Characterizing nicotianamine aminotransferase: Improving its assay system and details of the regulation of its activity by Fe nutrition status

Under iron deficient conditions, graminaceous plants secrete mugineic acid family phytosiderophores (MAs) from their roots to dissolve sparingly soluble iron compounds in the rhizosphere, and take up iron in the form of an Fe³⁺-MAs complex (Takagi 1976). A good correlation has been reported between the tolerance of Fe-deficiency and the amount of secreted MAs (Takagi 1993). Therefore, by using the genes involved in MAs biosynthesis, molecular breeding might produce transgenic plants tolerant to Fe-deficiency with a high level of MAs secretion. The biosynthetic pathway of MAs from L-methionine has been clarified (Fig. 1) and the enzymes participating in this process are now being investigated to isolate the genes responsible. Nicotianamine aminotransferase (NAAT) catalyzes the amino group transfer between nicotianamine (NA) and 2-oxoglutaric acid (Fig. 1). In order to purify NAAT, enzyme assay methods for NAAT have been developed and modified (Shojima et al. 1990; Ohata et al. 1993; Kanazawa et al. 1994). Some characteristics of NAAT have been reported using these enzyme assay methods (Kanazawa et al. 1994, 1995). Here, we further investigate some characteristics of this enzyme to improve the enzyme assay method, namely 1) the effect of K⁺ and Mg²⁺ on NAAT activity in vitro, and 2) the direct influence of MAs, Fe³⁺, and Fe²⁺ on NAAT activity. In addition, based on these results, the induction of enzyme activity by Fe-deficiency and suppression of the activity by Fe-resupply was investigated, by applying the new enzyme assay method.     

A new method for measuring microbial biomass nitrogen in soils: Direct extraction after toluene treatment

Abstract

Phenolic compounds occurring naturally are reducing agents which react with hydrous Fe oxide and Mn oxides (Lehmann et al. 1987). Diphenols such as hydroquinone, resorcinol, and catechol were oxidatively darkened by the presence of hydrous Fe oxide though the degree of darkening was much less pronounced than that by Mn oxides (Shindo and Huang 1984). Okazaki et al. (1976) suggested that polyphenols are one of the materials responsible for the dissolution of Mn in a paddy soil under reducing conditions.     

Effect of rice straw application on CH4 emission from paddy fields

The effect of rice straw (RS) incorporated at the time of plowing in the previous cr–p season on CH₄ emission from rice paddies was investigated in a pot experiment. Rice straw that incorporated just before transplanting of rice seedlings (June) into a paddy field was collected after the harvest (October) and at the beginning of the next cropping period (May). Methane emission rates from the rice-planted pots with the application of fresh RS, RS collected in October. and RS collected in Mayas well as the pots without RS application were measured using the chamber method. The composition of organic constituents in the three kinds of RS was estimated by the proximate analysis. The cumulative amount of CH₄ emitted during the first 50–d period was lower in the order of the pots with RS collected in May, pots with RS collected in October, and pots with fresh RS. The cumulative amount of CH₄ emitted throughout the rice growth period from the pots with fresh RS and with RS collected in October was significantly larger while that from the pots with RS collected in May did not differ statistically compared with the total CH₄ emission from the pots without RS. These results suggested that there was an overall decrease in the amount of organic constituents in RS based on the large differences in T-C content and similar composition of organic constituents between the fresh RS and RS collected in May. Significant effect of RS continuously applied during the previous cropping period on the increase in CH₄ emission was discussed.     

Response of differentiated and degenerated spikelets to top-dressing,shading and day/night temperature treatments in rice cultivars with large panicles

The presence of large panicles enables to reach a sufficient number of surviving spikelets per unit area for further increase of rice yield. However, the response of differentiated and degenerated spikelets to environmental factors in terms of number in cultivars with large panicles has not been elucidated. By applying top-dressing at different rates and frequencies, shading, and day/night temperature treatments in pot experiments, we observed the response of the main stem in two cultivars with large panicles: Yangdao 4 (Chinese indica) and Akenohoshi (Japanese japonica-indica hybrid). The results showed that top-dressing increased the number of differentiated spikelets per panicle and decreased the percentage of degenerated spikelets. Consequently, the number of surviving spikelets per panicle increased. Shading increased the percentage of degenerated spikelets, resulting in the decrease of the number of surviving spikelets per panicle. Higher day/night temperature treatments led to a higher percentage of degenerated spikelets. The treatments affected mainly the spikelets on the secondary branches, while the number of spikelets on the primary branches did not change appreciably, irrespective of the presence of differentiated, degenerated, and surviving spikelets. The influence on the number of surviving spikelets per panicle depended on the number of differentiated spikelets. Shading effect on the percentage of degenerated spikelets also depended on the number of differentiated spikelets per panicle. The decrease of the percentage of degenerated spikelets was associated with the increase of the shoot dry weight (SDW) at heading and the ratio of SDW-differentiated spikelets. The SDW and N absorption at heading affected more significantly the number of surviving spikelets compared to the ratio of surviving spikelets-SDW and surviving spikelets-N, respectively. The above results were similar in Akenohoshi and Yangdao 4. In Akenohoshi, the percentage of degenerated spikelets responded much more conspicuously to the change of SDW or the ratio of SDW -differentiated spikelets than that in Yangdao 4. So did the number of surviving spikelets to SDW and N absorption at heading. Akenohoshi showed a higher ratio of surviving spikelets-N than Yangdao 4. These differences may account for the fact that Akenohoshi showed a larger number of surviving spikelets per panicle than Yangdao 4, even at the same levels of SDW and N absorption.     

Nutrient availability and response of sago palm (Metroxylon sago Rottb.) to controlled release N fertilizer on coastal lowland peat in the tropics

Sago palms (Metroxylon sagu Rottb.) growing on peat soils were found to grow more slowly and to show a lower production than palms growing on mineral soils. This difference was related to the physical and chemical constraints of peat soils, which include low bulk density, high acidity, and low N, P, K, Ca, Zn, and Cu levels. In coastal lowland peat soils, the distance from the sea has been found to be an important determinant of soil elemental composition. We predicted that a sufficient supply of N at the rosette stage would improve sago palm growth and that the availability of N in soil to which controlled release N fertilizer was applied might be higher than that in soil treated with soluble fertilizer. To investigate the changes in the nutrient composition of peat soils at various distances from the sea and the effect on sago palm growth, we studied sago palm areas in Indonesia and Malaysia. To observe the influence of N on the growth performance, we also conducted a fertilizer experiment on coastal lowland peat soil in Indonesia. Distance from the sea had no significant effect on the cation concentration in the soil solution (with the exception of Mg) or on the levels of soil-exchangeable cations. No significant differences were observed between the concentrations of exchangeable cations in surface peat soils and those in mature leaves. However, the concentrations of K, Na, and Ca in mature leaves increased significantly with their concentrations in the soil solution. This finding implies that the concentrations of cations in sago palm leaves depend directly on the concentrations of cations in the soil solution. No significant effect of N fertilizers on plant height and leaf formation was observed. N fertilizers applied twice a year did not affect appreciably the foliar concentration of N determined in December 1998 (5 months after the initial application) and December 1999. In June 2000, we detected a significantly higher concentration of N (p < 0.01) in young leaves of the palms treated with LP-100 or urea than in control leaves. However, no significant difference was detected between the LP-100 and urea treatments in the concentration of N in both mature and young leaves. This finding indicated that the concentration of N in sago palm leaves increased with the level of soil-applied N, regardless of whether N was applied as controlled release fertilizer or in the soluble form. We anticipate that a significant difference in the effects of these N fertilizers may occur during the next rainy season, when there should be a considerable loss of soluble N.     

Influence of surface printing materials on the degradability of biodegradable plastic films in soil

Effect of surface printing on the biodegradability of plastic films was studied. Biodegradable films (polybutylene-succinate (PBS)) printed with four kinds of gravure inks were placed in soil for 1 year. The inks consisted of carbon black-pigment with four kinds of resins: poly-(ε-caprolactone) (PCL), nitrocellulose-polyamide blended resin (NT), polyvinyl chloride-vinyl acetate copolymer (V), and nitrocellulose (NC). Degradation of film specimens printed on one side and both sides as well as the control film without printing was monitored every 3 months by collecting sample specimens for the measurement of weight loss. No appreciable degradation was observed until 6 months after placement in soil for the control specimens and until 9 months for the printed specimens. And the degradation of the PCL- and NC-printed specimens with one-side printing and V-printed specimens with both-side printing was significantly slower than that of the control specimens without printing after 9-month placement at p < 0.05. Only after 12 months of placement, was the degradation significantly faster for the specimens printed on one side than for those printed on both sides except of the specimens printed with NC. There was no difference in biodegradability among PCL, NT, NC, and V resins. Specimens printed on both sides did not show any appreciable weight loss after 1 year in soil (percentage of maintenance of weight exceeding 98%). Microscopic observation indicated that the degradation mainly proceeded from the non-printed side to the printed side cross-sectionally.     

Identification of the strong vasorelaxing substance scirpusin B, a dimer of piceatannol, from passion fruit (Passiflora edulis) seeds

Piceatannol is present in passion fruit (Passiflora edulis) seeds in high amounts. In this study, we isolated the second major polyphenolic compound of passion fruit seeds and identified it as scirpusin B, which is a dimer of piceatannol. We investigated the antioxidant activities and vasorelaxing effects of these polyphenols. Their antioxidant effects were measured using an in vitro 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, and their vasorelaxant effects were determined ex vivo in rat thoracic aorta. Both polyphenolic compounds exhibited potent antioxidant activities and significant vasorelaxant effects in endothelium-intact aortas. More specifically, scirpusin B exerted a greater antioxidant activity and vasorelaxant effect compared with that of piceatannol. Additionally, the vasorelaxation effects of the compounds were induced via the NO derived from the endothelium. This study provides the possibility that polyphenols in passion fruit seeds are effective against cardiovascular diseases (CVDs).     

Chemical characteristics and potential source of fulvic acids leached from the plow layer of paddy soil

To understand better the chemical characteristics and source of dissolved organic matter (DOM) leached from the plow layer of rice paddies, a lysimeter study was conducted, which simulated submerged paddy topsoil during rice growth. The fulvic acid (FA) fraction in the percolation water from the lysimeter was collected by adsorption onto insoluble polyvinylpyrrolidone (PVP), and the temporal variations in its elemental composition, molecular size distribution, IR spectra, ¹³C CPMAS NMR spectra, and δ¹³C values were investigated. The proportion of the FA fraction to bulk DOM varied greatly, but the chemical characteristics of the FA fraction did not change appreciably during the experimental period. Thus, it is considered that the changes in the DOM composition in percolation water were mainly due to the differing contributions of the FA fraction. Further, to investigate the source of the FA fraction in the leachate, the chemical characteristics of the FA fraction in the leachate were compared with those extracted from the plow layer soil. A sequential extraction of the FA fraction was conducted using a sequence of water, 0.25 M Na₂SO₄, 0.1 M Na₄P₂O₇ (pH 7.0), 0.1 M Na₄P₂O₇ (pH 10.5), and NaBH₄+0.1 M Na₄P₂O₇ (pH 10.5). It was found that the water- and 0.25 M Na₂SO₄-extractable fractions, which were most mobile, were not the only source of the FA fraction in the leachate. The small molecular size sub-fraction of the NaBH₄+0.1 M Na₄P₂O₇ (pH 10.5)-extractable FAs, most of which are probably bound mainly to iron oxides, are considered to be another source of the FA fraction leached from the plow layer of paddy fields.     

Determination of organic acids by high-performance liquid chromatography with electrochemical detection during wine brewing

Voltammetric determination of acids by means of the electrochemical reduction of quinone was applied to high-performance liquid chromatography (HPLC) with electrochemical detection (ED) for determining organic acids in fruit wines. A two-channel HPLC-ED system was fabricated by use of an ion-exclusion column and an electrochemical detector with a glassy carbon working electrode. Aqueous solution of 0.1 mM HClO(4) and ethanol containing 2-methyl-1,4-naphthoquinone served as a mobile phase and reagent solution, respectively. Determination of acetic, citric, lactic, malic, succinic, and tartaric acids was made by measuring the peak areas of the flow signals due to the reduction current of quinone caused by the eluted acids. The peak area was found to be linearly related to the acid amount ranging from 0.1 to 40 nmol per 20 microL injection. The present method was characterized by reproducibility with the simple and rapid procedure without derivatization of analytes. The method was shown as an effective means for following acid contents in fruit juices during fermentation with wine yeast.     

RFLPs of chloroplast and mitochondrial DNA in wild soybean, Glycine soja, growing in China

RFLPs of chloroplast (cp) and mitochondrial (mt) DNA have been used to characterize the cytoplasmic genome of wild soybean, Glycine soja, growing in China. We have found from the RFLPs of that the cytoplasmic genomes of most Chinese wild soybeans have a combination of cpIII with mt-a or a combination of cpII with mt-b accompanied by mtIV or mtV according to our classification. CpII was not observed in combination with mt-a and cpIII was not observed in combination with mt-b. The regional distribution of these two types showed clines with opposite directions. The Yangtze River Valley had the greatest diversity in each of cp and two mt profiles detected in this study and the cytoplasmic genome combining these three profiles showed the highest degree of polymorphism in this region. The Yangtze River Valley may be a center of cytoplasmic diversity of wild soybean and may contain various genetic resources of soybean.