Analysis of the nitrogen nutritional requirements of the opaque-6 mutant

--C. Balconi, E. Rizzi, F. Salamini, R. Thompson and M. Motto

The opaque-6 (o6) mutation of maize, which causes seedling lethality and interferes with the synthesis of zein and b-32 proteins in the endosperm of developing kernels, has been demonstrated to be a proline requiring mutant functionally allelic to proline-1 (Manzocchi et al., Theor. Appl. Genet. 72:778-781, 1986). We have further analysed, by in vitro culture, the growth requirements of the wildtype and mutant o6 embryos of the B37 inbred line. Mature wildtype and mutant seeds were sterilized and germinated in Petri dishes for 48h at 27 C. Embryos were then dissected and placed in test tubes containing different media, as described in Table 1. On the "basic" growth medium (without amino acid) the wildtype seedlings showed a normal phenotype, whereas the mutant seedlings exhibited the described abnormalities of leaves, reduced growth and lethality at the second leaf stage (Manzocchi et al., Theor. Appl. Genet. 72:778-781, 196). o6 mutant embryos cultivated on basic medium (N) added with several specific amino acids (Table 1) showed a complete recovery of the normal phenotype, suggesting that this mutation is possibly related to a defect affecting not only proline biosynthesis but more generally all N-metabolism of the seed.

Table 1. Growth of excised embryos of mutants o6 o6 B37 and wildtype O6 O6 B37 on basic (salts as described in Nitsch and Nitsch, Science 163:85-87, 1969) and enriched media (basic medium added with 320mg/l amino acid). All media contained 0.4mg/l thiamine, 100mg/l inositol, 20g/l sucrose and 8g/l agar.
 
Growth medium Mutant Wildtype
Basic (N) - +
N + Arg (Arginine) + +
N + Asn (Asparagine) + +
N + Gly (Glycine) + +
N + Leu (Leucine) + +
N + Met (Methionine) + +
N + Pro (Proline) + +
N + Trp (Tryptophan) + +
N + Ala (Alanine) - +
N + Asp (Aspartic Acid) - +
N + Cys (Cystein) - +
N + Gln (Glutamine) - +
N + Glu (Glutamic Acid) - +
N + His (Histidine) - +
N + Ile (Isoleucine) - +
N + Lys (Lysine) - +
N + Phe (Phenylalanine) - +
N + Ser (Serine) - +
N + Thr (Threonine) - +
N + Tyr (Tyrosine) - +
N + Val (Valine) - +

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