Nucleotide Sequence of Arabidopsis thaliana Arginase Expressed in Yeast

Arginase (L-Arg amidinohydrolase, EC 3.5.3.1) catalyzes the hydrolysis of L-Arg to urea and L-Orn. Orn is a precursor of Pro and polyamines and urea N is recycled by urease-catalyzed hydrolysis to ammonia (Polacco and Holland, 1993). Many plant species store much seed protein N in Arg (VanEtten et al., 1963), significant quantities of which are released and catabolized during germination (Polacco and Holland, 1993). In soybean axes arginase activity increases sharply during germination (Kang and Cho, 1990), consistent with considerable urea accumulation in urease-negative soybean seedlings (Stebbins et al., 1991). The Arubidopsis 12s globulin (Pang et al., 1988) and 2s albumin (Krebbers et al., 1988) seed storage proteins have a deduced Arg content of 7.0 and 6.9 mo1 %, respectively, whereas the Arg content of an “average” protein is 3.0 mo1 % (VanEtten et al., 1963). Conversion of Arubidopsis seed storage protein to seedling protein potentially involves Arg breakdown, consistent with a 10-fold increase in seedling arginase activity during the Oto 6-d after germination interval (Zonia et al., 1995). We report the nucleotide sequence of an Arubidopsis arginase cDNA recovered by its complementation of a yeast (Succkuromyces cerevisiue) arginase-deficient (curl) mutant (Table I). The pertinent genotype of the yeast host was curl,durl,ura3-52, a nonreverting uracil auxotroph (uru3-52) unable to utilize either Arg (curl) or urea (dur l ) as sole N source. URA+ transformants (recovered via the method of Gietz et al., 1992) harboring members of a two-leaf-stage Arubidopsis cDNA library (in yeast-Esckerickiu cozi shuttle vector pFL61; Minet et al., 1992) were replica plated to medium with Arg as sole N source. One URA’ isolate grew consistently on this medium and its plasmid transmitted this trait to new host cells. We concluded that this isolate expressed an arginase because it accumulated urea in the presence of Arg and because its cDNA insert showed size and sequence sim-