium, glutamine, or urea were supplied from 12 DAP until 17 DAP. Repression of nodule growth and nitrogen fixation activity at 17 DAP were observed by ammonium, urea, and glutamine like nitrate, although the inhibitory effects were milder than nitrate. The removal of nitrogen from the culture solutions after nitrogen treatments resulted in a recovery of the nodule growth. It was found that the glutamine treatment followed by N-free cultivation gave highest nitrogen fixation activity about two times of the control. Tracer experiments with 15N and 13C were performed to evaluate the translocation of N and C to the different tissues. Culture solutions containing a 15N-labeled nitrogen source were supplied from 21 DAP, and the whole shoots were exposed to 13CO2 for 60 min on 23 DAP, and plants were harvested on 24 DAP. The percentage distribution of 15N in nodules was highest for ammonium (1.4%) followed by glutamine (0.78%), urea (0.32%) and nitrate (0.25%). The percentage distribution of 13C in the nodules was highest for the control (11.5%) followed by urea (5.8%), glutamine (2.6%), ammonium (2.3%), and nitrate (2.3%). The inhibitory effects of nitrogen compounds appeared to be related to a decrease in photoassimilate partitioning in the nodules, rather than 15N transport into the nodules. The free amino acid concentrations after nitrogen treatments were increased in the nodules and leaves by nitrate, in the roots by ammonium, in the stems by urea, and the roots, stems, and leaves by glutamine treatment. The concentrations of asparagine, aspartate, and glutamine were increased after nitrogen treatments. By the long-term supply of nitrogen for 2-weeks, nitrate significantly increased the lateral roots and leaf growth. The long-term supply of urea and glutamine also promoted the lateral roots and leaf growth, but ammonium suppressed them.

DOI： 10.3389/fpls.2019.00131

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DOI： 10.1007/s13580-018-0071-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Leguminous plants form root nodules with rhizobia that fix atmospheric dinitrogen (N2) for the nitrogen (N) nutrient. Combined nitrogen sources, particular nitrate, severely repress nodule growth and nitrogen fixation activity in soybeans (Glycine max [L.] Merr.). A microarray-based transcriptome analysis and the metabolome analysis were carried out for the roots and nodules of hydroponically grown soybean plants treated with 5 mM of nitrate for 24 h and compared with control without nitrate. Gene expression ratios of nitrate vs. the control were highly enhanced for those probesets related to nitrate transport and assimilation and carbon metabolism in the roots, but much less so in the nodules, except for the nitrate transport and asparagine synthetase. From the metabolome analysis, the concentration ratios of metabolites for the nitrate treatment vs. the control indicated that most of the amino acids, phosphorous-compounds and organic acids in roots were increased about twofold in the roots, whereas in the nodules most of the concentrations of the amino acids, P-compounds and organic acids were decreased while asparagine increased exceptionally. These results may support the hypothesis that nitrate primarily promotes nitrogen and carbon metabolism in the roots, but mainly represses this metabolism in the nodules.

DOI： 10.3390/plants7020032

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

An adequate supply of nitrogen (N) is important for patumma growth and flower quality. This study aimed to compare the uptake and translocation of N by foliar and root application. Fertilization with (15) nitrate (NO3)-N via roots or leaves was carried out at four stages, at the 1st to 4th fully expanded leaf (FEL) stages, and the plants were sampled at each successive stage. The uptake and translocation of N-15 from foliar or root applications showed relatively similar patterns at all stages. Although the N fertilizer utilization rate by roots was higher than that via leaves, the foliar application stimulated reproductive growth by earlier flowering. The N supplied at the 1st FEL and the 2nd FEL was utilized mainly in leaves, whereas supplying N at the 3rd and 4th FEL promoted flower quality. Fertilizer application method and stage of application influence the utilization rate and translocation of N to the sink organs.

DOI： 10.1080/01904167.2016.1264597

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HEADLEY BROTHERS LTD  

Curcuma alismatifolia rhizomes are exported to world markets for ornamental use. Rhizome size and the number of storage roots affected shoot sprouting, plant growth, and inflorescence yield and quality, indicating an important role for storage food reserves such as the carbohydrates and proteins in these organs. An understanding of the proteins used for growth and development in rhizomes and in storage roots is lacking, so changes in the storage proteins in these organs were investigated at the planting stage, the sprouting stage, the second-leaf stage, the flowering stage, and the dormancy stage. The protein concentrations in both these organs decreased rapidly until the flowering stage in order to support the initial growth of roots and shoots. The intensity of Coomassie Brilliant Blue staining of tissues was stronger in rhizomes than in storage roots. Protein staining was present in the cytosol and in the cell walls in rhizomes, and was intense in small particle-like protein bodies in the cytosol. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles showed that peptides of 10.6 kDa and 12.0 kDa were the two major soluble proteins in rhizomes and were different from the proteins in storage roots. The 10.6 kDa and 12.0 kDa bands contained five peptides and one peptide, respectively, when separated by 2D-PAGE. The amino acid sequences of these six peptides were not homologous to any known proteins using BLASTP or FASTA analysis. The N-terminal amino acid sequences of these two polypeptides did not start with methionine, indicating that they had undergone posttranslational modification. Knowledge of the major storage proteins in the rhizomes and storage roots of C. alismatifolia may provide information to improve N-fertilisation.

DOI： 10.1080/14620316.2014.11513112

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The application of combined nitrogen, especially nitrate, to soybean plants is known to strongly inhibit nodule formation, growth and nitrogen fixation. In the present study, we measured the effects of supplying 5 mM nitrate on the growth of nodules, primary root, and lateral roots under light at 28 degrees C or dark at 18 degrees C conditions. Photographs of the nodulated roots were periodically taken by a digital camera at 1-h intervals, and the size of the nodules was measured with newly developed computer software. Nodule growth was depressed approximately 7 h after the addition of nitrate under light conditions. The nodule growth rate under dark conditions was almost half that under light conditions, and nodule growth was further suppressed by the addition of 5 mM nitrate. Similar results were observed for the extending growth rate of the primary root as those for nodule growth supplied with 5 mM nitrate under light/dark conditions. In contrast, the growth of lateral roots was promoted by the addition of 5 mM nitrate. The 2D-PAGE profiles of nodule protein showed similar patterns between the 0 and 5 mM nitrate treatments, which suggested that metabolic integrity may be maintained with the 5 mM nitrate treatment. Further studies are required to confirm whether light or temperature condition may give the primary effect on the growth of nodules and roots.

DOI： 10.3390/ijms15034464

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The quantitative analysis of the initial transport of fixed isotope 15-nitrogen (N-15) in intact nodulated soybean plants (Glycine max [L.] Merr. cv. Williams) was investigated at the vegetative stage (36 days after planting, DAP) and pod-filling stage (91 DAP) by the N-15 pulse-chase experiment. The nodulated roots were exposed to N-2 gas labeled with a stable isotope N-15 for 1h, followed by 0, 1, 3 and 7h of exposure with normal air. Plant roots and shoots were separated into three sections (basal, middle and distal parts) with the same length of the main stem or primary root. Approximately 80 and 92% of fixed N was distributed in the basal part of the nodulated roots at the vegetative and pod-filling stages by the end of 1h of N-15(2) exposure, respectively. In addition, about 90% of fixed N-15 was retained in the nodules and 10% was exported to root and shoot after 1 h of N-15(2) exposure at 91 DAP. The percentage distribution of N-15 in the nodules at the pod-filling stage decreased from 90% to 7% during the 7h of the chase period, and increased in the roots (14%), stems (54%), leaves (12%), pods (10%) and seeds (4%). The N-15 distribution was negligible in the distal root segment, suggesting that N fixation activity was negligible and recycling fixed N from the shoot to the roots was very low in the initially short time of the experiment.

DOI： 10.1080/00380768.2013.838742

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Tulip (Tulipa gesneriana, cultivar Oxford) bulbs were planted in October and grown in culture solution in a glasshouse under natural conditions. One group was cultivated in a solution containing nitrogen (N) (+N treatment: nitrate-N (NO3--N) 12.0, ammonium-N (NH4+-N) 3.0 mg L-1). Another group was cultivated in a nitrogen-free solution (-N treatment). In the first experiment, the transverse accumulation of free amino acids in the cortex and stele of the tulip roots was measured. In the cortex, the total concentration of free amino acids amounted to 39.4 mg Ng(-1) dry weight (DW) in the +N treatment, but only 5.3 mg Ng(-1) DW in the -N treatment. In the stele, the concentration was 14.4 and 5.8 mg Ng(-1) DW, respectively. Glutamine was the predominant amino acid in the cortex in the +N treatment, reaching 18.1 mg Ng(-1) DW. The concentrations of asparagine and arginine were also higher after the +N than -N treatment. On the other hand, the concentration of 4-methyleneglutamine was not so different between the treatments in either the cortex or stele. In the second experiment, the accumulation of free amino acids at five vertical positions from the base to root tip was analyzed. The total concentration of free amino acids was not so different among the positions. According to statistics, the concentrations of glutamine and asparagine were relatively constant between the +/-N treatments. In the -N treatment, the concentration of 4-methyleneglutamine gradually decreased from the base toward the root tip, and it tended to be higher in the -N treatment than that in the +N treatment.

DOI： 10.1080/00380768.2012.669352

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The balance between carbon (C) and nitrogen (N) availability is an important determinant for various phases of plant growth; however, the detailed mechanisms regulating the C/N response are not well understood. We previously described two related ubiquitin ligases, ATL31 and ATL6, that function in the C/N response in Arabidopsis thaliana. Here, we used FLAG tag affinity purification and MS analysis to identify proteins targeted by ATL31, and thus likely to be involved in regulating the phase transition checkpoint based on C/N status. This analysis revealed that 14-3-3 proteins were associated with ATL31, and one of these, 14-3-3 chi, was selected for detailed characterization. The interaction between ATL31 and 14-3-3 chi was confirmed by yeast two-hybrid and co-immunoprecipitation analyses. In vitro assays showed that ubiquitination of 14-3-3 chi is catalyzed by ATL31. Degradation of 14-3-3 chi in vivo was shown to be correlated with ATL31 activity, and to occur in a proteasome-dependent manner. Furthermore, 14-3-3 protein accumulation was induced by a shift to high-C/N stress conditions in Arabidopsis seedlings, and this regulated response required both ATL31 and ATL6. It was also shown that over-expression of 14-3-3 chi leads to hypersensitivity of Arabidopsis seedlings to C/N stress conditions. These results indicate that ATL31 targets and ubiquitinates 14-3-3 proteins for degradation via the ubiquitin-proteasome system during the response to cellular C/N status.

DOI： 10.1111/j.1365-313X.2011.04673.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Society for Horticultural Science  

Curcuma alismatifolia Gagnep. rhizomes were planted on 15 May 2007. At 10 weeks after planting, at the termination of the new leaf formation stage, the plants were supplied with 15N-labeled culture solution (3.81 mM NH4 + + 8.96 mM NO3 -: 10.6 atom% 15N) twice at 3 and 1 days before 13CO2 feeding. Then 13C-labeled 13CO2 (99 atom% 13C) was exposed to the shoot. The plants were harvested at four different stages of growth. The 15N and 13C abundance in each part were determined to investigate the characteristics of N and C translocation and distribution in curcuma. The total dry weights of the plant increased continuously during the termination of the new leaf formation stage (stage 1), the flowering stage (stage 2) and the rhizome storage period (stage 3) and especially rapidly increased during stage 2 to stage 3. The DW was relatively constant thereafter until the start of the dormancy period (stage 4). The total N content of the plant rapidly increased after stage 2 until stage 4. At stage 1, the total N was distributed mostly to the old storage roots, leaves and old rhizome just as the dry weight. At stage 2, the dry weights of new organs were mostly increased as well as the N content. At stage 3 and stage 4, the total N content of the new storage organs (new rhizomes and new storage roots) was highly increased, particularly in the new rhizome. The old rhizome and the old storage roots gave very low 15N and 13C in abundance. At stage 1, leaves are the main N sink and about 5.17 mg labeled N (LN) assimilated to this organ. In this stage, translocation of the newly assimilated N to the new rhizome already started. At stage 2, the distribution of LN was increased in flower and flower stalk but it was less than in the leaves that are also the main sink of N organ. At stage 3 the distribution of LN to the aboveground part organs (leaves, flowers and flower stalks) was decreased. This contrasted to the underground part organs (new rhizome, new storage roots and fibrous roots) where LN increased especially in the new rhizome. At stage 4, the 15N was mostly distributed to the storage organ (new rhizome and new storage roots) about 8.85 mg LN and 3.59 mg LN, respectively.

DOI： 10.17660/ActaHortic.2011.886.16

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Society for Horticultural Science  

Freesia corms were grown in substrate culture using sand: rice husk-charcoal: coconut fiber at the ratio of 1:1:1. The optimum levels of NPK in the nutrient solution were studied using different combinations of nitrogen at 100 and 200 mg/L, phosphorus at 50 and 100 mg/L combined with potassium at 100, 200 and 300 mg/L. The results showed that the minimum level of NPK at 100:50:200 mg/L was good enough for growing Freesia in substrate culture. Tubers of Gloriosa rothschildiana (10-25 g FW) were grown in substrate culture. The -N, -P, -K, -Mg and -Ca treatments compared with complete nutrient formula were supplied to the tubers. The results showed that nutrient deficiency reduced plant height, number of nodes, tuber fresh weight and tuber length. The optimum level of NPK was further studied by supplying tubers with different combinations of nitrogen at 100, 200 and 300 mg/L , phosphorus at 50 and 100 mg/L combined with potassium at 100, 200 and 300 mg/L. The results showed that the NPK combination at 200:100:200 mg/L was optimal for growing Gloriosa rothschildiana in substrate culture.

DOI： 10.17660/ActaHortic.2011.886.29

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Curcuma or Siam tulip (Curcuma alismatifolia Gagnep.) is an ornamental flowering plant with two underground storage organs, rhizomes and storage roots. Characteristics of N and C assimilation and transport in curcuma were investigated. The plants were treated with 15NH4+ + 15NO3- and 13CO2 at 10, 13 or 21 weeks after planting. Plants were sampled at several stages up to 32 weeks. The C stored in old storage roots was used rapidly during the first 10 weeks
after which N stored in old rhizomes and old storage roots were used. The daily gain in C depending on photosynthesis was remarkably high between 10 and 21 weeks. However, the daily gain in N was relatively constant throughout the growth period. The 15N absorbed at 10 weeks was initially accumulated in leaves and roots, but some was transported to flowering organs at 13 weeks. At harvest, 41% of 15N was recovered in new rhizomes and 17% in new storage roots. After 13CO2 exposure at 10 and 13 weeks, the distribution of 13C among organs was relatively constant in subsequent stages. When given 13CO2 at 21 weeks, a large amount of labelled C was recovered in new storage roots and new rhizomes at harvest. Both new rhizomes and new storage roots stored N and C, however, rhizomes played a more important role in supplying N, while storage roots provided C. © 2009 German Botanical Society and The Royal Botanical Society of the Netherlands.

DOI： 10.1111/j.1438-8677.2009.00229.x

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Rice (Oryza sativa cv. Koshihikari) seedlings were grown in a sandy dune soil in pots with a basal dressing of N (0.5 g N), P and K. Two N treatments were applied, a +N treatment in which a top dressing of 15N-labeled 0.5 g N was supplied on July 20 and a -N treatment in which no additional fertilizer was supplied. During the grain-filling stage from August 6 to 13, plants were subjected to one of three temperature treatments
controlled low temperature, LT (daynight 2823 °C), controlled high temperature, HT (3530 °C) and uncontrolled glasshouse temperature, UT (daynight averages, 3826 °C). All plants were then exposed to 13CO2 for 1 h on August 11 in a growth chamber at 25 °C. On August 13, all plants were harvested and the 13C and 15N abundances and starch and sugar concentrations in the ears, shoots and roots were determined. The 13C content of the ear was lower in UT than in LT irrespective of the +N or -N treatment. The translocation of 15N to the ears was also slightly depressed in UT compared with LT. Under high-temperature conditions (HT and UT), the starch content per plant was reduced for -N, but for +N, it was not significantly different among the temperature treatments. A high accumulation of sucrose was observed in all plant parts under UT conditions. It is suggested that extreme high day temperatures during the grain-filling period may reduce starch synthesis in the grains and, especially so under N-deficient conditions. High temperatures also induce an accumulation of sucrose and a decrease in carbon and nitrogen transport from the shoots to the ears via the phloem. © 2009 Blackwell Verlag GmbH.

DOI： 10.1111/j.1439-037X.2009.00376.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

A rapid and simple method for determination of cyanamide in fertilizer, soil and plants has been developed. In this method, cyanamide is extracted with 2% acetic acid and the extract separated by centrifugation. It is then purified by passing through a membrane filter. The extract was derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and the derivatized compound separated by ultra-high-pressure liquid chromatography. it is then detected with a UV detector at 260 nm by the same method as is used for amino acid analysis. The proposed method is fast, simple and cheap and also has good selectivity and sensitivity for the determination of cyanamide in a wide range of biotic and abiotic materials. (C) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.chroma.2009.05.067

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT CELL & MOLECULAR BIOL  

A high affinity transport system (HATS) for nitrate in plants is operated by a two-component NRT2/NAR2 transport system. However, the regulation and localization of NRT2 and NAR2 at protein level are largely unknown and especially so in crop plant species. In this study with barley (Hordeum vulgare), membrane localization, protein expression in the roots, and a direct protein-protein interaction of HvNRT2 and HvNAR2 proteins were investigated. Immunochemical analysis showed that both HvNRT2 and HvNAR2 proteins were co-localized in the plasma membrane of the roots. Expression of HvNRT2 and HvNAR2 proteins was more strongly induced by treatment with higher concentrations of external nitrate, while HATS activity and transcripts for HvNRT2 and HvNAR2 were markedly repressed. An affinity Column binding analysis using recombinant proteins suggests that the C-terminus of HvNRT2.1 is possibly involved in its binding to the HvNAR2.3 central region and that the Ser463 present in the HvNRT2.1 C-terminus plays a role in the binding ability.

DOI： 10.5511/plantbiotechnology.26.197

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE  

The tracer (15)N(2) was used to investigate sites of N(2) fixation and the possible translocation of the fixed N. Young sugarcane plants (Saccharum officinarum L.) from a stem cutting were exposed to (15)N(2)-labeled air in a 500 mL plastic cylinder. Plants fed (15)N(2) for 7 days were grown in normal air for a further chase period. After 21 days, about half of the N originating in the stem cutting had been transported to the shoot and roots, suggesting that the cutting played a role in supplying N for growth. After 3 days of feeding, the percentage of N derived from (15)N(2) was higher in the roots (2.22%) and stem cutting (0.271%) than the shoot (0.027%). Most of the fixed N was distributed in the 80% ethanol-insoluble fractions in each plant part, and the (15)N fixed either in the roots or in the stem cutting remained there and was not appreciably transported to the shoot. The results were quite different from the fate of fixed N in soybean nodules, which is rapidly transported from nodules to roots and shoots.

DOI： 10.1264/jsme2.ME09105

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

A novel Arabidopsis thaliana mutant that shows a low concentration of nitrate in shoots was isolated from an ethyl-methanesulfonate-mutagenized M-2 population. In the mutant, the nitrate concentration in the shoots was reduced to 45-61% of that in the wild-type plants when plants were grown hydroponically at a different range of external nitrate. When grown in solid media containing 5 mmol L-1 nitrate, the fresh weights of shoots and roots in the mutant were approximately 62% and 67%, respectively, compared with those of the wild type. The mutant did not show any difference in in vitro nitrate reductase activity from wild-type plants and net nitrate uptake in the mutant was 22% lower than that in wild-type plants. Genetic mapping analysis revealed that the mutation was mapped near the bottom of chromosome 1.

DOI： 10.1111/j.1747-0765.2007.00211.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Cucumber (Cucumis sativus L.) plants were grown with hydroponics in a greenhouse and the absorption and translocation of nitrogen in the plants were investigated after the first fruit harvest using N-15-labeled nitrogen. The N-15-labeled nutrient solution, which contained 13 mmol L-1 nitrate, was supplied for 7 days from first fruit harvest on 21 September, 2005. Following 7 days of N-15 treatment, these plants received the same composition of non-labeled nitrogen for 16 days (chase period). Commercial-sized fruits, approximately 20 cm in length, were harvested every day. The whole plants were harvested three times at the beginning (21 September) and at the end (28 September) of the N-15 treatment period, and at the end of the chase period on 14 October. The average nitrogen absorption rate was 196 mg plant(-1) per day during the N-15 treatment period and 181 mg plant(-1) per day during the chase period. The labeled nitrogen was detected in the fruits within 1 day of the N-15 treatment, and the percentage of nitrogen from labeled nitrogen in the harvested fruits increased up to approximately 50% at the end of the N-15 treatment period. The percentage of labeled nitrogen in the fruits quickly decreased after changing from N-15 solution to the non-labeled conditions during the chase period. At the end of the N-15 treatment period on 28 September, the percentage distribution of labeled nitrogen was 46% in the leaves, 24% in the harvested fruits, 13% in the stems, 9% in the roots and 9% in the small fruits. After 16 days of chase period, the distribution of labeled nitrogen decreased in the leaves (33%), stems (6.5%), roots (3.6%) and small fruits (1%), but 56% of labeled nitrogen was distributed in the harvested fruits. Based on the result obtained, the nitrate absorbed from the medium is rapidly transported to young fruits as well as to leaves and stems. The nitrogen once assimilated or stored in the leaves, stems and roots is gradually redistributed to the fruits for supporting their rapid growth. To maximize the yield of cucumber fruits, it is necessary to supply sufficient levels of nitrogen continuously after the first harvest.

DOI： 10.1111/j.1747-0765.2007.00213.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

To understand the autoregulation of nodulation (AON) system, in which leguminous plants control the nodule number, we examined the details of the characteristics of hypernodulation soybean mutants NOD1-3 and NOD3-7. A microscopic study showed that NOD1-3 and NOD3-7 produced small-size leaves due to the smaller number of leaf cells, compared with the Williams parent. These phenotypes were not affected by inoculation with bradyrhizobia or nitrate supply. The AON signaling might be related to the control system of leaf cell proliferation. This hypothesis was strongly supported by the finding that activation of AON in wild types by inoculation leads to an increase in the cell number of leaves.

DOI： 10.1093/pcp/pcm161

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER  

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：International Society for Horticultural Science  

The effects of red light on carbohydrate content in Curcuma alismatifolia Gagnep. were studied. Plants grown under two artificial light sources: T1) red light fluorescent lamp (Philips TLD 36W/15
632-660 nm) and T2) cool daylight source (Philips TLD 36W/865
405-812 nm) at 27±2°C, 70-80% RH and 60 μmol m-2s-1 PAR of light intensity compared with growing under natural condition (T3). Each plant were supply with 100 ml of nutrient solution comprised of 200 mg N, 50 mg P, 200 mg K, 65 mg Ca, 20 mg Mg, 0.22 mg B, 0.54 mg Mn, 0.26 mg Zn, 0.04 mg Mo and 0.45 mg Fe per litre, three times a week. The starch content in new rhizome of plants growing under red light was the lowest. Total soluble sugar, fructose, glucose and sucrose of plants growing under natural light were the highest. The contents of starch and total soluble sugar in storage roots of plant grown under natural light were the highest.

DOI： 10.17660/ActaHortic.2008.788.15

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

In the present study, the effect and utilization of deep placement N-15-labeled coated urea (CU) and lime nitrogen (LN) were investigated at various growth stages of soybean (Glycine max [L.]). Soybean (cv. Enrei) plants were planted in a rotated paddy field in Niigata, Japan. N-15-labeled CU or LN (100 kg N ha(-1)) was supplied separately at a depth of 20 cm just below the seeding line. Deep placement of LN and CU significantly enhanced the dry weight and N content of the plants compared with the control treatment (Cont) without deep placement. As a result, the seed yield per plant in CU (67.2 g) and LN (70.6 g) was much higher than the Cont (37.1 g). N-15 analysis of plants showed that the pattern of labeled N absorption tended to be lower with LN than CU at the R3 and R5 stages, but the recovery rate at R7 was higher in LN (70%) than CU (61%). Combining the N-15 analysis with a relative ureide analysis, the N derived from nitrogen fixation (Ndfa), from fertilizer (Ndff) and from soil (Ndfs) were evaluated. At the R7 stage, the amount of Ndfa was higher in CU (32.1 g m(-2)) and LN (31.1 g m(-2)) than in Cont (21.4 g m(-2)). This positive response for N-2 fixation by the deep placement of CU and LN may result from the continuous supply of N from the lower parts of the roots, which promotes shoot growth and extends the photosynthetic activity of the green leaves, resulting in the promotion of nitrogen fixation and seed yield.

DOI： 10.1111/j.1747-0765.2007.00194.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

The characteristics of plant growth of hypernodulation soybean mutant lines NOD1-3, NOD2-4 and NOD3-7 were compared with their parent cv. Williams. The plants were grown using hydroponics in an illuminated growth chamber with or without seed inoculation of bradyrhizobia, and in the absence or presence of nitrate in the culture solution. When the plants were grown without inoculation, the total dry weight of all mutant lines was not different statistically from Williams, both in the absence and presence of nitrate. When they were grown with inoculation of bradyrhizobia, however, the total dry weight of each mutant line was significantly lower than that of Williams, both in the absence and presence of nitrate. These results indicate that the reduced accumulation of total dry matter of hypernodulation mutant lines compared with the wild type may be a secondary effect passively resulting from the large number of nodules, while the hypernodulation trait is a primary effect of the mutated gene. When the plants were grown with inoculation, the nodule number was decreased by the presence of nitrate in Williams, NOD1-3 and NOD2-4, but not in NOD3-7. NOD3-7 may be the most tolerant to nitrate inhibition of nodulation among the NOD mutant lines. In contrast, leaf growth of NOD3-7 and NOD1-3 was different from the wild type; the expanded leaf was smaller, but the leaf emergence rate was faster compared with Williams under all conditions. This indicates that NOD3-7 and NOD1-3 might decrease the ability for leaf expansion or may have a faster leaf emergence rate.

DOI： 10.1111/j.1747-0765.2007.00113.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

A new fertilization method with deep placement of slow-release N fertilizers, such as coated urea and lime nitrogen (LN) (calcium cyanamide) at 20 cm depth was found to promote soy bean seed yield. In the present study, the effect of deep placement of LN was investigated on different parameters such as growth, N accumulation, NZ fixation activity and yield of soy bean by applying LN at different rates in the rotated paddy field of Niigata, Japan. In addition to the basal fertilizer, ammonium sulphate (16 kg N ha(-1)), deep placement of LN was conducted by applying various amounts such as 50 kg N ha(-1) (A50), 100 kg N ha(-1) (A100) and 200 kg N ha(-1) (A200) at 20 cm depth in separate plots. A N-15-labelled LN fertilizer was also employed for each of the above treatments to calculate N utilization from LN in separate plots. Soya bean plant growth and N-2 fixation activity were periodically analysed. Both plant growth and N accumulation were found to increase with LN treatment compared with control plants. An increase in N-2 fixation activity was found in the A100 plots. The total seed yield was the highest in the deep placement of LN with A100 (73 g per plant) compared with other treatments. The visual quality of harvested seeds also showed that A100 enhanced the quality of seeds compared with other treatments. Thus, it is suggested that N fertilization management with particular reference to optimum amount of fertilizers is important for maximum growth. N-2 fixation and enhancement of seed yield of soy bean.

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Curcuma (Curcuma alismatifolia cv. Gagnep.), a tropical flowering plant known as "Siam tulip", were cultivated in a pot with vermiculite and supplied with different levels of nitrogen (N). Rhizomes with storage roots were harvested at 215 days after planting. Results indicated that a high level of N supply increased flower numbers and promoted continuous new rhizome formation, but storage root growth was depressed. The N supply to the plants increased the N concentrations both in the rhizomes and in the storage roots. The predominant nitrogenous compounds related to total N increase were proteins in the rhizomes. The N of the insoluble fraction of 80% ethanol or the N of the soluble fraction of 10% trichloroacetic acid was the predominant fraction of N that accumulated in the storage roots. A lack of N supply increased the starch concentration both in the rhizomes and in the storage roots. These results suggested that a high level of N supply to the curcuma plant increased new rhizome formation because of increased flower numbers, but depressed new storage root formation because of reduced starch accumulation.

DOI： 10.1111/j.1747-0765.2006.00094.x

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Language：Japanese   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/kagakutoseibutsu1962.44.752

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

We have shown in previous reports that a new fertilization method with deep placement of slow-release N fertilizers, such as coated urea and lime nitrogen (calcium cyanamide), at a depth of 20 cm promoted soybean growth and seed yield. In the present study, the effect of deep placement of lime nitrogen (which gave the highest yield in our previous experiment) was compared at various depths in a rotated paddy field at the Niigata Agricultural Research Institute. The objective of the present study was to analyze growth, N-2 fixation activity and seed yield, and to examine visually the quality of soybean seeds cultivated at different depths with the slow-release fertilizer, lime nitrogen. In addition to the conventional basal application of mixed chemical fertilizer (including ammonium sulfate 16 kg N ha(-1)) in 0-10 cm soil layers, deep placement of lime nitrogen was conducted by applying at various depths (10 cm [D10], 15 cm [D15] and 20 cm [D20]) the same amount (100 kg N ha(-1)) in separate plots. N-15-labeled lime nitrogen fertilizers were also used for each of the above treatments to calculate N absorption in relation to fertilization depth. Soybean plant growth was periodically analyzed and the quality of harvested seeds was also visually examined (hereafter referred to as "visual quality"). The total seed yield was highest in the deep placement of lime nitrogen (100 kg N ha(-1)) at D20 (69 g plant(-1)) compared with D15 (59 g plant(-1)), D10 (57 g plant(-1)) and the control (48 g plant(-1)). The visual quality of harvested seeds showed that lime nitrogen (20 cm depth and 100 kg N ha(-1)) enhanced the quality of seeds compared with the other treatments, in which the good quality seeds (hereafter referred to as "good seeds") based on dry weight were 24 g plant(-1) (control), 37 g plant(-1) (D10), 33 g plant(-1) (D15) and 45 g plant(-1) (D20). Thus, it appears that N fertilization management with particular reference to the depth of fertilizer placement is important for maximum yield and enhancement of seed quality of soybean.

DOI： 10.1111/j.1747-0765.2006.00056.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

A time-course study examining the current photosynthate allocation of soybean (Glycine max [L.] Merr.) cv. Williams was conducted in relation to nodule initiation. Whole shoots were exposed to (CO2)-C-14 for 120 min and the distribution of radioactivity in each organ was determined. During the early stages of nodule formation (i.e. 4, 6 and 8 days after inoculation) the C-14 distribution to the inoculated roots did not increase when compared with uninoculated control roots. In addition, the C-14 respired by underground parts was similar in both the inoculated and the control roots. Eight days after inoculation, the accumulation of starch and sugar was similar in both inoculated and uninoculated plants. These results indicate that photosynthate allocation for nodule initiation does not increase markedly during the early stages of nodule formation. After the emergence of the nodules, photosynthate allocation to the inoculated roots gradually increased. In addition, the consumption of current photosynthate by the respiration of underground parts increased at day 12 after inoculation, but did not increase at day 8 after inoculation.

DOI： 10.1111/j.1747-0765.2006.00051.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Rhizomes of Curcuma alismatifolia Gagnep. were planted in a soil-less culture medium composed of sand: perlite mixed at a ratio of 1:1. Three groups of plants were fed with a N-15 ((NO3-)-N-15 40 mg N L-1 + (NH4+)-N-15 10 mg N L-1)-labeled culture solution over three different periods: (1.) Period 1: from root emergence (2 weeks after planting [WAP]) to the two expanded leaf stage (Stage 1: 6 WAP), (2) Period 2: from Stage 1. to the first floret opening stage (Stage 2: 9 WAP), (3) Period 3: from Stage 2 to harvest during dormancy (Stage 3: 24 WAP). Plants received the same composition of non-labeled N from 2 WAP to 24 WAP, except for the N-15-labeling period. The fourth group of plants was cultured in a nitrogen-free solution and the fifth group was cultured in a complete solution (referred to as control treatment) from planting until harvest and sampled to determine the changes in total N content. Nitrogen content and N-15 abundance were determined at different stages of growth. Average absorption of N-15 from the culture solution was 23.5 mg N plant(-1) during Period 1, 97.8 mg N plant(-1) during Period 2 and 78.7 mg N plant(-1) during Period 3. Daily N absorption rates per plant were 0.84 mg N during Period 1, 4.66 mg N during Period 2 and 0.75 mg N during Period 3. A large amount of absorbed nitrogen was translocated to the compact spike, particularly in Period 2. At harvest the nitrogen accumulation was partitioned almost equally between new rhizomes and storage roots. Most of the absorbed nitrogen in the new rhizomes and storage roots was derived from that received during Period 3.

DOI： 10.1111/j.1747-0765.2006.00027.x

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Japanese Society for Plant Cell and Molecular Biology  

Hypocotyl sections of Rhaphiolepis umbellata (Thunb.) Makino were transformed by Agrobacterium tumefaciens bearing a binary vector pIG121-AtNiR, which contains cDNA of the nitrite reductase gene from Arabidopsis thaliana (Atni) under the control of a modified cauliflower mosaic virus 35S promoter and chimeric hygromycin phosphotransferase gene (hph). A 4% of the hypocotyl explants transfected with Agrobacterium formed hygromycin resistant adventitious shoots, and most of them rooted upon root induction treatment. The presence and expression of the introduced transgene in putative transgenic plants (12 months after transfection) were respectively confirmed by polymerase chain reaction (PCR) analysis using primers specific to Atni and by western blot analysis using anti NiR antibody. A total of 37 transgenic plant lines were obtained. Plants 33 months after the transfection were fumigated with 200±50 ppb 15NO2 under the natural light for one week in the fumigation chamber in a confined glasshouse. The amount of total nitrogen derived from NO2 (reflecting uptake of NO2) and that of Kjeldahl nitrogen derived from NO2 (reflecting assimilation of NO2) were determined using a mass spectrometer. One of the 9 transgenic plants tested was 1.6-2.0 times higher both in the uptake and assimilation of NO2 than non-transformed wild-type ones.

DOI： 10.5511/plantbiotechnology.23.111

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC SOIL SCIENCE PLANT NUTRITION  

The long-term effect of the concentration and duration of application of nitrate from the lower part of soybean roots on the nodulation and nitrogen fixation in the upper part of roots was investigated using a two-layered pot system separating the upper roots growing in a vermiculite medium and the lower roots growing in a nutrient solution. Continuous absence of nitrate (hereafter referred to as "0-0 treatment"), and continuous 1 mm (1-1 treatment) and 5 mm (5-5 treatment) nitrate treatments were imposed in the lower pot from transplanting to the beginning of the maturity stage. In addition, 5 mm nitrate was supplied partially from the beginning of the pod stage till the beginning of the maturity stage (0-5 treatment) or from transplanting till the beginning of the pod stage (5-0 treatment). The values of the total plant dry weight and seed dry weight were highest in the 5-5 treatment, intermediate in the 1-1, 5-0, 0-5 treatments, and lowest in the 0-0 treatment. The values of the nodule dry weight and nitrogen fixation activity (acetylene reduction activity) were lowest in the 5-5 treatment. The value of the nodule dry weight in the upper roots was highest in the plants subjected to the 1-1 treatment and exceeded that in the 0-0 treatment. Total nitrogen fixation activity of the upper nodules per plant at the beginning of the pod stage was also highest in the 1-1 treatment. These results indicated that long-term supply of a low level of nitrate from the lower roots could promote nodulation and nitrogen fixation in the upper part of roots. Withdrawal of 5 mm nitrate after the beginning of the pod stage (5-0 treatment) markedly enhanced nodule growth and ARA per plant in the upper roots at the beginning of the maturity stage when the values of both parameters decreased in the other treatments. The nitrate concentration in the nodules attached to the upper roots was low, including the 5-5 treatment regardless of the stages of growth. This indicated that the inhibitory effect of 5 mm nitrate or promotive effect of 1 mm nitrate supplied from the lower roots was not directly controlled by nitrate itself, but was mediated by some systemic regulation, possibly by the C or/and N requirement of the whole plant.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC SOIL SCIENCE PLANT NUTRITION  

It is well known that nodulation and nitrogen fixation of soybean are generally depressed by the application of N fertilizers. We previously reported that a new fertilization method consisting of deep placement of slow-release N fertilizers, such as coated urea and lime nitrogen (calcium cyanamide) at a 20 cm depth enhanced soybean growth and seed yield. In the present study, the effect of deep placement of various N-15-labeled N fertilizers (100 kg N ha(-1)) was compared in converted paddy fields located at Niigata Agricultural Research Institute, in Nagaoka. Fertilizers consisting of N-15-labeled ammonium sulfate (AS), urea (U), coated urea (CU) or lime nitrogen (LN) were applied at a 20 cm depth for nodulated soybean (cv. Enrei) and for a non-nodulated isogenic line (En1282). The plants without deep placement of N fertilizer were used as control (Cont) for both Enrei and En1282. Whole plants were sampled at maturing stage, and N-15 abundance and N concentration in each part were determined. The evaluation of the amounts of Ndfa, Ndfs and Ndff was conducted by the N-15 dilution method using En1282 as a reference plant. Also the amounts of Ndfa and Ndfs+Ndff were estimated by the N balance method for comparing Cont and the other treatments. In all the deep placement treatments, the non-nodulated line exhibited only 36-40% of total dry weight and 16-30% of total N accumulation compared with the nodulated line, due to N deficiency associated with the lack of nitrogen fixation. In both nodulated and non-nodulated soybeans, plant growth and the total amount of accumulated N showed the highest values by the application of LN, followed by CU, U, AS and Cont, respectively: The value of the seed dry weight of Enrei was highest in the LN (73 g plant(-1)), followed by CU (63 g plant(-1)), U (47 g plant(-1)), AS (37 g plant(-1)) and Cont (26 g plant(-1)) treatments. The value of Ndfa estimated by the N-15 dilution method was higher in the LN (3.6 g N plant(-1)) and CU (2.8 g N plant(-1)) treatments than in the U (2.3 g N plant(-1)) and AS (2.2 g N plant(-1)) treatments. In addition, the value of Ndfa estimated by the N-balance method was also higher in the LN (4.1 g N plant(-1)), CU (3.9 g N plant(-1)), U (2.8 g N plant(-1)) and AS (2.6 g N plant(-1)) treatments than in the Cont treatment (1.5 g N plant(-1)). The recovery rate of N fertilizers in Enrei was higher in the LN (43%) and CU (36%) treatments than in the U (21%) treatment and lowest in the AS (8.5%) treatment. These results indicate that deep placement of LN and CU is effective to improve soybean growth and seed yield. The combination of "deep placement" and "slow-release N fertilizers" is important to supply N until the maturation stage, resulting in the increase of seed yield.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC SOIL SCIENCE PLANT NUTRITION  

The objective of the present study was to record the seed yield and to examine visually the quality of soybean seeds cultivated under different types and placements of urea fertilizers. In addition to the conventional fertilizer application (including ammonium sulfate 16 kg N ha(-1)), broadcasting (100 kg N ha(-1)) of urea (UB) and 100-d type coated urea CU-100 (CUB), and deep placement (100 kg N ha(-1)) of urea (UD) and 100-d type coated urea CU-100 (CUD) was conducted in separate plots in a paddy field converted to an upland field located at Shindori Experimental Station of Niigata University. Soybean plant growth was periodically analyzed and the quality of harvested seeds was also visually examined (hereafter referred to as "visual quality"). It was found that the deep placement treatments were more conducive to nitrogen (N,) fixation, based on the relative ureide N concentration in the xylem sap, which is a good indicator of N, fixation by soybean. Also the total seed yield was the highest in CUD (82 g plant(-1)) and UD (81 g plant(-1)), compared to the control (62 g plant(-1)), UB (68 g plant(-1)), and CUB (68 g plant(-1)). The visual quality of harvested seeds showed that CUD enhanced the quality of seeds compared to the other treatments, in which the percentage of good quality seeds, hereafter referred to as "good seeds," based on the dry weight was 51 (control), 65 (UB), 61 (CUB), 61 (UD), and 66% (CUD). In terms of diseased seeds, the percentage of turtle wrinkle and broken seed coats was found to decrease by N application compared to the control. Thus, it is suggested that N fertilization management is important for maximum yield of soybean as well as for the enhancement of seed quality.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Barley (Hordeum vulgare L.) has two nitrate reductase (NR) isozymes, namely NADH-specific NR and NAD(P)H-bispecific NR. To determine the effect of NADH-specific NR deficiency on in vivo nitrate reduction and distribution of reduced N to shoots and roots, the N-15-incorporation model was applied to NADH-specific NR-deficient mutant (Az12) seedlings of barley. The N-deprived seedlings were treated with a nutrient solution containing 2.3 mm NO3- and 0.2 mm NO2-, and were labeled with different amounts of 15 N for 48 It under continuous illumination. In Az12, the total in vitro NR activity derived from the NAD(P)H-bispecific NR was only 10% of that of the wild type (cv. Steptoe). In Az12, the total (NO3-)-N-15 incorporation and translocation of absorbed (NO3-)-N-15 to shoots were about 25% higher than those of Steptoe. Nitrate reduction in the Az12 roots was 2 times higher than that in Steptoe during the first period (0-24 h) and 1.3 times higher during the second period (24-48 h). However, nitrate reduction in the Az12 shoots was 10 to 30% lower than that in the Steptoe shoots. As a result, nitrate markedly accumulated in the Az12 shoots. Accumulation of reduced N-15 in the Az12 roots was 2 times higher than that in the Steptoe roots, but 10% lower in the Az12 shoots than in the Steptoe shoots at the end of the experiment. Upward transport of reduced N-15 via the xylem in Az12 was nearly 2 times more active than that of Steptoe throughout the experiment. This result suggested that the derepression of the NAD(P)H-NR isozyme in the Az12 shoots could compensate for the absence of the NADH-NR isozyme. Furthermore, increased levels of root nitrate reduction seemed to make up for the limited nitrate assimilation in the Az12 shoots.

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In this report, we determined whether liquid chromatography mass spectrometry (LC-MS) using the atmospheric pressure chemical ionization (APCI) method could be applied for isotopic analysis of the high polar compounds, allantoin, and allantoic acid, which are the major transport forms of N from soybean nodules. The peak areas of the ion chromatogram of authentic allantoin and allantoic acid obtained by selected ion monitoring (SIM) were proportional to the contents of the compounds. The data obtained from the ion intensity using the scan mode and the area of the ion chromatogram using the SIM mode were entirely consistent with the isotope ratio of the compounds. The allantoin and allantoic acid concentrations in the xylem sap of soybeans at various growth stages were measured by the following two methods using LC-MS; an absolute calibration method and an isotope-labeled internal standard method. The values estimated by the isotope-labeled internal standard method were more consistent with those obtained by the colorimetric method. Furthermore, amino acids could be detected under the same analytical conditions. The N-15 ratio of asparagine and aspartic acid in the non-nodulating soybean xylem sap which had been supplied with N-15-labeled nitrate could be measured. These results indicate that LC-MS (APCI) is a suitable method for isotopic analysis of high polar compounds, including allantoin and allantoic acid in plant xylem sap and tissues.

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Language：English   Publisher：新潟大学  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL VERLAG GMBH  

The effects of deep placement (supplied at 20 cm depth from soil surface below plants) of 100 kg N ha(-1) of N fertilizers, urea, coated urea or calcium cyanamide (lime nitrogen) on the growth, nitrogen fixation activity, nitrogen absorption rate and seed yield of soybean (Glycine max L. Merr.) plants were examined by comparing them with control plots without deep placement of N fertilizer in sandy dune field. In addition, three different inoculation methods of bradyrhizobia were used for each N treatment: (1) transplantation of 10-day-old seedling in a paper pot with vermiculite inoculated with Bradyrhizobium japonicum USDA110, (2) direct transplantation of inoculated 10-day-old seedlings, and (3) transplantation of 10-day-old seedlings in a non-inoculated paper pot. The deep placement of N fertilizers, especially calcium cyanamide and coated urea, markedly increased the growth and total N accumulation in shoot, roots and nodules, which resulted in an increase in seed yield. Daily N-2 fixation activity and N absorption rate were estimated by relative abundance of ureide-N analysed from the concentration of N constituents (ureide-N, amide-N and nitrate-N) in root bleeding xylem sap and increase in total N accumulation in whole plants at R1, R3, R5 and R7 stages. The total amount of N-2 fixation was about 50 % higher in the plants with calcium cyanamide and coated urea deep placements compared with control plants. Deep placement of slow release fertilizers kept nodule dry weight higher in the maturing stage of seed, possibly through abundant supply of photoassimilate to the nodules by supporting leaf area and activity until late reproductive stages. The results indicate that deep placement of calcium cyanamide or coated urea enhances N-2 fixation activity, which ultimately increases the seed yield. The promotive effect was observed with the seedlings transplanted in paper pot with inoculum of bradyrhizobia within any treatments, although nodulation by indigenous rhizobia was observed in the plants transplanted with non-inoculated paper pot.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The effect of nitrate placement on the nodule number, nodule growth, and N-2 fixation activity in soybean plant was elucidated by using a two-layered pot system separating the upper roots grown in vermiculite medium from the lower roots cultured in hydroponics. Four treatments were imposed i.e. 0/0, 0/5, 5/0, and 5/5, with the 0 or 5 mm nitrate treatment in the upper pot/lower pot, respectively. The plants were harvested at the flowering stage and initial pod setting stage. Systemic and local inhibition on nodule number did not occur in the upper nodules in vermiculite. On the other hand, systemic inhibition on the nodule growth and N-2 fixation activity in the upper pot was apparent at pod setting stage. In the lower pot where the nodules were in direct contact with 5 mm nitrate, inhibition on nodule number, nodule size, and N-2 fixation activity were conspicuous. Systemic inhibition on the number and dry weight of lower nodules was also observed. Root growth was promoted with the root part in direct contact with 5 mm nitrate, irrespective of upper or lower roots. Nitrate accumulation was observed only in the part of roots and nodules in direct contact with 5 mm nitrate. The results indicated that systemic nitrate inhibition occurred both on N-2 fixation activity and on nodule growth but not on the upper nodule number after long-term application of 5 mm nitrate. Milder inhibition on upper nodules compared with the lower nodules may be due to the rapid depletion of nitrate from the vermiculite medium compared with the culture solution in the lower pot.

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Nodule growth of a hypernodulating soybean (Glycine max (L.) Merr.) mutant line NOD1-3 was compared to that of its wild-type parent cv. Williams from 14 to 18 days after planting (DAP) in the absence of nitrate treatment (hereafter referred to as "0 mm treatment") or with 5 mm nitrate treatment. The growth rate determined by increase in the diameter of the nodules was relatively lower in the mutant NOD1-3 than that of the parent Williams under nitrogen-free conditions (0 mm nitrate). The inhibition of nodule growth by 5 mm nitrate started at 1 d after the onset of the nitrate treatment in Williams, while the inhibition did not occur before the application of the nitrate treatment for 2 d in NOD1-3. The nodule growth was completely inhibited after 2 d in Williams and after 3 d in NOD1-3 during the 5 mm nitrate treatment period. After 4 d of 5 mm nitrate treatment, the nodule dry weight decreased by 22% in NOD1-3 and by 58% in Williams, respectively. The treatment with 5 mm nitrate decreased the acetylene reduction activity (ARA) in NOD1-3 by 60% per plant and by 50% per nodule g DW and these parameters were less sensitive to the treatment than those in Williams in which the inhibition rate was 90% per plant and 80% per nodule g DW. These results indicate that NOD1-3 is partially nitrate-tolerant in terms of individual nodule growth as well as total nodule dry weight and N-2 fixation activity. A whole shoot of Williams and NOD1-3 plants was exposed to Co-14(2) for 120 min followed by 0 or 5 mm nitrate treatment for 2 d, and the partitioning of the photoassimilates among the organs was analyzed. Under 0 mm nitrate treatment, the percentages of the distribution of 14 C radioactivity between the nodules and roots were 63 and 37% in Williams and 89 and 11% in NOD1-3. Under the 5 mm nitrate conditions, the percentages of the distribution of C-14 between the nodules and roots changed to 14 and 86% in Williams and 39 and 61% in NOD1-3, respectively. These results indicated that the hypernodulating mutant NOD1-3 supplied a larger amount of photoassimilates to the nodules than to the roots under nitrogen-free conditions, and that the nitrate depression of photoassimilate transport to the nodules was less sensitive than that of the parent line.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC SOIL SCIENCE PLANT NUTRITION  

It was found that tulip (Tulipa gesneriana L.) plants accumulate a large amount of nitrogen in their roots during the winter season, which is efficiently used for rapid shoot growth after sprouting in spring. In the present paper, the site and origin of nitrogen accumulation in tulip roots were investigated in a vertical split-root experiment. Tulip roots were separated into upper and lower half parts in a two-compartment tray, and the culture solution was separately supplied for a week. Culture solution with N-free medium or that containing N-15-labeled nitrate (2 mm (NO3-)-N-15 + 0.5 mm NH4+) or N-15-labeled ammonium (2 mm NO3- + 0.5 mm (NH4+)-N-15) was supplied to individual trays to determine the metabolic fate of nitrate and ammonium nitrogen in either part of the roots. The results indicated that nitrate and ammonium absorption activities were similar between the lower and upper half-parts of the tulip roots. The absorbed N-15 predominantly accumulated either in the upper or lower part of the roots in direct contact with N-15-labeled medium, and only a small portion of N-15 was translocated to the other part. In the root part treated with N-15-labeled medium, most of the absorbed nitrogen was in ethanol soluble forms, and free glutamine and glutamic acid were intensely labeled with N-15. Also in the root parts directly in contact with nitrogen, the glutamine concentration in either the upper or the lower roots was considerably enhanced. On the other hand, nitrogen application did not affect the 4-methyleneglutamine concentration. Sucrose and glucose were the major soluble sugars in tulip roots, and the concentration was reduced at the nitrogen absorption site, possibly due to energy and carbon consumption for the absorption and assimilation of nitrogen.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The upper part of a nodulated soybean root hydroponically cultured in a glass bottle was monitored using a computer microscope under controlled environmental conditions, and the diameter of individual nodules was measured from 10-24 d after planting. The diameter of a root nodule attached to the primary root increased from 1 mm to 6 mm for 2 weeks under nitrogen-free conditions. The increase in diameter of the nodules was almost completely stopped after 1 d of supplying 5 mM nitrate, and was due to the cessation of nodule cell expansion. However, nodule growth quickly returned to the normal growth rate following withdrawal of nitrate from the solution. The reversible depression of nodule growth by nitrate was similar to the restriction of photoassimilate supply by continuous dark treatment for 2 d followed by normal light/dark conditions. In addition, the inhibitory effect of nitrate was partially alleviated by the addition of 3% (w/v) sucrose to the medium. Plant leaves were exposed to C-11 or C-14-labelled carbon dioxide to investigate the effects of 5 mM nitrate on the translocation and distribution of photosynthates to nodules and roots. Supplying 5 mM nitrate stimulated the translocation rate and the distribution of labelled C in nitrate-fed parts of the roots. However, the C-14 partitioning to nodules decreased from 9% to 4% of total C-14 under conditions of 5 mM nitrate supply. These results indicate that the decrease in photoassimilate supply to nodules may be involved in the quick and reversible nitrate inhibition of soybean nodule growth.

DOI： 10.1093/jxb/erg147

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC SOIL SCIENCE PLANT NUTRITION  

The main objective of this study was to increase the productivity of soybean [Glycine max (L.) Merr. cv, Enrei] seed by deep placement of controlled release nitrogen fertilizers and by the application of different., methods of inoculation of bradyrhizobia. Ten days old seedlings in an inoculated paper.,pot (IPP), in a non-inoculated paper pot (NIPP), and those grown in a vermiculite bed without paper pot (DT) were transplanted to an upland field converted from a drained paddy field in Nagaoka. In addition to the basal application of 16 kg N ha(-1) in the surface layer (Control), deep placement of 100 kg N ha-1 of urea (Urea), 100-day type coated urea (CU-100), and calcium cyanamide (CaCN2,) treatments were applied at the depth of 20 cm. In the IPP method, a significantly higher seed yield was obtained with the deep placement of CaCN2 and CU-100 compared with the Urea and Control treatments. A similar tendency was observed for the DT and NIPP methods. Among the same N fertilizer treatments, the seed yield for IPP and DT tended to exceed that for NIPP, although the NIPP roots also showed nodulation probably due to infection with indigenous bradyrhizobia. The percentage of nitrogen derived from atmospheric N, estimated by the simple relative ureide method was higher in the plants with CU-100 and CaCN2 compared with those with the Urea and Control treatments at the R1 stage, suggesting that the basal deep placement of CaCN2 or CU-100 for soybean cultivation enabled the supply of N without concomitant depression of N-2 fixation. Thus the deep placement of cheaper CaCN2 was found to be as effective as that of CU-100 for enhancing the soybean seed yield.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL MUNKSGAARD  

Although it has been shown that leaf nitrate reductase (NR: EC 1.6.6.1) is phosphorylated by subjecting plants to darkness, there is no evidence for the existence of dark-activated or dark-induced NR kinase. This study was undertaken to investigate the occurrence of a protein kinase phosphorylating NR in response to dark treatments. Immediately after transferring Komatsuna (Brassica campestris L.) plants to darkness, we observed rapid increases in the phosphorylating activity of the synthetic peptide, which is designed for the amino acid sequence surrounding the regulatory serine residue of the hinge 1 region of Komatsuna NR, in crude extracts from leaves. The activity reached a maximum after 10 min of darkness. Inactivation states of NR estimated from relative activities with or without Mg2+ were correlated to activities of the putative dark-activated protein kinase. Using the synthetic peptide as a substrate, we purified a protein kinase from dark-treated leaves by means of successive chromatographies on Q-Sepharose, Blue Sepharose, FPLC Q-Sepharose, and ATP-gamma-Sepharose columns. The purified kinase had an apparent molecular mass of 150 kDa with a catalytic subunit of 55 kDa, and it was Ca2+-independent. The purified kinase phosphorylated a recombinant cytochrome c reductase protein, a partial protein of NR, and holo NR, and inactivated NR in the presence of both 14-3-3 protein and Mg2+. The kinase also phosphorylated synthetic peptide substrates designed for sucrose phosphate synthase and 3-hydroxy-3-methylglutaryl-Coenzyme A reductase. Among inhibitors tested, only K252a, a potent and specific serine/threonine kinase inhibitor, completely inhibited the activity of the dark-activated kinase. The activity of the purified kinase was also specifically inhibited by K252a. Taken together with these findings, results obtained suggest that the putative dark-activated protein kinase may be the purified kinase itself, and may be responsible for in vivo phosphorylation of NR and its inactivation during darkness.

DOI： 10.1034/j.1399-3054.2002.1150403.x

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Publishing type：Research paper (scientific journal)   Publisher：Informa UK Limited  

DOI： 10.1080/00380768.2002.10409219

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC SOIL SCIENCE PLANT NUTRITION  

Nodulated soybean (Glycine max. (L) Merr. cv. Williams) plants were hydroponically cultured, and various combinations of I-week culture with 5 or 0 mm nitrate were applied using 13-d-old soybean seedlings during three successive weeks. The treatments were designated as 0-0-0, 5-5-5, 5-5-0, 5-0-0, 5-0-5, 0-5-5, and 0-0-5, where the three sequential numbers denote the nitrate concentration (mm) applied in the first-second-third weeks. The size of the individual nodule was measured periodically using a slide caliper. All the plants were harvested after measurement of the acetylene reduction activity (ARA) at the end of the treatments. In the 0-0-0 treatment, the nodules grew continuously during the treatment period. Individual nodule growth was immediately suppressed after 5 mm nitrate supply. However, the nodule growth rapidly recovered by changing the 5 mm nitrate solution to a 0 mm nitrate solution in the 5-0-0 and 5-5-0 treatments. In the 5-0-5 treatment, nodule growth was completely inhibited in the first and the third weeks with 5 mm nitrate, but the nodule growth was enhanced in the second week with 0 mm nitrate. The nodule growth response to 5 mm nitrate was similar between small and large size nodules. After the 5-5-5, 5-0-5, 0-0-5, and 0-5-5 treatments, where the plants were cultured with 5 mm nitrate in the last third week, the ARA per plant was significantly lower compared with the 0-0-0 treatment. On the other hand, the ARA after the 5-0-0 and 5-5-0 treatments was relatively higher than that after the 0-0-0 treatment, possibly due to the higher photosynthate supply associated with the vigorous vegetative growth of the plants supplemented with nitrate nitrogen. It is concluded that both soybean nodule growth and N-2 fixation activity sensitively responded to the external nitrate level, and that these parameters were reversibly regulated by the current status of nitrate in the culture solution, possibly through sensing of the nitrate concentration in roots and / or nodules.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

In cucumber (Cucumis sativus L.) leaves at different ontogenic stages, a differential appearance of three major endopeptidases was observed by employing activity staining using gelatin as a substrate. On the basis of this observation, we discussed their physiological roles in senescing leaves. The most active endopeptidase in young mature leaves was a glutamyl endopeptidase with a pI of 4.5. It might be involved in active protein catabolism in young leaves because its activity became maximal just after the leaf had fully expanded and when protein and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) contents rapidly decreased. An endopeptidase with a pI of 4.3 was not observed in young leaves, however, it was highly active in senescing leaves. Interestingly, its activity in cotyledons was eliminated when the upper metabolically active leaves were removed. This implies that the appearance of this enzyme is regulated by the presence of sink tissues, and it is involved in the degradation of protein in senescing leaves facilitating N transfer to upper developing leaves. A trypsin-like endopeptidase with a pI of 5.0 showed relatively constant activity during the whole period. This endopeptidase has been shown to be inhibited by arginine, guanidino compounds and Mg2+, therefore, it might exist constitutively and its activity might be regulated mainly at a post-translational level responding to nutrient and environmental conditions. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

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The effects of anion channel blockers, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and anthracene-9-carboxylic acid (A-9-C), on the uptake and xylem transport of nitrate were investigated in intact barley (Hordeum vulgare L.) seedlings using (NO3-)-N-13 and (NO3-)-N-15 as tracers. The seedling roots were pretreated with either blocker for 1 h and then (NO3-)-N-13 or (NO3-)-N-15 was supplied to the medium. Real-time images of N-13 accumulation in shoots were monitored during the first 30 min using Positron Emitting Tracer Imaging Systems (PETIS). The radioactivity in the shoots of the plants treated with either blocker was about half of that in the shoots of the control plants. Analysis of the distribution of N-13 in whole seedlings using a Bioimaging Analyzer System (BAS) showed a N-13-accumulation in both shoots and roots of the control plants but reduced N-13-levels in the shoots of the plants treated with either blocker. Nitrate concentrations in the xylem sap were significantly reduced by the application of either blocker while the net uptake of (NO3-)-N-15 was not or slightly influenced by the treatment with blockers. The translocation and uptake of chloride were also significantly reduced by the treatment with A-9-C but not with DIDS. These results suggested that anion channels contributed to xylem loading of nitrate in barley plants.

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It has been reported that the accumulation of the beta-subunit of beta-conglycinin, a storage protein of soybean (Glycine max L.) seeds, was suppressed by nitrogen (N)-deficiency. In this report we attempted to determine which compound(s) may play a key role in regulating the accumulation of beta-subunit mRNA and protein in intact seeds and in an in vitro cotyledon culture system. Non-nodulating soybean plants were cultivated under N-deficient (0.5 mm NO3-) conditions, and transferred to N-sufficient medium (5 mm NO3-) when the plants reached the pod filling stage. The beta-subunit mRNA and the protein were detected in immature seeds within 2 d after transfer to 5 mm NO3- medium. Among the free amino acids in the immature seeds, asparagine concentration increased rapidly within 2 d. In the in vitro cotyledon culture system, the application of glutamine induced the accumulation of beta-subunit mRNA within 12 h, while asparagine did not induce the accumulation of beta-subunit mRNA for 7 d. An inhibitor of transaminases, (aminooxy)acetic acid, enhanced beta-subunit mRNA accumulation without Gln accumulation when asparagine was the sole nitrogen source. Although the asparagine concentration in the seed reflected the nitrogen statues in whole plant, the accumulation of beta-subunit mRNA and continuous protein storage in immature seeds appeared to be regulated by glutamine, its metabolites or related compounds.

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The role of xylem sap nitrate in the regulation of nitrate reductase (NR) in leaves was investigated in barley (Hordeum vulgare L.) seedlings. Upon the exposure of barley roots to nitrate at concentrations ranging from 0.1 to 5 mm, rapid accumulation of NADH-specific NR mRNA was observed in both leaves and roots. In the same treatment, the concentration of xylem sap nitrate also rapidly increased during the early feeding period and reached a steady state at concentration ranging from 15 to 50 mm. Patterns of NR mRNA accumulation in leaves were closely linked with the increase in the nitrate concentration in the xylem sap, but not with the amount of nitrate stored in the leaf tissues. High levels of NR mRNA in intact leaves were observed only when the xylem sap nitrate reached mm levels. Supply of different concentrations of nitrate to detached leaves also indicated that mM levels of nitrate supplied through the xylem were required for adequate accumulation of NR mRNA in leaves. When nitrate-grown seedlings were transferred to a nitrate-free medium, a parallel decline in both the concentration of xylem sap nitrate and NR mRNA level was observed, while the nitrate concentrations in leaf tissues remained constant. These results indicate that continuous supply of mM levels of nitrate through the xylem is important for maintaining the high levels of NR mRNA in barley leaves.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL MUNKSGAARD  

The expression of asparagine synthetase (AS; EC 6.3.5.4) in response to externally supplied nitrogen was investigated with respect to enzyme activity and protein levels as detected immunologically in rice (Oryza sativa) seedlings. The asparagine content was very low in leaves and roots of nitrogen-starved rice plants but increased significantly after the supply of 1 mM NH4+ to the nutrient solution. While neither AS activity nor AS protein could be detected in leaves and roots prior to the supply of nitrogen, levels became detectable in roots but not in leaves within 12 h of the supply of I mM NH4+ Or 10 mM glutamine. Other nitrogen compounds, such as nitrate, glutamate, aspartate and asparagine had no effect. Methionine sulfoximine completely inhibited the NH4+-induced accumulation of AS protein but did not affect the glutamine-induced accumulation of the enzyme. The results suggested that glutamine or glutamine-derived metabolites regulate AS expression in rice roots.

DOI： 10.1034/j.1399-3054.2002.1140107.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

An endopeptidase was purified and characterized from green leaves of cucumber (Cucumis sativus L. suyo). The purified enzyme, a basic amino acid-specific endopeptidase with a pI of 5.0, was a monomeric protein of 80 kDa whose pH optimum was 9.5. Inhibitor analysis suggested that it was a serine endopeptidase and contained sulfhydryl groups essential for catalytic activity. Analysis of internal amino acid sequences of the endopeptidase showed no significant similarity to other proteins. Its activity was inhibited by L-Arg and guanidino compounds having high hydrophobicity, as well as divalent cations such as Mg2+ and Ca2+. The K-i values of L-Arg and Mg2+, which are also likely in vivo inhibitors, were 3.5 and 10 mM, respectively. Inhibition by L-Arg and Mg2+ was additive, and more than 70% of the activity was reversibly inhibited under their physiologically significant concentrations. These results suggest that the enzyme is possibly regulated by L-Arg and/or guanidino compounds, and by divalent cations in vivo. (C) 2001 Elsevier Science Ltd. All rights reserved.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

We purified a glutamyl endopeptidase that is a major foliar endopeptidase in cucumber. The endopeptidase had a molecular mass of 400 kDa, consisted of four subunits of 97 kDa, and was inactivated by SH-modifying reagents. Its optimum pH and optimum temperature were 8.0 and 30-37 degreesC, respectively. An internal amino acid sequence of the endopeptidase was highly homologous to a partial sequence of unidentified proteins deduced from genetic information for Arabidopsis thaliana, soybean and rice, but not to the sequences of bacterial glutamyl endopeptidases or animal proteases. Therefore, the unidentified proteins might be glutamyl endopeptidases and be widely distributed only among plant species. The activity of the cucumber glutamyl endopeptidase was inhibited by at least three inhibitors existing in cucumber leaves. One of the inhibitors was a competitive inhibitor of 25 kDa, which did not significantly inhibit commercial endopeptidases derived from animals and microorganisms. This suggests that the cucumber glutamyl endopeptidase might be controlled by endogenous inhibitors in vivo.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC SOIL SCIENCE PLANT NUTRITION  

The accumulation of the beta -subunit of beta -conglycinin, a major soybean storage protein, in seeds is controlled by the nitrogen (N) status of the plant. When non-nodulating soybean mutants (T201) were hydroponically cultivated under N-sufficient conditions (supply of 5 mM NaNO3 to the medium), beta -subunit accumulation was first detected at 30 d after flowering (DAF). In N-deficient plants (supply of 1 mM NaNO3), the accumulation of the beta -subunit was strongly inhibited. When 5 mM N-15-labeled NO3- was supplied for 1 week to N-deficient T201 plants before 28 DAF, the beta -subunit protein did not accumulate, but the application of 5 mM NO3- after 28 DAF, led to a substantial accumulation of the beta -subunit, The total N-15 percentage in mature seeds was similar, irrespective of the time of feeding, regardless of the accumulation of the beta -subunit. The 7 S (mostly composed of beta -conglycinin) and 11 S globulins (mostly glycinin, which was another major component of storage protein) contained N from 5 mM (NO3-)-N-15 at a similar level, about 60% of total N, These results indicate that a) the temporary supply of a high level of NO3- effectively promoted beta -subunit accumulation in N-deficient plants when feeding was carried out after 28 DAF, and b) incorporation of fed NO3- was similar between 7 S and 11 S globulins.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Non-nodulated soybean (Glycine max (L,) Merr,) plants were cultivated hydroponically under N-sufficient (5 mM NaNO3) or N-deficient (0.5 mM NaNO3) conditions. N-13- Or N-15-labelled nitrate was fed to the cut end of the stems, and the accumulation of nitrate-derived N in the pods, nodes and stems was compared. Real-time images of N-13 distribution in stems, petioles and pods were obtained using a Positron Emitting Tracer Imaging System for a period of 40 min. The results indicated that the radioactivity in the pods of N-deficient plants was about 10 times higher than that of N-sufficient plants, although radioactivity in the stems and nodes of N-deficient versus N-sufficient plants was not different. A similar result was obtained by supplying (NO3-)-N-15 to cut soybean shoots for 1 h, The fact that the N translocation into the pods from NO3- fed to the stem base was much faster in N-deficient plants may be due to the strong sink activity of the pods in N-deficient plants. Alternatively, the redistribution of N from the leaves to the pods via the phloem may be accelerated in N-deficient plants. The temporal accumulation of (NO3-)-N-13 in nodes was suggested in both N-sufficient and N-deficient plants. In one (NO3-)-N-13 pulse-chase experiment, radioactivity in the stem declined rapidly after transferring the shoot from the (NO3-)-N-13 solution to non-labelled NO3; in contrast, the radioactivity in the node declined minimally during the same time period.

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Soybean nodules contain four major leghemoglobin (Lb) components, Lba, Lbc1, Lbc2 and Lbc3. A sensitive and selective method for quantitative analysis of the four Lb components was devised with capillary isoelectric focusing (CIEF). The changes in the concentrations of four Lb components in nodules during the initial stages of development were compared between hypernodulating soybean mutant NOD1-3 and its parent cv. Williams. The hydroponically cultivated soybean plants were periodically sampled. All the visible nodules were collected from the roots, and then the four Lb components in the largest nodules were analyzed with the CIEF method. In NOD1-3 Lbs were initially detected at 19 days after sowing (DAS), a few days earlier than in Williams at 22 DAS. The Lbcs (Lbc1, Lbc2 and Lbc3) were the main component at the earliest nodule growth stage, and the relative proportion of Lba increased with nodule growth in both NOD1-3 and Williams. This result is in agreement with previous observation, and the CIEF method is considered to be useful for Lb components analysis to define their function and gene expression.

DOI： 10.1023/A:1013317219871

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC SOIL SCIENCE PLANT NUTRITION  

Nitrate-induced accumulation of mRNAs for nitrate reductase and nitrite reductase was strongly inhibited by treatment with inhibitors of Ca2+ channels, protein phosphatases, and tyrosine protein kinases in excised barley leaves. It was shown that these components may be involved in the nitrate-response signaling pathways for the expression of nitrate and nitrite reductase genes in barley leaves.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MUNKSGAARD INT PUBL LTD  

We investigated the cause and effect relationships among ethylene, polyamines, and K+ in barley (Hordeum vulgare L. cv. Amagi) seedlings, Application of 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene, to the growth medium caused a decrease in K+ concentration in roots and an increase in shoots. Addition of ACC induced putrescine accumulation in roots, while spermidine and spermine levels remained unchanged. Exogenous supply of putrescine led to putrescine accumulation and reduced K+ concentration. Application of Co2+, an inhibitor of ethylene biosynthesis, together with ACC, inhibited putrescine accumulation with a decrease in K+ concentration in roots, ACC-treated roots showed K+ uptake capacity equivalent to that of control roots, implying that the majority of K+ is translocated to shoots. These results suggest that ethylene regulates K+ partitioning between roots and shoots through the level of accumulation of putrescine in barley seedlings.

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The hypernodulating mutant of soybean En6500 has a larger number of small nodules than its parent, cv. Enrei, and the shoot size is relatively small at the same time. It was shown that some characteristics of the nodules of En6500 were different from those of the parent nodules such as, nodule size distribution, acetylene reduction activity (ARA), concentration of leghemoglobin (Lb) components and proportion of the infected region in the nodules. In this study, we attempted to identify the main factor(s) which control(s) the changes in the characteristics of mutant nodules, by manipulating the source-sink relationship in relation to the photosynthetic supply.
By decreasing the infection dose of Bradyrhizobium japonicum, the nodule number of En6500 could be reduced to the level of nodulation normally observed in Enrei. In addition a part of Enrei shoot was cut off for equalizing the shoot size of Enrei with that of En6500 at 16 d after sowing (DAS). The plants were hydroponically cultured until 30 DAS with or without 5 mM NO3- for the last 14 d. ARA, bacteroid density, concentration of Lb components (Lba, Lbc1, Lbc2, and Lbc3), and volume ratio of the infected region in the nodules were determined.
When the hypernodulating mutant En6500 which was treated to obtain a similar nodule number to that of Enrei was compared with the parent Enrei which was treated to obtain the same shoot size as that of En6500, nodule characteristics such as, ARA per nodule dry weight, Lb concentration, Lb component ratios and volume ratio of the infected region in the nodules, were found to be similar in both En6500 and Enrei. These results suggest that the specific characteristics of the nodules of hypernodulating mutant, such as low ARA, low Lb concentration and small infected region, may be caused by the insufficient supply of photosynthates to each nodule. It is concluded that many of the specific characteristics described above in the nodules of hypernodulating En6500 are not directly related to genetic factors induced by chemical mutation, but that a secondary effect is involved in that the mutants have an excess number of nodules with a relatively small shoot. On the other hand, the partial tolerance of nodule growth and ARA per plant to nitrate may be involved in another mechanism than the secondary effect of photosynthate deficiency.
In soybean, autoregulatory control may play an important role in optimizing the nodule number where the nodule growth and total N-2 fixation activity can be maximized.

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Language：Japanese   Publisher：一般社団法人日本土壌肥料学会  

DOI： 10.20710/dohikouen.45.0_402_2

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Language：Japanese   Publisher：一般社団法人日本土壌肥料学会  

DOI： 10.20710/dohikouen.45.0_124_2

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Language：Japanese   Publisher：一般社団法人日本土壌肥料学会  

DOI： 10.20710/dohikouen.45.0_123_1

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Language：Japanese   Publisher：一般社団法人日本土壌肥料学会  

DOI： 10.20710/dohikouen.45.0_401_2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：GUSTAV FISCHER VERLAG  

The factor that inactivates the phosphorylated nitrate reductase was purified 870-fold from leaves of Brassica campestris L. ssp. apifera by column chromatography oil DEAE-Toyopearl, Phenyl Sepharose, Sephadex G-200 and Mono Q. Final preparation of the purified factor was nearly homogenous as revealed by polyacrylamide gel electrophoresis and its molecular weight was estimated to be 70 ku using Superose 12 gel filtration column. SDS-polyacrylamide gel analysis of the final preparation showed two protein bands with molecular weight of 30 and 33 ku suggesting that the inactivation factor was composed of two heterogenous subunits. The inactivation of phosphorylated nitrate reductase by the purified protein was neither rime dependent, nor due to degradation of nitrate reductase, suggesting chat the inactivation factor exhibits its effect by binding to the phosphorylated nitrate reductase.

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The hypernodulation soybean mutant lines (NOD1-3, NOD2-4, NOD3-7) and their parent Williams, and the mutant En6500 and its parent Enrei were cultivated in a sandy dune field in Niigata, and the nodules and root bleeding xylem sap were sampled at 50, 70, 90, and 120 d after planting (DAP). The nodule size distribution patterns and concentration of leghemoglobin components were determined. The number of nodules of the hypernodulation mutant lines was about two to three times higher than that of the parent lines irrespective of the sampling date. At 50 DAP the nodule size was relatively smaller in the hypernodulation mutant lines, and the total dry weight of the nodules was almost the same in the mutant lines and their parents. At 70 DAP and 90 DAP, the size distribution of the hypernodulation mutant nodules became.almost the same as that of the parent lines, and both the number and total dry weight of the nodules were higher than those of the parent lines. The concentration of four Lb components was separately measured by capillary electrophoresis. The concentration of the Lb components in the hypernodulation mutant lines tended to be lower than in the parents, but the component ratios were not different between the hypernodulation mutants and their parents. Under field conditions, plant growth and nodulation characteristics were more similar between mutants and parents than in the hydroponic culture reported previously, although the mutants did exhibit hypernodulation traits. These findings suggest that the decrease in the Lb concentration and the different Lb components ratios in the mutants may be caused by secondary effects of excess nodulation, such as photosynthate deficiency, rather than by a genetic defect in mutation. The concentration of major nitrogenous compounds (allantoic acid, allantoin, asparagine, aspartic acid, and nitrate) in the xylem sap was also measured by capillary electrophoresis. The concentration of ureides and nitrate in xylem sap decreased with the plant age, but the asparagine concentration increased during the same period. The concentrations of ureides and asparagine were higher, and the nitrate concentration was lower in the mutant lines than in their parents, possibly due to the higher dependence on N2 fixation than N03 ™ utilization. In the xylem sap, nitrate was the major inorganic anion followed by phosphate, sulfate, and chloride, and potassium was the major cation followed by calcium or magnesium and sodium. © 1998, Taylor &amp
Francis Group, LLC.

DOI： 10.1080/00380768.1998.10414487

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARCEL DEKKER INC  

Application of nitrogen (N) fertilizer at the flowering stage changed the contents of storage compounds in seeds of Soybean (Glycine max L. cvs Enrei and Tamahomare). The effects of the N application on the maturation of soybean seeds were examined by comparing changes in the contents of amino acids, sugars, water, protein, and oil in seeds from N-dressed plants (NDS) with those from undressed plants (UDS) during maturation. The application resulted in a decrease of contents of total and some amino acids (glutamine and asparagine) in developing seeds except for at the early maturation stage and in a decreased protein content of mature seeds. On the other hand, the N application led to faster accumulation of oil in developing seeds and to an increased oil content of mature seeds. Based on these results, it was concluded that the N application at the flowering stage changed the composition of solutes imported by developing seeds and resulted in variations in the contents of storage compounds. The results indicate that it is possible to improve seed quality by fine control of N application.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC SOIL SCIENCE PLANT NUTRITION  

In vivo Fe3+-chelate reductase (FeR) activity in cucumber roots (Cucumis sativus L,) increased by transferring the plants from Fe-sufficient (+Fe) conditions to Fe-deficient (-Fe) conditions, This increase was inhibited by the protein synthesis inhibitor, cycloheximide, The plasma membranes were isolated from the +Fe and -Fe roots and then the enzymatic properties of plasma membrane-bound FeR were characterized, The FeR in the plasma membranes from both the +Fe and -Fe roots reduced Fe3+-citrate using NADH as an electron donor in the presence of Triton X-100, Plasma membrane-bound FeR from both types of roots showed similar K-m values for Fe3+-citrate and NADH at 70 and 100 mu M, respectively, whereas V-max of the enzyme from -Fe roots was three-fold higher than that of the enzyme from +Fe roots, The enzyme was solubilized from plasma membranes with 1.0% Triton X-100 and subsequently analyzed on an isoelectric focusing gel, The activity staining of the gel after electrophoresis showed that four FeR isozymes with different pls of 5.3, 6.8, 7.5, and 8.7 were present on the plasma membranes of both the +Fe and -Fe roots, Only the intensity of the pi 7.5 band was enhanced in the -Fe roots, These results suggested that the increase of FeR activity in the -Fe roots resulted from the increased synthesis of the FeR isoform which is constitutively present in the plasma membranes of +Fe roots.

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Language：Japanese   Publisher：一般社団法人日本土壌肥料学会  

DOI： 10.20710/dohikouen.43.0_104_1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT PHYSIOLOGISTS  

The degradation of storage protein in germinating cucumber seeds was shown to proceed via two distinct steps. First, several proteases with acidic isoelectric points (pIs) were involved in solubilization and partial degradation of 11S globulin, Treatment of seedlings with cycloheximide inhibited this step and the expression of these proteases. Thus, the first step appeared to be governed by these proteases, which were synthesized de novo after imbibition, The first step was observed in dark-grown cotyledons, but the complete degradation of 11S globulin did not occur in the absence of illumination, An additional protease, with a pI of 4.5, was induced by illumination, and it was involved in the further cleavage of the partially degraded products of 11S globulin. Thus, the complete degradation of the storage protein proceeded via a two-step process in illuminated germinating seedlings, Light is needed to induce the second step in the degradation of 11S globulin that supplies the nitrogen required for development of the photosynthetic apparatus in the greening cotyledon.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT PHYSIOLOGISTS  

Barley (Hordeum vulgare L.) has two, differentially regulated, nitrate reductase (NR) genes, one encoding the NADH-specific NR (Nar1) and the other encoding the NAD(P)H-bispecific NR (Nar7). Regulation of the two NR genes by nitrate was investigated in wild-type Steptoe and in an NADH-specific NR structural gene mutant (Az12). Gene-specific probes were used to estimate NADH and NAD(P)H NR mRNAs. The kinetics of induction by nitrate were similar for the two NR genes; expression was generally below the limits of detection prior to induction, reached maximum levels after 1 to 2 h of induction in roots and 4 to 8 h of induction in leaves, and then declined to steady-state levels. Derepression of the NAD(P)H NR gene in leaves of the NADH-specific NR gene mutant Az12 did not appear to be associated with changes in nitrate assimilation products or nitrate flux. Nitrate deprivation resulted in rapid decreases in NADH and NAD(P)H NR mRNAs in seedling roots and leaves and equally rapid decreases in the concentration of nitrate in the xylem sap. These results indicate that factors affecting nitrate uptake and transport could have a direct influence on NR expression in barley leaves.

DOI： 10.1104/pp.107.4.1303

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Language：Japanese   Publisher：日本土壌肥料學會  

The effects of various ATPase inhibitors and ionophores on both H^+-K^+ exchange by excised rice roots, ATPase activity, and formation of pH gradient in the reconstituted plasma membrane vesicles were investigated. The inhibitors and the ionophores completely inhibited H^+-K^+ exchange, but partially both ATPase activity and formation of pH gradient. In some cases, these reagents resulted in reverse H^+-K^+ exchange that mediated influx of H^+ and efflux of K^+. These results suggest that the electrochemical gradient across the plasma membrane controls the H^+-K^+ exchange in point of the rate and the direction. Rb^+ and NH_4^+ were also effective for the exchange K^+, while Na^+, Li^+ and Cs^+ were ineffective.

DOI： 10.20710/dojo.63.1_39

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Plasma membrane vesicles were purified from rice (Oryza sativa L. cv. Nipponbare) roots using aqueous two-phase partitioning. The purified vesicles were solubilized with deoxycholate and reconstituted with soybean phospholipids by gel filtration. Ionophores stimulated the ATPase activity of the reconstituted vesicles. Inhibition of the ATPase activity by vanadate increased from 77 to 93% by the reconstitution. These results indicate that the reconstituted vesicles were sealed and the vanadate-sensitive ATPase activity was purified during the reconstitution. The ATPase activity of the reconstituted vesicles was characterized. Only ATP was effectively hydrolyzed. Apparent K(m) value was 1.26 mm. Divalent cations were necessary for the activity, and their stimulation was in the following order: Mg2+ > Fe2+ > Mn2+, Co2+ >> Ca2+. Half-maximal inhibition by vanadate occurred at 5-mu-M. Monovalent ions stimulated the activity, and cations and anions were effective in the order of NH4+ > K+, Rb+ > Na+ > Li+ > choline+ and NO3- > Cl-, Br- > I- > SO42-, respectively. Optimum pH was 6.5.

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Language：Japanese   Publisher：日本土壌肥料學會  

Membrane vesicles were isolated from homogenates of barley roots by a combination of differential and dextran density gradient centrifugation. The vesicles possessed ATP-dependent and PP_i-dependent proton transport activities inhibited by nitrate and fluoride, respectively, suggesting that the vesicles derive from tonoplast membrane. In tonoplast vesicles, PP_i hydrolysis and PP_i-dependent proton transport showed an absolute requirement for K^+. Rb^+ was able to substitute K^+ in both activities, but Na^+ or Li^+ was not. PP_i hydrolysis was unaffected by anions tested, while PP_i-dependent proton transport was significantly affected. Permeant anions, like NO_3^-, Cl^- and Br^-, were necessary to support proton transport. Stimulation of proton transport by NO_3^- and Cl^- showed saturation kinetics. The result indicates the presence of anion transport systems on the tonoplast.

DOI： 10.20710/dojo.62.4_393

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Language：Japanese   Publisher：一般社団法人日本土壌肥料学会  

DOI： 10.20710/dohikouen.37.0_99_1

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Language：Japanese   Publisher：一般社団法人日本土壌肥料学会  

DOI： 10.20710/dohikouen.37.0_73_2

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Language：Japanese   Publisher：一般社団法人日本土壌肥料学会  

DOI： 10.20710/dohikouen.37.0_340_1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC BIOSCI BIOTECHN AGROCHEM  

Degradation intermediates of nitrate reductase (NR) were extracted from fresh spinach leaves in the presence of protease inhibitors, and concentrated by chromatography on an anti-NR IgG-conjugated Sepharose-4B column. Seven polypeptides, 120 (that of the intact subunit), 110, 100, 76, 64, 62, and 44 kDa, were obtained. Their cross-reactivities to mono-specific polyclonal antibody and monoclonal antibody prepared against NR subunit were assessed by an immunoblotting method. All polypeptides were recognized with mono-specific polyclonal antibody. On the other hand, four of them, at 120, 110, 100, and 44 kDa, were recognized with monoclonal antibody which specifically binds to the NADH-ferricyanide reductase domain of NR. The possibility that these polypeptides were artifacts in preparation was ruled out by an internal tracer experiment. Thus, it is concluded that spinach leaf NR <i>in vivo</i> degraded <i>via</i> putative intermediates catabolic products of 110, 100, 76, 64, 62, and 44 kDa.

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Two monoclonal antibodies, 17(3)9 and 36(79)4, were prepared against nitrate reductase from Spinacia oleracea L. leaves. An enzyme-linked immunosorbent assay showed that 17(3)9, but not 36(79)4, reacted more strongly to heat-denatured than native antigen. These antibodies inhibited NADH-nitrate reductase as well as its various partial activities including reduced methyl vilogen-nitrate reductase, reduced flavin mononucleotide-nitrate reductase and NADH-cytochrome c reductase activities, but not NADH-ferricyanide reductase activity. Immunoblotting after electrophoretic separation of nitrate reductase fragments obtained by Staphyrococcus aureus V8 protease digestion of native enzyme revealed that the two monoclonal antibodies bind to different epitopes located on the 28 kDa of the NADH-ferricyanide reductase domain.

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Extracts from brains of mouse, rat, rabbit, pig, chicken, and duck were assayed for peptidylarginine deiminase, which catalyzes the deimination of arginyl residues. There was peptidylarginine deiminase in the extracts from all the brains that we tested, suggesting the widespread occurrence of this enzyme in vertebrate brains. The levels of the activity in the brains of 8 different inbred strains of mice could be divided into two groups. There is a difference of over 300% between the high and low groups. Furthermore peptidylarginine deiminase purified from the brains of mouse, pig, and chicken showed that there is no great difference in their MW and the optimal conditions for the activity. However, the substrate specificity of the enzyme from chicken brain was somewhat different from those of the enzymes from mammalian brains.

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Language：Japanese   Publisher：一般社団法人日本土壌肥料学会  

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ダイズは，豆腐，味噌，醤油，納豆など我が国の伝統食品の原料作物であるとともに，世界的にも油料および飼料用穀物として生産が増加している。我が国ではダイズは年間約400万トンが消費されているが，国内生産高は約20万トンと低く，平均収量も10aあたり170kg(農林水産省大豆関連データ)と世界平均248kg(FAOSTAT 2013)と比べても低迷している。ダイズは種子にタンパク質を約35-40%と高濃度に含み，開花期以降も多量の窒素を必要とする(大山 2001)。ダイズは，根に土壌微生物である根粒菌を共生し，空中窒素を固定利用できる。また，ダイズは他の作物と同様に肥料窒素や土壌窒素も利用できるが，速効性窒素肥料を多量に施用すると，根粒の着生および窒素固定活性を抑制することが知られている。したがって，ダイズの施肥や栽培技術の改善のためには，根粒による窒素固定と根からの化合態窒素の吸収割合を調べることにより有用な情報が得られる。

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Other Link： http://hdl.handle.net/10191/30053

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Language：English   Publisher：新潟大学農学部  

施肥窒素供給期間の違いによるサトウキビ(Saccharum officinarum L., 品種農林8号)の窒素固定寄与率を15N希釈法で調べる目的で新潟大学の温室内でポット試験を実施した。サトウキビの種茎を水中で発芽させたのち，バーミキュライトを充填した1/5000aのワグネルポットで栽培した。窒素施肥処理は，3区設けた。1)N0区:無窒素培養液のみ供与，2)N100:15N標識の硫酸アンモニウム水溶液(100mgN)を週1回ポットに添加，3)N100N0:移植から移植後6週目まで，15N標識の硫酸アンモニウム水溶液(100mgN)を週1回ポットに添加し，その後無窒素栽培した。移植後植物の地上部の長さと葉数を毎週測定した。また，移植12週間後と20週間後に植物を採取し，各部位の窒素含有量，15N濃度とアセチレン還元活性を測定した。N0区の植物はほぼ生長が停止し，窒素固定だけでは生育を支えるには不十分であることが確認された。N100区とN100N0区の植物は，地上部の長さや葉の数は，大きな差はなかったが，N100N0区の葉は葉色が薄く，窒素含有量もN100区の半分程度であった。移植12週間後には，N100区とN100N0区の株あたり全窒素含有率は，それぞれ408mgNと286mgNであった。15N希釈法で推定した窒素固定量は，N100区とN100N0区でそれぞれ株あたり87mgN(窒素固定由来窒素割合%Ndfa，21%)，48mgN(17%Ndfa)であった。移植20週間後では，N100区とN100N0区の株あたり全窒素含有率は，それぞれ569mgNと292mgNであった。窒素固定推定量は，N100区とN100N0区でそれぞれ株あたり87mgN(%Ndfa，21%)，57mgN(33%Ndfa)であった。器官別窒素固定割合では，枯葉，種茎が高く，緑葉，茎が低い傾向がみられた。

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Other Link： http://hdl.handle.net/10191/23967

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Other Link： http://hdl.handle.net/10191/23964

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サトウキビ(Saccharum officinarum L., 品種農林8号)の茎から分離された内生窒素固定菌(JA1株，JA2株，菌の性質からGluconacetobacter diazotrophicusと予測された)の15N2固定活性とアセチレン還元活性(ARA) を調べた。試験管内で固形LGIP培地または液体LGIP培地で培養した菌を，酸素濃度を0から20%まで変化させて，24時間15N2固定活性を測定し，その後同じ試験管で1時間ARAを測定した。固形培地では，O2 0.4%で最大活性を示したが，液体培地では，O2 0%で最大活性を示した。どちらも，O2 20%まで，酸素濃度が高まるにつれて活性が低下した。JA1株の生育密度(吸光度)とARAをLGIP液体培地に植継いでから10日後まで毎日測定した。菌密度は，植継1日後に急速に高まり6日後までは増加したが，それ以降10日目までは増加が抑制された。JA1株の増殖抑制はクオラムセンシング機構によると予想される。ARAも植継5日目までは上昇したが，6日目以降急激に低下した。7日目から10日目までは，ほとんど活性を示さなかった。この結果は，本菌の窒素固定活性は，菌の増殖中にのみ発現し，増殖が止まると窒素固定活性も停止することが示唆された。このことが，サトウキビ植物体内でも同様であるとすると，内生菌が増殖を続けることが，窒素固定の維持に必要であると推定される。内生菌から，植物への窒素の放出は，サトウキビの生育促進に重要である。液体培養したJA1株は，24時間の15N2固定直後では，大部分の固定窒素が菌体内に残存しており，培地中には，固定窒素総量の4%程度のみ放出していた。一方，24時間15N2を固定させた後，酸素濃度0-20%で10日間培養を続けたところ，酸素濃度が高い程，放出15N量の割合が増加し，O2 20%では，固定窒素総量の40%が培地に放出されていた。これらの結果は，JA1株は，窒素固定停止後，増殖を停止し，菌の死に伴う分解により，窒素を放出するのではと予想された。

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Other Link： http://agriknowledge.affrc.go.jp/RN/2010700658

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Population of GUS marked bradyrhizobia inoculated in five different soils including calcined vermiculite increased over 10 times in a week. Each soybean seed was planted in a paper pot filled with vermiculite inoculated with or without bradyrhizobia, and the 10-d-old seedlings were transplanted with a paper pot to the first cropping reclaimed field in Niigata. In addition to the conventional fertilization (1.6 g N m^<-2>), deep placement of urea, 100 d type coated urea, or calcium cyanamide (10 g N m^<-2>) was applied. In conventional fertilization the soybean plants transplanted with non-inoculated paper pot did not nodulate due to the absence of indigenous bradyrhizobia, and the seed dry weight was lowest, about 9 g plant^<-1>. Seed dry weight of the plants either with paper pot inoculation or seed inoculation was higher, about 22 g plant^<-1>. Soybean plants with deep placement of calcium cyanamide exhibited prolonged leaf activity. With deep placement of urea, coated urea and calcium cyanamide, seed dry weight increased to 32-37, 40-44 and 38-47g plant^<-1> respectively. The highest average seed yield was obtained in the inoculated paper pot transplantation with calcium cyanamide.

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