Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme in nitrogen metabolism; it catalyzes the critical incorporation of inorganic ammonium into glutamine. Two full-length cDNAs that encode the rice (Oryza sativa) cytosolic glutamine synthetase1 genes (OsGS1;1 and OsGS1;2) were isolated from a Minghui 63 normalized cDNA library, and glnA encoding GS in Escherichia coli was isolated by PCR amplification. Transformants for GS gene (GS1;1, GS1;2, and glnA) in rice were produced by an Agrobacterium tumefaciens-mediated transformation method, and transcripts of GS gene accumulated at higher levels in the primary transgenic plants. Our results indicated an increased metabolic level in GS-overexpressed plants, which showed higher total GS activities and soluble protein concentrations in leaves and higher total amino acids and total nitrogen content in the whole plant. Decreases in both grain yield production and total amino acids were observed in seeds of GS-overexpressed plants compared with wild-type plants. In addition, GS1;2-overexpressed plants exhibited resistance to Basta selection and higher sensitivity to salt, drought, and cold stress conditions, whereas the other two types of GS-overexpressed plants failed to show any significant changes for these stress conditions compared with wild-type plants.

Overexpressed glutamine synthetase gene modifies nitrogen metabolism and abiotic stress responses in rice

Aspartate aminotransferase (AAT) is a key enzyme in the synthesis of amino acids. It plays an important role in regulating carbon and nitrogen metabolism in almost all organisms. In this study, we over-expressed in rice separately all three AAT genes from rice (OsAAT1~3) and one AAT gene from Escherichia coli (EcAAT). Over-expression was driven by the CaMV 35S promoter and constructs were introduced into rice by Agrobacterium tumefaciens-mediated transformation. Compared with control plants, the transformants showed significantly increased leaf AAT activity and greater seed amino acid and protein contents. No other phenotypic changes were observed. The total leaf AAT activities in plants over-expressing OsAAT1, OsAAT2, and EcAAT were 26.6, 23.6, and 19.6 A min−1 mg−1 FW (A: units of activity, defined as increase of absorbency per min per mg; FW: fresh weight), which were significantly higher than that in the wild-type control (17.7 A min−1 mg−1 FW). The amino acid content in seeds of transgenic plants over-expressing OsAAT1, OsAAT2, and EcAAT was 119.36, 115.36, and 113.72 mg g−1, respectively, which were 16.1, 12.0, and 5.4% higher, respectively, than that in the control plants. The transgenic plants over-expressing OsAAT1, OsAAT2, and EcAAT had significantly higher protein contents (increased 22.2, 21.1, and 11.1%, respectively) than wild-type plants. No significant changes were found in leaf AAT activity, seed amino acid content or protein content in OsAAT3 over-expressed plants. The expression patterns of the three OsAAT genes and their different functions are also discussed.

Over-expression of aspartate aminotransferase genes in rice resulted in altered nitrogen metabolism and increased amino acid content in seeds

Urea production and disposal Urea production is used by the body to remove potentially toxic products of nitrogen metabolism and may also have evolved for the disposal of hydrogen ions. Ammonia is a product of amino acid metabolism and the major source of circulating ammonia is the gastrointestinal tract. 2 It is neurotoxic, possibly due to its effect on the glutamate dehydrogenase pathway causing a reduction in the a-ketoglutarate available to the citric acid cycle." Other mechanisms are possible, but the overall effect is interference with preand post-synaptic function. Animals must therefore remove ammonia from the body. This may be direct (ammonotelic) as in fish, via uric acid (uricotelic) as in frogs, or via urea (ureotelic) as in mammals. Urea accounts for 80-90010 of nitrogen excretion in man; assuming a diet containing 100 g protein/day, this amounts to 25-35 g urea daily." Urea exceeds creatinine as a component of urinary nitrogen by a factor of up to 30. Urea synthesis from ammonia occurs only in the liver, by means of a cyclical process in which

https://journals.sagepub.com/doi/pdf/10.1177/000456329202900301

Analytical reviews in clinical biochemistry: the estimation of urea.

A virulent strain of Legionella pneumophila serogroup 1, subgroup Pontiac, was grown in continuous culture at a constant growth rate under iron-replete and iron-limited conditions. Iron limitation was achieved by the removal of ferrous sulfate and hemin from the chemically defined medium. Residual contaminating iron, 0.45 microM, was sufficient to support iron-limited growth. Typical iron-replete cultures metabolized 3.3 microM iron. Serine provided the principal source of carbon and energy for both cultures, although iron-replete cultures also depleted a number of other amino acids. There was a 40% decrease in culture biomass under iron-restricted conditions. Iron limitation did not significantly affect carbohydrate metabolism, with the molar growth yield for carbon (Ycarbon) comparable for both cultures. However, under iron-limited conditions a sixfold increase in Yiron correlated with a significant decrease in the iron content of the biomass, as the culture utilized the available iron more efficiently. Highly pleomorphic iron-replete cultures became uniform cultures of short fine rods when adapted to iron-deficient conditions. In addition to the morphological and physiological changes, iron limitation had a critical effect on culture virulence. The virulence of this strain was significantly (P < 0.05) reduced when the culture was subjected to iron-limited conditions. This phenomenon was reversible, with a significant increase in culture virulence upon reversion to iron-replete conditions. When compared in an in vitro macrophage assay, the number of culturable avirulent iron-limited cells located intracellularly after infection was significantly lower than for the virulent replete and control cultures. These results further support the role of environmental parameters in regulating the virulence of L. pneumophila.

Influence of iron-limited continuous culture on physiology and virulence of Legionella pneumophila.

Phomopsis liquidambari can establish a mutualistic symbiotic relationship with rice. It promotes the growth and yield of the host plant and reduces the amount of nitrogen (N) fertilizer required for plant growth. However, the mechanisms responsible for the effects of the fungal endophyte on N use in rice are largely unknown. We conducted a hydroponic experiment to investigate the effects of P. liquidambari on N uptake and N metabolism in rice plants. Rice plants were cultivated in the presence or absence of P. liquidambari under three N levels. Under the low-N treatment, fungal infection significantly increased the biomass, and the total N, soluble protein, free amino acid, free NH4
 +, and chlorophyll contents of rice roots and shoots. The activities of nitrate reductase and glutamine synthetase were increased in infected rice plants. Some genes related to N uptake (OsAMT1;1, OsAMT1;3, OsAMT2;2, OsAMT3;2, OsAMT3;3, OsNRT2;1) and N metabolism (OsNR1, OsGS1, OsGS2, OsNADH-GOGAT) were also up-regulated in infected plants under the low-N treatment. However, these effects gradually weakened as the N level increased. The colonization rate of the endophyte substantially decreased with increasing N levels. Taken together, these results suggest that low-N fertilization induces a physiological state in rice that is favorable for the P. liquidambari symbiosis. The greater extent of P. liquidambari colonization under low-N conditions stimulated the expression of several genes involved in N uptake and N metabolism in rice, thereby enhancing N utilization. These results have implications for enhancing plant growth in low-input systems at nutrient-poor sites.

Effects of the fungal endophyte Phomopsis liquidambari on nitrogen uptake and metabolism in rice

A technique for optimising reagent concentrations on the AutoAnalyzer has been applied to the estimation of ammonia by the Berthelot reaction in the determination of urea and organic nitrogen. Comparison of the use of phenol and salicylate revealed that the optimum concentration of the latter is about four times that of the former. The optimum concentration of hypochlorite is five times greater with salicylate than with phenol, and for the catalyst, sodium nitroprusside, the factor is two. The precision obtained with the different methods is similar.

https://journals.sagepub.com/doi/pdf/10.1177/000456327801500164

Sharon A. Gordon

Papers

Optimal conditions for the estimation of ammonium by the Berthelot reaction.