**3.3 Loss of chemical protection**

As described in the next section, *G. max* contains two different isozymes of urease: the enzyme which is ubiquitous is made in all the examined tissues, more so, embryo-specific synthesis of urease is dependent to the embryo that is developing and it is conserved in the seed that is mature where its specific activity is about 1000-fold higher than that of the ubiquitous urease in any tissue [61, 62]. Since mutants that lacks the embryo-specific urease do not display any of the abnormalities related with loss of the ubiquitous enzyme necrotic leaf tips, accumulation of urea in leaves or seeds, retarded germination [41] it was concluded that this enzyme has no important physiological use. *In vitro* culture of developing cotyledons of pea [63] and *G. max* [41, 64] indicates that ureases play little or no role in embryo nutrition since urea was an extremely poor source of nitrogen. Indeed, urea is not normally generated within the developing *glycine max* cotyledon *in vivo* [41], in agreement with the lack of ureide delivery to the legume embryo from maternal tissues [65, 66]. The obvious question from the observations of the previous paragraph is why would the developing soybean embryo and those of jackbean, watermelon, and many other members of Leguminosae and Cucurbitaceae [67] invest in a very active ureolytic activity when it never "sees" urea. Although much urea is generated upon germination [41] and although much of the embryo-specific urease is retained in seedling cotyledons and roots [68], the loss of the embryo-specific urease causes no discernible increase in seedling urea levels over those of wild type [41]. It is suggested, therefore, that the embryo-specific urease plays no urea assimilatory role but rather that of chemical defense in seeds. To draw two parallels, at least, with the pathogenic effects of bacterial urease on vertebrates, active seed urease could cause

either hepatic coma by subversion of the urea cycle or peptic ulceration by localized increases in NH+ and OH-ions (urea + 3H2O + 2NH4 + + HCO− + OH− ). A micro aerophilic, bacterium, *Helicobacter pylori,* can colonize gastric epithelium because its active urease creates a more basic micro environment in this acidic milieu [69, 70]. Arguments summarized by [71] link urease from bacteria to ulceration of the gastric mucosa either by cytotoxicity of ammonia directly or by its prevention of proton flux from gastric glands to the gastric lumen leading in a back-diffusion of protons. Hepatic coma results when intestinally derived nitrogenous compounds, e.g., ammonia, bypass the liver and get to the brain. Administration of urease inhibitors has proven effective in reducing hyperammonemia [72, 73]. It is easy to visualize an active seed urease mimicking these bacterial effects (the bacterial and plant seed ureases have >50% amino acid identity) [74], especially urease aided by other cytotoxic components in the seed i.e. protease inhibitors, lectins that disrupt intestinal brush borders [75], etc. Another postulated chemical defense for seed urease is its induction of a hostile environment upon microbial and perhaps insect attack. With this second model, wounding or infection of the immature embryo will lead to the release of arginase from mitochondria that is ruptured. Cytoplasmic arginase would generate urea from the large pool of arginine which is at least 50% of free amino acid nitrogen [27, 28] and cytoplasmic urease would rapidly convert urea to ammonia. It was observed that cultured cotyledons containing the embryo specific urease commit suicide (probably by the combined effects of ammonia toxicity and medium alkalinization) in the presence of urea (20 mM), whereas those containing only the ubiquitous urease isozyme survive and utilize this urea, albeit poorly [41]. The assessment of pest resistance and herbivore avoidance of soybean cultivars lacking the embryo-specific urease requires extensive field testing of isogenic lines exposed to a variety of pathogens and pests. In addition to the abundant storage proteins, seeds contain several moderate- to high-abundance proteins with enzymatic or other biological activity. It is easy to invoke a plant protection role for many of these proteins: phytohemagglutinins (lectins) [75], lipoxygenases [76], ribosomal inactivators [77] and inhibitors of amylase [78], and animal proteases [79]. The lack of an essential physiological role for many of these proteins in *plant i*s suggested by the relatively high frequency of cultivars and varieties lacking one of the urease [61], lipoxygenases [80–82], etc. Lack of an essential physiological role for the seed (embryo specific) urease is further suggested by the high prevalence of seed urease nulls in Japanese populations of *Glycine soja* [83], a sexually compatible close relative of cultivated soybean (*Glycine max*).
