**5. The Physiological Role of Soybean Ureases**

After carbon, nitrogen is the main limiting element for plant performance [62], and there is a constant pressure on plants for efficient use of N leading to the development of efficient mechanisms for N uptake and metabolic pathways for N remobilization [37, 56]. Such a pressure even led to a reduction of N content of plant proteins [63]. Urea is an important primary source of N for plants. The action of arginase is the only confirmed pathway for urea generation *in vivo*; urea could also be generated by the degradation of purines and ure‐ ides [64], although this later pathway is very controversial. Urea can only be assimilated af‐ ter its hydrolysis into ammonia and carbon dioxide by urease [65], and that is the main physiological role attributed to ureases in plants [37]. Ammonia will then be re-assimilated by glutamine synthetase using glutamate as substrate [66]. Urease activity is present virtual‐ ly in all plant species and is ubiquitously distributed in all plant tissues [18, 31, 67], which is indicative of the great importance of its physiological role for the whole plant. For a long time, the relevance of plant urease-mediated metabolism of urea was considered not signifi‐ cant, since it was assumed that urea was hardly taken up by the plants, but instead degrad‐ ed by microorganisms in the soil and then the ammonia and nitrate were absorbed. Today, it is well known that plants can actively import urea from the soil, through the activity of dedicated urea transporters [68] and can also efficiently process soil-imported urea, even at high concentrations. These findings point at urease as a target for studies on improving plant N metabolism based on urea, the most used N fertilizer in the world [69].

Soybean makes a very interesting model for studies on the physiological role of urease in plants, since it is so far the only genome-sequenced plant that presents more than one isoform of the enzyme. ub-SBU has long been known to be the isoform responsible for recycling all metabolically derived urea [19, 70, 71]. This has been demonstrated since mutants lacking es-SBU do not accumulate urea in any tissue and do not have any impairment on the use of urea as sole nitrogen source, even though ub-SBU is present at levels only 0.1 to 0.3% that of es-SBU [19, 72]. Soybean mutants lacking ub-SBU activity present a characteristic phenotype consist‐ ing of necrosis of leaf tips, due to urea "burn", and accumulation of urea in many tissues [32, 36]. Urease is the only Ni2+ dependent enzyme yet identified in plants and the same pheno‐ type, early mentioned, is observed for plants grown under Ni2+ deprivation [27].

though the Ch14UreF is present and is supposed to activate urease. Another mutant, in which the expression of Ch02UreF was impaired, presented 5-10 % of the wild type urease activity. The authors presented two possible explanations for the results. The first one con‐ sidered that Ch02UreF could spoil activation by Ch14UreF because of a higher affinity for the activation complex. The second explanation, favoured in their work, proposed that Ch02UreF is more abundant than Ch14UreF, which would be less efficiently translated. Therefore, in *Eu2* mutants, the missense mutants of Ch02UreF block the access of Ch14UreF

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

As pointed out here, although accessory proteins differ widely according to their source, the process of urease activation seems to be very well conserved. Among plants, the urease acti‐ vation complex seems to be structurally very similar, since accessory proteins from different plants are able to functionally complement each other. Rice urease, for instance, can be acti‐ vated by *Arabidopsis* bulk of accessory proteins, and UreD and UreG from rice can replace the native accessory protein in mutants of Arabidopsis [56]. The similarities of the activation process seem also to break the kingdom barrier. Soybean UreF has been shown to comple‐ ment the *Saccharomyces pombe* accessory set [39], and UreG from potato (*Solanum tuberosum*)

After carbon, nitrogen is the main limiting element for plant performance [62], and there is a constant pressure on plants for efficient use of N leading to the development of efficient mechanisms for N uptake and metabolic pathways for N remobilization [37, 56]. Such a pressure even led to a reduction of N content of plant proteins [63]. Urea is an important primary source of N for plants. The action of arginase is the only confirmed pathway for urea generation *in vivo*; urea could also be generated by the degradation of purines and ure‐ ides [64], although this later pathway is very controversial. Urea can only be assimilated af‐ ter its hydrolysis into ammonia and carbon dioxide by urease [65], and that is the main physiological role attributed to ureases in plants [37]. Ammonia will then be re-assimilated by glutamine synthetase using glutamate as substrate [66]. Urease activity is present virtual‐ ly in all plant species and is ubiquitously distributed in all plant tissues [18, 31, 67], which is indicative of the great importance of its physiological role for the whole plant. For a long time, the relevance of plant urease-mediated metabolism of urea was considered not signifi‐ cant, since it was assumed that urea was hardly taken up by the plants, but instead degrad‐ ed by microorganisms in the soil and then the ammonia and nitrate were absorbed. Today, it is well known that plants can actively import urea from the soil, through the activity of dedicated urea transporters [68] and can also efficiently process soil-imported urea, even at high concentrations. These findings point at urease as a target for studies on improving

plant N metabolism based on urea, the most used N fertilizer in the world [69].

Soybean makes a very interesting model for studies on the physiological role of urease in plants, since it is so far the only genome-sequenced plant that presents more than one isoform

to the urease activation complex, preventing activation [40].

**5. The Physiological Role of Soybean Ureases**

complements the *K. aerogenes* operon [61].

Relationships

324

Interestingly, no physiological role, being it assimilatory or of any other nature, could be dem‐ onstrated for the very abundant es-SBU. In fact, wild-type cultured cotyledons could not grow in the presence of urea, due to a sudden pH increase resultant of an uncontrolled ammonia re‐ lease. The same effect was not observed for mutants that have only ub-SBU [36]. It was inferred that es-SBU could be involved in plant defense against predators. A chemical protection was postulated for the case of microbial or insect attack. By this model, wounding or infection of the immature embryo would lead to arginase release from ruptured mitochondria which would generate urea from the large pool of arginine and cytoplasmic urease would rapidly convert urea to ammonia [32]. This hypothesis still waits demonstration, but it has been reported that mutants lacking urease activity were more susceptible to microbial infections [73]. As it will be discussed in the next section, es-SBU can be involved in plant defense not only by conferring chemical protection, but also ureolysis-independent mechanisms, including the generation of toxic peptides. On the other hand, it is tempting to propose that the third urease found in the soy‐ bean genome (Glyma08g10850), that apparently has no enzymatic activity, can also be involved in other physiological roles in the plant, such as plant defense, and some indications of that have already been reported (see the next section).
