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

Functional test of rice urease and UAPs expressed in N. benthamiana. A, Western blot loaded with 40 μg of protein (prot.) per lane and probed with anti-urease antibody (top) and urease activity quantification (bottom) using leaf extracts from N. benthamiana after 5 d of transient coexpression of different combinations of OsUrease, OsUreD, OsUreF, OsUreG (lanes I–V) and Atallantoinase (lane VI) as a control. Uninfected leaves were used as an additional control (lane VII). Each urease activity was quantified using three independent leaves from different plants (n = 3). Error bars indicate sd. As indicated by lowercase letters, only activity I is different from all others with statistical significance (P < 0.01). B, Western blot and urease activity as in A from leaves coexpressing either OsUrease, OsUreD, OsUreF, OsUreG (gray columns) or OsUrease, OsUreD, OsUreG, but not OsUreF (black columns). Activities were assessed in a time course from 3 to 6 d after infiltration of the plants with Agrobacterium. Error bars indicate sd (n = 3 independent leaves).

Structural comparison of plant and bacterial UreD and UreF proteins, and protein interactions of rice UAPs with urease. A, Schematic representation of secondary structure elements of UreF from rice (Os), from H. pylori (Hp), and from K. aerogenes (Ka). Proteins are drawn to scale; α-helices are displayed as gray boxes and β-elements as black boxes. The structure for the rice protein was predicted using the Jpred3 server with a multiple sequence alignment of 19 manually curated plant UreF proteins (Supplemental Fig. S3). The mean prediction confidence for each α- or β-element is given as a numerical value (scale 0–9, where 9 = highest confidence). The H. pylori partial UreF protein structure is based on experimental data but lacks the N and C termini of the protein. The K. aerogenes UreF protein structure was predicted by Salomone-Stagni et al. (2007). B, Schematic representation of secondary structure elements of UreD from rice (Os) and K. aerogenes (Ka). The structure for the rice protein was predicted using the Jpred3 server with a multiple sequence alignment of 16 manually curated plant UreD proteins (Supplemental Fig. S2). For the prediction of the K. aerogenes UreD structure, an alignment of 100 bacterial UreD sequences was used. C, Western blots of a copurification experiment. Rice urease (U) and N-terminally StrepII-tagged OsUAPs UreD, UreF, and UreG (D, F, and G) were coexpressed as indicated in leaves of N. benthamiana and affinity purified from extracts. Panels 1 and 3 show detection with StrepTactin alkaline phosphatase conjugate in crude extracts (1; input) and after affinity purification (3; output). Panels 2 and 4 show detection with anti-urease antibodies in crude extracts (2; input) and after purification (4; output). Stars label unspecific signals.

Structure of the transcript of plant ureF and splicing efficiency in rice and Arabidopsis. A, Schematic overview of the plant ureF transcript with the conserved intron in the 5′ leader sequence and primer positions used to investigate intron splicing in rice and Arabidopsis. B, RT-PCR products resolved on agarose gels. The left panel shows RT-PCR using Arabidopsis leaf RNA and primers 2438 and 1021, and the right panel shows RT-PCR using rice leaf RNA and primers 2440 and 2439 (lane 1) or 2441 and 2439 (lane 2).

Characterization of rice urease and arginase. A, Michaelis-Menten graph (left axis, squares; r² = 0.990) and corresponding Hanes plot (right axis, circles; r² = 0.998) for rice urease. Error bars indicate sd (n = 3). prot., Protein. B, Western blot (left panel) documenting the StrepII tag-mediated affinity purification of rice arginase from leaf extracts of N. benthamiana after transient expression. Lane 1, Extract of soluble proteins; lane 2, proteins not bound after incubation with StrepTactin affinity matrix; lane 3, protein in the fifth wash supernatant; lane 4, pool of eluted protein; lane 5, affinity matrix boiled in SDS loading buffer after elution. Silver gel (right panel) to visualize the purity of rice arginase. Lane 1, Marker proteins; lane 2, affinity-eluted arginase (10 μL loaded). C, Michaelis-Menten graph (left axis, squares; r² = 0.977) and corresponding Hanes plot (right axis, circles; r² = 0.995) for purified rice arginase. Activity with reference to volume assay mixture is displayed. Error bars indicate sd (n = 4). D, Activation of purified arginase by manganese. Error bars indicate sd (n = 3).

Changes of urease and arginase activity during rice germination. Urease activity (top) and arginase activity (bottom) are shown relative to total protein (prot.) in a time course taken during germination of subspecies Hwayoung from day 0 (dry seed) to day 6. Error bars indicate sd (n = 3).