In Arabidopsis thaliana, urease transcript levels increased sharply between 2 and 4 d after germination (DAC) and were maintained at maximal levels until at least 8 DAC. Seed urease specific activity declined upon germination but began to increase in seedlings 2 DAC, reaching approximately 75% of seed activity by 8 DAC. Urea levels showed a small transient increase 1 DAC and then approximately paralleled urease activity, reaching maximal levels at approximately 9 DAC. Urease inhibition with phenylphosphorodiamidate resulted in a 2-to 4-fold increase in urea levels throughout seedling development. Arginine pools (0-8 DAC) changed approximately in parallel with the urea pool. Consistent with arginine being a major source of urea, arginase activity increased 10-fold in the interval O to 6 DAC. Allopurinol, a xanthine dehydrogenase inhibitor, had no effect on urea levels up to 3 DAC but reduced the urea pool by 30 to 40% during the interval 5 to 8 DAC, suggesting that purine degradation contributed to the urea pool well after germination, if at all. In aged Arabidopsis seeds, there was a correlation between phenylphosphorodiamidate inactivation of urease and germination inhibition, the latter overcome by NH,NO, or amino acids. Since urease activity, urea precursor, and urea increase in young seedlings, and since urease inactivation results in a nitrogen-reversible inhibition of germination, we propose that urease recycles urea-nitrogen in the seedling.

We assayed the in vivo activity of the ureases of soybean (Glycine max) embryos by genetically eliminating the abundant embryo-specific urease, the ubiquitous urease, or a background urease. Mutant embryos accumulated urea (250-fold over progenitor) only when lacking all three ureases and only when developed on plants lacking the ubiquitous urease. Thus, embryo urea is generated in matemal tissue where its accumulation is not mitigated by the background urease. However, the background urease can hydrolyze virtually all urea delivered to the developing embryo. Radicles of 2-day-old germinants accumulated urea in the presence or absence of the embryo-specific urease (2 micromoles per gram dry weight radicle). However, mutants lacking the ubiquitous urease exhibited increased accumulation of urea (to 4-5 micromoles urea per gram dry weight radicle). Thus, the ubiquitous and not the embryo-specific urease hydrolyzes urea generated during germination. In the absence of both of these ureases, the background urease activity (4% of ubiquitous urease) may hydrolyze most of the urea generated. A pleiotropic mutant lacking all urease accumulated 34 micromoles urea per gram dry weight radicle (increasing 2.5-fold at 3 days after germination). Urea (20 millimolar) was toxic to in vitrocultured cotyledons which contained active embryo-specific urease. Cotyledons lacking the embryo-specific urease accumulated more protein when grown with urea than with no nitrogen source. Among cotyledons lacking the embryo-specific urease, fresh weight increases were virtually unchanged whether grown on urea or on no nitrogen and whether in the presence or absence of the ubiquitous urease. However, elimination of the ubiquitous urease reduced protein deposition on urea-N, and elimination of both the ubiquitous and background ureases further reduced urea-derived protein. The evidence is consistent with the lack of a role in urea hydrolysis for the embryo-specific urease in developing embryos or germinating seeds. Because the embryo-specific urease is deleterious to cotyledons cultured in vitro on urea-N, its role may be to hydrolyze urea in wounded or infected embryos, creating a hostile environment for pest or pathogen. While the ubiquitous urease is operative in leaves and in seedlings, all or most of its function can be assumed by the background urease in embryos and in seedlings. ' Supported in part by the Missouri Agricultural Experiment Station and by grants from the National Science Foundation (DCB-8718314 and DCB-8804778) and the U. S. Department ofAgriculture (59-2291-1-1-672-0). This is journal paper No. 11,463 In this communication, we describe a biochemical genetic approach to examine three aspects of urea metabolism in the developing and germinating soybean embryo: (a) the origin (maternal versus embryonic) of the urea accumulating in the embryos of urease-negative plants, (b) the roles of each of the urease isozymes in hydrolyzing urea accumulating in the preand postdormancy embryo, and (c) the effectiveness of urea as...

The hypothesis that soybean (Clycine max 1. [Merrill]) catabolizes ureides to urea to a physiologically significant extent was tested and rejected. Urease-negative (eu3-el/eu3-el) plants were supported by fixed N, or by 2 mM NH,NO,, so that xylem-borne nitrogen contained predominantly ureides (allantoin and allantoic acid) or amide amino acids, respectively. Seed nitrogen yield was equal on either nitrogen regime, although 35-d-old fixing plants accumulated about 6 times more leaf urea. In callus, lack of an active urease reduced growth on either arginine or allantoin as the sole nitrogen source, but the reduction was greater on arginine (73%) than on allantoin (39%). Furthermore, urease-negative cells accumulated 17 times more urea than urease-positive cells on arginine; for allantoin the ratio was 1.8. Urease-negative callus accumulated urea at 3 % the rate of seedlings. To test whether urea accumulating in urease-negative seedlings was derived from ureides, seeds were first allowed to imbibe in 1 mM allopurinol, an inhibitor of ureide formation. Seedling ureides were decreased by 90%, but urea levels were unchanged. Thus, ureides are poor precursors of urea, which was confirmed in seedlings that converted no more than 5% of seed-absorbed [' 4C-ureido]

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