Hiroyuki Fujikake

Rapid and reversible nitrate inhibition of nodule growth and N2 fixation activity in soybean (Glycine max (L.) Merr.)

Abstract Nodulated soybean (Glycine max. (L) Merr. cv. Williams) plants were hydroponically cultured, and various combinations of 1-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 N2 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.

Effect of exogenous salicylic acid supply on nodule formation of hypernodulating mutant and wild type of soybean

Abstract Soybean plant is characterized by a systemic autoregulatory control system of nodulation (autoregulation) by initial infection with rhizobia, and plants commonly display a systemic acquired resistance (SAR) to pathogenic microbe infection related to salicylic acid (SA) signal transmission. We investigated the effect of exogenous SA supply on soybean nodulation to determine whether SA affects the autoregulation of nodulation. Seedlings of the hypernodulating mutants NOD1-3, NOD2-4 and their parent cv. Williams were treated or not treated (control) with a 100 μmS-SA solution at 5 d before the inoculation of Bradyrhizobium japonicum strain USDA110. The nodule dry weight and the number of nodules of the wild type soybean Williams exhibiting autoregulation drastically decreased by the addition of 100 μm SA. The decrease in the nodule number was not caused by the reduction of the rhizobium number in the medium. Salicylic acid inhibited only early nodule formation and did not affect the growth of formed nodules. The inhibitory effect of SA on the nodulation of NOD1-3 and NOD2-4 was significantly less pronounced than that in Williams. These results indicate that SA is directly involved in signal transmission in the autoregulation, and that SA or the SAR induced by SA stimulates the autoregulation of nodulation in soybean.

Effect of deep placement of calcium cyanamide, coated urea, and urea on soybean (Glycine max (L.) Merr.) seed yield in relation to different inoculation methods

Abstract 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 N2 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 RI stage, suggesting that the basal deep placement of CaCN2 or CU-100 for soybean cultivation enabled the supply of N without concomitant depression of N2 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.

Effect of Nitrate on Nodulation and Nitrogen Fixation of Soybean

1.1 Biological nitrogen fixation and nitrogen nutrition in soybean plants Biological nitrogen fixation is one of the most important processes for ecosystem to access available N for all living organisms. Although N2 consists 78% of atmosphere, but the triple bond between two N atoms is very stable, and only a few group of prokaryotes can fix N2 to ammonia by the enzyme nitrogenase. Annual rate of natural nitrogen fixation is estimated about 232 x 106 t, and the 97% depends on biological nitrogen fixation (Bloom, 2011). This exceeds the rate of chemical nitrogen fertilizer uses about 100 x 106 t฀in 2009. Soybean can use N2, though symbiosis with nitrogen fixing soil bacteria, rhizobia, to make root nodules for harboring them. Soybean (Glycine max [L.] Merr.) is a major grain legume crop for feeding humans and livestock. It serves as an important oil and protein source for large population residing in Asia and the American continents. The current global soybean production was 231 x 106 t in 2008 (FAOSTAT). It is a crop predominantly cultivated in U.S.A., Brazil, Argentina and China, which together contribute nearly 87 percent of the total world produce in 2008. Soybean has become the raw materials for diversity of agricultural and industrial uses. Soybean seeds contain a high proportion of protein, about 40% based on dry weight, therefore, they require a large amount of nitrogen to get a high yield. About 8 kg N is required for 100 kg of soybean seed production. Soybean can use atmospheric dinitrogen (N2) by nitrogen fixation of root nodules associated with soil bacteria, rhizobia. Soybean plants can absorb combined nitrogen such as nitrate for their nutrition either from soil mineralized N or fertilizer N. It is well known that heavy supply of nitrogen fertilizer often causes the inhibition of nodulation and nitrogen fixation. Therefore, only a little or no nitrogen fertilizer is

Systemic and local effects of long-term application of nitrate on nodule growth and N2 fixation in soybean (Glycine max [L.] Merr.)

Abstract The effect of nitrate placement on the nodule number, nodule growth, and N2 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 N2 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 N2 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 N2 fixation activity and on nodule growth but not on the upper nodule number after longterm 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.

Photoassimilate partitioning in hypernodulation mutant of soybean (Glycine max (L.) Merr.) NOD1-3 and its parent Williams in relation to nitrate inhibition of nodule growth

Abstract 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 Nz fixation activity. A whole shoot of Williams and NOD1-3 plants was exposed to 14CO2 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 14C 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 14C 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.

Changes in four leghemoglobin components in nodules of hypernodulating soybean (Glycine max [L] Merr.) mutant and its parent in the early nodule developmental stage

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.

Long-Term Effect of Nitrate Application from Lower Part of Roots on Nodulation and N2 Fixation in Upper Part of Roots of Soybean (Glycine max (L.) Merr.) in Two-Layered Pot Experiment

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.

Effects of anion channel blockers on xylem nitrate transport in barley seedlings

Abstract 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 13NO3 − and 15NO3 − as tracers. The seedling roots were pretreated with either blocker for 1 h and then 13NO3 − or 15NO3 − was supplied to the medium. Real-time images of 13N 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 13N in whole seedlings using a Bioimaging Analyzer System (BAS) showed a 13N-accumulation in both shoots and roots of the control plants but reduced 13N-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 15NO3 − 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.

Effect of Short-Term Application of Nitrogen on the Accumulation of β-Subunit of β-Conglycinin in Nitrogen-Starved Soybean(Glycine max L.) Developing Seeds

Abstrct It has been reported that the accumulation of the β-subunit of β-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 playa key role in regulating the accumulation of β-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 β-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 β-subunit mRNA within 12 h, while asparagine did not induce the accumulation of β-subunit mRNA for 7 d. An inhibitor of transaminases, (aminooxy)acetic acid, enhanced β-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 β-subunit mRNA and continuous protein storage in immature seeds appeared to be regulated by glutamine, its metabolites or related compounds.