Last year (C. Tonelli et al., MNL 60:100) we reported that the pro1 mutant is functionally allelic to o6; accordingly, o6 seedlings recover when supplemented with l-proline in the growing medium.
This summer we tested the effect of l-proline on young developing o6 endosperms. On account of the relationship between o2 and o6 (both repress zein accumulation and suppress or reduce b32 protein in the endosperm: L. Manzocchi et al., TAG 72:778-781, 1986), we also included o2 developing endosperms in the test.
A69Y normal, o2 and + /o6 plants were selfed, and the endosperms were collected ten days after pollination and cultivated in vitro. Ears segregating + and o6 seeds were recognized by leaving half of the ear on the plant till maturation and saving the results only for those endosperms deriving from segregating ears. The endosperms were cultivated on agar medium with MS salts, sucrose (3%), asparagine (0.2%) and in the presence or absence of 2 mM l-proline. After 6 days at 26 C, the fresh and dry weight and the content of salt and ethanol soluble proteins were determined. In addition, total proteins were analyzed by SDS-PAGE on pooled normal and o2 endosperms, and on individual endosperms in + /o6.
Presence or absence of proline did not affect the growth of the endosperms, which was about ten-fold the original weight. In the absence of proline, wildtype endosperms accumulated large amounts of zeins, while in o2 zeins were drastically reduced; endosperms from + /o6 plants segregated, as expected, high zein/low zein phenotypes, as judged by their electrophoretic patterns. In the presence of proline, essentially the same results were obtained, in particular for the endosperms from + /o6 plants, where a segregation for high/low zein phenotypes was still observed.
The results suggest that in our culture conditions 2 mM l-proline (a concentration restoring a normal phenotype in o6 seedlings) does not promote restoration of o6 endosperms.
Lucia A. Manzocchi and Carlo Soave
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