A polypeptide screening in 7% polyacrylamide gels by the technique of Davis (Ann. N. Y. Acad. Sci. 121:404-427) was carried out with the proteins of the shoots derived from embryo cultures of the mutant floury-a line 79-7660 (a lysine-threonine 1mM insensitive line) and normal BP line 79-7511 (a lysine-threonine 1 mM partially insensitive line).
The embryos were cultured in tubes containing the Murashige-Skoog medium with (or without = control) lysine-threonine 2.5 mM (strong inhibitory medium). After 10 days of culture at 28 C in the dark, the soluble proteins of the shoots were extracted in a chilled mortar and pestle using two volumes of buffer tris-glycine 0.1 M, pH 8.3, containing 40% sucrose. The homogenates were centrifuged at 44,630 xg, 20 min. All the procedures were at 4 C. A sample of 0.3 ml of the supernatant (approximately 120 mg of fresh material) was taken and poured into each acrylamide tube.
Other embryos of the same lines were subjected to the same kind and time of inhibition and then were transferred to a control medium, lysine-threonine free, and were analyzed by the same means after 10 days in such "rescue" medium.
Comparing the polypeptide pattern of control with treated derived shoots we have found in both maize lines several alterations. Although the pattern of alteration was different in both lines, the phenomena of lack, unfold and synthesis of polypeptides in the treated derived shoots were usually observed. The extent and kind of alteration in each line was exactly the same in the five repetitions of the experiment carried out. But the most important fact is that several polypeptides altered by the effect of lysine-threonine reverted to the control pattern when the seedlings were transferred to a "rescue" medium (lysine-threonine free). The results shown in Figure 1 were one of the most frequently observed. This result is shown here because in both lines, floury-a and BP, it was observed jointly.
The densitometer tracings of the Rf region from 0.40 to 0.60 of the treated shoots differ from those of the control in the unfold of the polypeptide located between the two major polypeptides of the soluble shoot extract, at Rf = 0.52. The densitometer tracing of the shoots derived from seedlings transferred to a "rescue" medium has shown a pattern similar to that of the control, with the re-synthesis of the unfold polypeptide.
These results were also observed in other Rf regions of the gel, in which the polypeptide pattern of the shoots derived from control and "rescue" seedlings were equal, and both were different from the polypeptide pattern of the shoots derived from treated seedlings.
The soluble protein alterations caused by lysine-threonine would probably have an epigenetic basis, and would not be heritable. However, we suggest that the extent of the alteration is genotype dependent. We have suggested previously (see MNL 55:54-55) that the lysine-threonine effect has secondary effects on several metabolic processes. The soluble protein alterations shown here would be one of such effects. In the same communication we also suggested that a genetic material inducible (or selectable) by the primary lysine-threonine effect but stable to the secondary lysine-threonine effects would be the best for selection purposes.
The results obtained by Phillips et al. (Crop Sci. 21:601-607) in their "rescue test" could also be genotype dependent. Those materials inhibited or not in lysine-threonine medium and recovered when transferred to lysine-threonine free medium would be the best, according to our point of view, for selection of possible variants resistant to lysine-threonine.
We suggest that by means of the electrophoretic analysis of the shoot and/or root soluble proteins of the control and treated derived seedlings and using an appropriate similarity index (S), it would be possible to predict the behavior of any strain in the "rescue" attempts. Those lines with higher "S" between the electrophoretic pattern of the soluble proteins derived from control and treated seedlings would be recovered in the "rescue" attempts. Such hypothesis is to be tested with large maize strains in our laboratory.
Miguel Angel Rapela
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