--Carol Rivin and Timothy Grudt
Abscisic acid (ABA) plays a dual role in embryo development, inhibiting germination and stimulating the accumulation of maturation proteins. Using viviparous mutants, we have been investigating the relationship of these two effects. Proteins from W22 embryos at developmental stages 1 through 6 were compared by 2D SDS-PAGE. About 40 polypeptides identified as characterizing embryo maturation were shown to be modulated by ABA. These patterns were then compared with those of vp1 and vp5 mutant embryos (obtained from Don Robertson in a W22 background). Proteins were extracted from mutant embryos and their wildtype sibs developing in planta, in culture with or without exogenous ABA, or in culture with a high level of osmoticum to block precocious germination.
The vp5 and vp1 mutants germinate precociously on the ear and we found that they didn't make the set of maturation proteins in planta. Wildtype embryos and vp5 mutant embryos cultured with 10-5 M ABA are inhibited from germination and make a normal set of maturation proteins. The vp1 mutants are not inhibited from germination at physiological levels of ABA, and while their protein profile changes slightly in response to the hormone, they do not make a normal developmental response.
Blocking germination by growth in a high osmoticum prevented germination of wildtype and both mutant embryos and it was sufficient, by itself, to induce a large fraction of the maturation protein set in vp5 mutants and pre-maturation wildtype embryos. Interestingly, the vp1 mutants did not show this response.
Notably absent from the proteins induced by growth in high osmoticum are the major embryo storage globulins. This subset of the maturation proteins must require ABA itself and the vp1 gene product to initiate synthesis. However, ABA does not seem to be required for the continued synthesis and accumulation of these polypeptides. As long as germination was suppressed, embryos that had initiated globulin synthesis continued to accumulate them in the absence of ABA. Precocious germination of these embryos (with or without prior desiccation) led to a gradual decline in globulin synthesis after 12 hours. The accumulated globulins degraded rapidly beginning about 24 hours after the start of germination.
Our data suggest that ABA regulates maturation-phase polypeptides by two general pathways, both of which involve the vp1 gene product. A peak of ABA early in development appears to initiate the synthesis of the storage globulins and it also limits the embryo's capacity to take up water. This event seems to then stimulate accumulation of a second class of prominent proteins which can also be induced by growth in high osmoticum. The osmoticum treatment does not result in measurable increases in ABA levels in wildtype embryos and it is effective in vp5 mutant embryos which are deficient in ABA synthesis. Blocking germination also prevents degradation activity and keeps the synthesis rate high.
The vp1 mutant embryos show some changes in protein profile in
response to high osmoticum and to exogenous ABA, but neither class of normal
maturation phase polypeptides is produced, even when germination is inhibited.
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