Identification and Characterization of Proteins Involved in Rice Urea and Arginine Catabolism  

Abstract

Rice (Oryza sativa) production relies strongly on nitrogen (N) fertilization with urea, but the proteins involved in rice urea metabolism have not yet been characterized. Coding sequences for rice arginase, urease, and the urease accessory proteins D (UreD), F (UreF), and G (UreG) involved in urease activation were identified and cloned. The functionality of urease and the urease accessory proteins was demonstrated by complementing corresponding Arabidopsis (Arabidopsis thaliana) mutants and by multiple transient coexpression of the rice proteins in Nicotiana benthamiana. Secondary structure models of rice (plant) UreD and UreF proteins revealed a possible functional conservation to bacterial orthologs, especially for UreF. Using aminoterminally StrepII-tagged urease accessory proteins, an interaction between rice UreD and urease could be shown. Prokaryotic and eukaryotic urease activation complexes seem conserved despite limited protein sequence conservation for UreF and UreD. In plant metabolism, urea is generated by the arginase reaction. Rice arginase was transiently expressed as a carboxylterminally StrepII-tagged fusion protein in N. benthamiana, purified, and biochemically characterized (K m = 67 mM, k cat = 490 s 21 ). The activity depended on the presence of manganese (K d = 1.3 mM). In physiological experiments, urease and arginase activities were not influenced by the external N source, but sole urea nutrition imbalanced the plant amino acid profile, leading to the accumulation of asparagine and glutamine in the roots. Our data indicate that reduced plant performance with urea as N source is not a direct result of insufficient urea metabolism but may in part be caused by an imbalance of N distribution.

Rice DUR3 mediates high‐affinity urea transport and plays an effective role in improvement of urea acquisition and utilization when expressed in Arabidopsis

“…Rice ( Oryza sativa L.ssp. japonica cv Nipponbare or cv ZH11) seed surface sterilization and germination, and plant growth conditions as well as N‐free basic nutrients (plus 1 mM (NH 4 ) 2 SO 4 as N source) used for a normal rice culture (in hydroponics or on agarose‐medium) were as described in Cao et al. (2010).…”

Uric Acid Accumulation in an Arabidopsis Urate Oxidase Mutant Impairs Seedling Establishment by Blocking Peroxisome Maintenance

“…Complementation lines were generated by transforming pXS2-UOX (P93) for the expression of UOX under the transcriptional control of a 35S promoter into the uox mutant. The construct was obtained by cloning UOX cDNA (from leaf RNA using primers 1823 and 1824) into pXS2pat (Cao et al, 2010) via NcoI and XmaI restriction sites. Transgenic lines expressing roGFP2-SKL were generated by transformation of the respective genotypes with per-GRX1-roGFP2 (Rosenwasser et al, 2011).…”

Biochemical and structural studies on native and recombinant Glycine max UreG: a detailed characterization of a plant urease accessory protein

“…The exact role played by each of these duplicated genes is unknown. Arabidopsis thaliana (Witte et al 2005), Solanum tuberosum (Witte et al 2001), and Oryza sativa (Cao et al 2010) have also been studied in the search for urease accessory proteins. In plants, no UreE has ever been identified, and UreG possibly carries out two roles: Ni 2?…”