With the id1-CSH allele closely linked to the bz2-m2 allele, anthocyanin sectors in the aleurone can be used as a marker for Ac activity. The Ac element used in these experiments is located on chromosome 9 (Kelly Dawe, personal communication), and therefore segregates independently of the id1-CSH bz2-m2 loci. We wanted to determine if the Ac activity, which causes a variegated phenotype in the aleurone by somatic excision of the Ds2 element from the bz2-m2 allele, has a similar effect on the id1-CSH phenotype; i.e., does somatic excision of the Ds2 element from the id1-CSH allele restore Id function such that the flowering time for the id1-CSH mutants is reduced by Ac?

As described in the above report, 4 out of 336 families segregated id1-CSH mutant plants. Since only spotted kernels were planted, all id plants in each of the four families should be homozygous for the id1-CSH and bz2-m2 alleles, whereas the normal siblings (i.e., in this background, those that flower 9 to 11 weeks after planting) should be id1-CSH bz2-m2/Id1bz2 (except in rare cases where recombination or germinal excision results in an Id1 bz2-m2 constitution). The spotted kernels indicate that each plant should also carry at least one Ac element. Normal siblings from each family were selfed and out-crossed to an Id1 bz2/Id1 bz2 tester in the 1993 summer field. The frequency of spotted and bronze kernels on each ear was analyzed to determine the number of Ac elements. Ears derived from selfed plants with one Ac element segregated spotted and bronze kernels at a frequency of 9/16 and 7/16, respectively, and the same plant outcrossed to a bz2 tester had a spotted kernel to bronze kernel ratio of 1/4 to 3/4.

To compare flowering time of id1 mutants in the presence and absence of Ac activity, spotted and bronze kernels from each ear of 10 different families with a single unlinked Ac element were planted in the 1994 summer field. As expected, 1/3 of the spotted kernels from these ears segregated id mutant plants. The other 2/3 had normal flowering times and should be heterozygous plants; i.e., id1-CSHbz2-m2/Id1 bz2. The bronze kernels of the selfed progeny of id1-CSHbz2-m2/Id1 bz2, Ac/- heterozygous plants represent 7/16 of the total kernels on each ear. Of these bz kernels, 1/4 (4/16) should be homozygous for the tester allele, Id1 bz2/Id1 bz2. The other 3/16 represent kernels that carry the id1-CSH bz2-m2 allele, but have no Ac element and therefore are not spotted. Of these kernels, 1/3 should also be homozygous for id1-CSH bz2-m2, and therefore express the indeterminate mutant phenotype. Therefore, of the bronze kernels planted, 1/7 should segregate id mutants. Overall, the experiment involved comparing the flowering time of id plants grown from spotted kernels with that of id plants grown from bronze kernels. These were compared to the flowering time of normal siblings found in both populations.

All kernels were planted in the third week of May, 1994. Normal plants from both spotted and bronze kernels usually made between 11 and 13 leaves, and all shed pollen sometime between the third week of July and the first week of August when they were approximately 9 to 11 weeks old. From the 10 rows of spotted kernels planted, 47 clearly indeterminate plants were segregating; i.e., none of these had emerging ears or tassels by the time their normal siblings had shed out, and they all had more than 13 visible leaves. The 10 rows of bronze kernels yielded 17 indeterminate plants. These also showed no signs of flowering by the time their normal sibs had shed out, and they appeared to grow more slowly than the spotted kernel id plants; i.e., they had fewer leaves and they were shorter. By the first week of September, at about 17 weeks after planting, some of the spotted kernel id plants had tassels emerging and several had leafy ear shoots, whereas the bronze kernel id plants showed no signs of flowering. In mid-September, three of the spotted kernel id plants were shedding pollen and silking; these were either selfed or crossed to each other. These plants had between 15 and 17 leaves. Approximately half of the id plants from spotted kernels were showing tassels at this time, while the bronze kernel id plants continued to produce leaves but no flowers.

By mid-October, when the plants were about 21 weeks old, all of the spotted kernel id plants, with the exception of three plants in one family (see below), had either shed out or had tassels emerging, and all had from 17 to more than 20 visible leaves. Most of these did not make ears, and the few ears that did emerge had marked vegetative characteristics. All of the id plants from bronze kernels, however, continued to produce leaves, and they had neither tassels nor ears. These plants continued to grow vegetatively and make approximately 18 to 25 leaves until the first killing frost on November 13, 1994 when they were 25 weeks old. Several of these plants were dissected and found to have tassel primordia ranging from 0.5 to 1.0 cm in length. By comparison, several id1-R plants that were planted at the same time in a nearby field were dissected and found to have similar size tassel primordia; none of these id plants produced ears.

Since spotted kernels indicate the presence of Ac, it appears that id1-CSH plants that had an Ac element in the background flowered significantly earlier than plants from the same family that did not have an Ac element. In fact, the latter plants did not flower at all, and they made only tiny tassel primordia by the time they were six months old at season's end. This is what one would expect if the id1-CSH allele had a Ds2 element inserted into it which responded to Ac transposase; i.e., Ac induces the somatic excision of the Ds2 element, occasionally producing sectors of normal tissue which can synthesize functional Id1 gene product. It is interesting to note that mosaic plants were not seen in the id1-CSH plants that flowered earlier; i.e., sectors of normal tissue juxtaposed to mutant tissue were not apparent. Instead the plants had an intermediate phenotype. This might suggest that the Id1 gene product does not act cell autonomously, and that it is a diffusible substance or that it mediates the production of a diffusible product. On the other hand, it is difficult to predict what a plant composed of sectors of indeterminate and normal tissue would look like. Clonal analysis experiments are currently underway to address this question.

Finally, as mentioned above, of the 47 id plants derived from spotted kernels, all flowered before the end of the season except 3. These plants originated from the same family and, in general, resembled id1-CSH plants derived from bronze kernels in terms of growth rate, leaf number and inability to flower. DNA was prepared from each of these id plants and Southern blotting was performed using Ds2-flanking DNA as a probe. Unlike all id1-CSH plants examined so far, which contain a single 4.2 kb SacI fragment, all three of these plants had a 2.9 kb SacI band; i.e. similar in size to the SacI band found in plants with a normal Id1 allele (see above report). Primers flanking the Ds2 insertion of id1-CSH alleles were used to amplify and clone this region from one of the 3 mutants. Sequence analysis revealed that this plant differed from normal plants in this region by having a 5 bp insertion. This sequence appears to be the remnants of a target site duplication, initially caused by the Ds2 insertion. In effect, a stable allele of id1 was created by the imprecise excision of Ds2. The 5 bp DNA insertion causes a frame shift in the alleged open reading frame of the Id protein (see above report). As observed, this new indeterminate allele (termed id1-X) should not respond to the presence of an Ac element. This finding provides additional evidence that the earlier flowering phenotype of Ac-containing id1-CSH plants is the result of somatic excision of the Ds2 element during development. 

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