Kuni Sueyoshi

Soybean Seed Production and Nitrogen Nutrition

The world population is consistently increasing, and it is over 7 billion in 2012, while the land area for agricultural use is limited. Therefore, the increase in crop production per area is very important. Soybean (Glycine max (L.) Merr.) originates from East Asia, and soybean seed is one of the most important protein sources for human and livestock all over the world. Annual production of soybean (262 M (million) t in 2010) is the fourth of the major grain crops, after maize (844 M t), paddy rice (672 M t) and wheat (650 M t) [1]. In the whole world, over 85% of soybean is used for oil and the residue is used for animal feed. Annual soybean seed production has been steadily increasing for recent decades (91 M t in 1980, 109 M t in 1990, 161 M t in 2000, 262 M t in 2010) [1]. The cultivation area of soybean is 102 M ha in 2010. Major soybean production countries (annual production in 2010) are USA (90.6 M t), Brazil (68.5 M t), Argentina (52.7 M t), China (15.1 M t), and India (9.8 M t) in this sequence. Soybean production in Japan in 2010 was only 223,000t and it accounted for 5% of the total consumption in Japan. The world average seed yield is 2.56 t ha-1 in 2010, and is higher in the USA (2.92 t ha-1), Brazil (2.94 t ha-1), and Argentina (2.90 t ha-1) compared with China (1.77 t ha-1), Japan (1.62 t ha-1) and India (1.07 t ha-1) and other countries.

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

Appearance of endopeptidases during the senescence of cucumber leaves

Abstract 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 Mg 2+ , therefore, it might exist constitutively and its activity might be regulated mainly at a post-translational level responding to nutrient and environmental conditions.

Effect of nitrogen application at the flowering stage on the quality of soybean seeds

Abstract 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 r...

Characterization of plasma membrane-bound Fe3+-chelate reductase from Fe-deficient and Fe-sufficient cucumber roots

Abstract 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 μM, respectively, whereas Vmax 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 ...

Increase of PEPC activity in developing rice seeds with nitrogen application at flowering stage

The activity of phosphoenolpyruvate carboxylase (PEPC) in the developing seeds of rice plants (Oryza sativa L. cv Nipponbare) that received 0.5 mol m-2 ammonium sulfate at the flowering stage was 20 to 60% higher than that in plants without N application. This increase was apparent throughout grainfilling although the enzyme activity gradually decreased towards maturity regardless of N supply. The seeds from nitrogen-dressed plants contained higher levels of amino acids and protein than those from undressed plants, and the increase in protein content in mature rice grain was correlated with that of PEPC activity in developing seeds at mid-grainfilling stage for the 6 cultivars tested (R2 = 0.72).

A serine endopeptidase from cucumber leaves is inhibited by l-arginine, guanidino compounds and divalent cations

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.

Purification of inactivation factor of phosphorylated nitrate reductase from leaves of Brassica campestris

Summary The factor that inactivates the phosphorylated nitrate reductase was purified 870-fold from leaves of Brassica campestris L. ssp. apifera by column chromatography on 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 time dependent, nor due to degradation of nitrate reductase, suggesting that the inactivation factor exhibits its effect by binding to the phosphorylated nitrate reductase.

Transcriptome and Metabolome Analyses Reveal That Nitrate Strongly Promotes Nitrogen and Carbon Metabolism in Soybean Roots, but Tends to Repress It in Nodules

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.

Effects of amides on the expression of NADH and NAD(P)H nitrate reductase genes in barley seedlings

Barley has two nitrate reductase (NR) genes; one encoding the NADH-specific NR and the other encoding the NAD(P)H-bispecific NR. We examined the effects of exogenously supplied amides on the nitrate-induced expression of two NR genes in hydroponic cultured barley seedlings. In leaves and roots of barley seedlings pretreated with glutamine and asparagine and subsequently treated with nitrate, NR mRNAs were transiently accumulated and then rapidly declined. Total nitrate uptake was inhibited only 25 to 30% by treatment of amides indicating that the repression of NR gene expression by amides was not caused by the inhibition of nitrate uptake. Concentration of tissue amides were increased by externally supplied amides and well correlated with the levels of NR mRNAs in leaves and roots. These results suggested that amides act as a factor controlling NR gene expression in barley.