Tahei Kawachi

Role of xylem sap nitrate in the regulation of nitrate reductase gene expression in leaves of barley (Hordeum vulgare L.) seedlings

Abstract 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.

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.

Contribution of shoots and roots to in vivo nitrate reduction in NADH-specific nitrate reductase-deficient mutant seedlings of barley (Hordeum vulgare L.)

Abstract 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 15N-incorporation model was applied to NADH-specific NR-deficient mutant (AzI2) seedlings of barley. The N-deprived seedlings were treated with a nutrient solution containing 2.3 mM N03 - and 0.2 mM N02 -, and were labeled with different amount of 15N for 48 h 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 15N03 - incorporation and translocation of absorbed 15N03 - 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 15N 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 15N 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.

Management of nitrogen fertilizer application rates based on soil nitrogen fertility with the goal of lowering nitrate nitrogen concentrations in Sudangrass (Sorghum sudanense (Piper) Stapf)

Abstract High levels of nitrate nitrogen (NO3-N) in forage crops can poison ruminants. To promote stable production of Sudangrass (Sorghum sudanense (Piper) Stapf) with safe NO3-N levels, we investigated the influence of soil nitrogen (N) on Sudangrass yield and on NO3-N concentrations, and we propose a fertilizer management strategy based on soil N levels for a regime based on the application of cattle manure composted with sawdust. We used the available N content determined by incubation of fresh soil as an indicator of soil N. Both the yield and NO3-N concentration of Sudangrass increased with increasing available N. The NO3-N concentrations in the first grass harvest remained lower than 2 g kg−1 (dry-matter basis), which represents the critical level to avoid nitrate poisoning of ruminants in Japan, with an available N content of 40 mg kg−1 (dry-soil basis), the level where yield remained approximately stable without additional N fertilizer. The NO3-N concentration increased linearly with increasing available N and exceeded the critical level when available N exceeded 50 mg kg−1. Under low levels of N (less than 30 mg kg−1), the target yields for the first grass harvest, with safe levels of NO3-N, could be obtained with the application of 10 g N m−2 in fertilizer, lower than the recommended rate of 15 g N m−2. The NO3-N concentration was higher in the second grass harvest under the same levels of soil N with an available N of 40 mg kg−1 or more. The NO3-N concentrations in the second harvest exceeded the critical level when the available N was 40 mg kg−1. These results indicate that available N content is an effective indicator for the stable production of Sudangrass with acceptably low NO3-N concentrations.

Temporary application of nitrate to nitrogen-deficient soybean plants at the mid- to late-stages of seed development increased the accumulation of the β-subunit of β-conglycinin, a major seed storage protein

Abstract The accumulation of the β-subunit of β-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 NaN03 to the medium), β-subunit accumulation was first detected at 30 d after flowering (DAF). In N-deficient plants (supply of 1 mM NaNO3), the accumulation of the β-subunit was stronglyinhibited. When 5 mM 15N-labeled NO3 - was supplied for 1 week to N-deficient T201 plants before 28 DAF, the β-subunit protein did not accumulate, but the application of 5 mM NO3 - after 28 DAF, led to a substantial accumulation of the β-subunit. The total 15N percentage in mature seeds was similar, irrespective of the time of feeding, regardless of the accumulation of the β-subunit. The 7 S (mostly composed of β-conglycinin) and 11 S globulins (mostly glycinin, which was another major component of storage protein) contained N from 5 mM 15NO3 - 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 β-subunit accumulation in N-deficient plants when feeding was carried out after 28 DAF, and b) incorporation of fed NO3 - was similar between 7 Sand 11 S globulins.

Isolation and characterization of a novel Arabidopsis thaliana mutant that shows low nitrate concentration in shoots

Abstract A novel Arabidopsis thaliana mutant that shows a low concentration of nitrate in shoots was isolated from an ethyl-methanesulfonate-mutagenized M2 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.