Inheritance of Mutator Activity in ZEA MAYS as Assayed by Somatic Instability of the bz2-mu1 Allele

Mutator lines of maize were originally defined by their high forward mutation rate, now known to be caused by the transposition of numerous Mu elements. A high frequency of somatic instability, seen as a fine purple spotting pattern on the aleurone tissue, is characteristic of Mu-induced mutable alleles of genes of the anthocyanin pathway. Loss of such somatic instability has been correlated with the de novo, specific modification of Mu element DNA. In this report the presence or loss of somatic instability at the bz2-mu1 allele has been monitored to investigate the inheritance of the Mutator phenomenon. The active state is labile and may become weakly active (low fraction of spotted kernel progeny) or totally inactive (no spotted kernel progeny) during either outcrossing to non-Mutator lines or on self-pollination. In contrast, the inactive state is relatively permanent with rare reactivation in subsequent crosses to non-Mutator lines. Cryptic bz2-mu1 alleles in weakly active lines can be efficiently reactivated to somatic instability when crossed with an active line. However, in reciprocal crosses of active and totally inactive individuals, strong maternal effects were observed on the inactivation of a somatically unstable bz2-mu1 allele and on the reactivation of cryptic bz2-mu1 alleles. In general, the activity state of the female parent determines the mutability of the progeny.     

Characterization of bz1 mutants isolated from mutator stocks with high and low numbers of Mu1 elements

The high frequency of mutations in Mutator stocks of maize is the result of transposition of Mu elements. Nine different Mu elements that share the 220 bp Mu terminal inverted repeats have been described. Mu1 elements have been found inserted into most of the molecularly characterized mutant alleles isolated from Mutator stocks, and most Mutator stocks contain a high number of Mu1 elements (10-60). However, it is clear that additional Mu elements, which share the Mu1 termini but have unrelated internal sequences, can also transpose in Mutator stocks. We were interested in comparing the mutation frequency and type of elements that inserted into a particular locus when Mutator stocks with differing numbers of Mu1 elements were utilized. Furthermore, previous studies with Mu-induced mutations have demonstrated that the element that inserted most frequently was Mu1. Therefore, to try to obtain Mu elements different from Mu1 we utilized a stock that had a low number (3-6) of Mu1 elements as well as a Mutator stock with a more typical number of Mu1 elements (20-60). Utilizing both stocks, we isolated numerous mutants at one gene, Bronze 1 (Bz1), and compared the type of elements inserted. In this paper we report that both the high and low Mu1 stocks produced bz1 mutants at frequencies characteristic of Mutator stocks, 6.6 and 4.3 x 10(-5), respectively. We describe the isolation of 20 bz1 mutations, and the initial molecular characterization of eight unstable mutations: two from the high Mu1 stock and six from the low Mu1 stock. The six alleles isolated from the low Mu1 stock appear to contain deleted Mu1 elements, and the two alleles isolated from the high Mu1 stock contain elements very similar to Mu1. When the mutants from the low Mu1 stocks were examined, it was found that the Mu1-related elements increased from 3-6 copies to 9-20 copies in one generation. The high number of Mu1-related elements was maintained in subsequent outcrosses. This spontaneous activation and amplification of Mu1-related elements occurred in at least 1% of the low Mu1 plants.     

Somatic excision of the Mu1 transposable element of maize.

The Mu transposons of the Robertsons's Mutator transposable element system in maize are unusual in many respects, when compared to the other known plant transposon systems. The excision of these elements occurs late in somatic tissues and very rarely in the germ line. Unlike the other plant transposons, there is no experimental evidence directly linking Mu element excision and integration. We have analyzed the excision products generated by a Mu1 transposon inserted into the bronze 1 locus of maize. We find that the excision products or 'footprints' left by the Mu1 element resemble those of the other plant transposable elements, rather than those of the animal transposable element systems. We also find some novel types of footprints resembling recombinational events. We suggest that the Mu1 element can promote intrachromosomal crossovers and conversions near its site of insertion, and that this may be another mechanism by which transposons can accelerate the evolution of genomes.     

Reactivation of the Mutator transposable element system following gamma irradiation of seed

Summary The bz2-mu1 allele contains a 1.4 kb Mu element insertion in the open reading frame of the bronze-2 locus. This insertion suppresses gene activity. In an active Mutator line, however, the bz2-mu1 allele shows high somatic instability resulting in numerous purple spots of full gene activity against a beige background in the aleurone tissue of the kernel; restoration of gene activity results from excision of the Mu element. In contrast, in plants with an inactive Mutator system, uniformly bronze kernels are found, and the Mu element at bz2-mu1 is stabilized. Accompanying a loss of somatic instability, this Mu element, as well as the Mu elements elsewhere in the genome, have an increased level of DNA modification. Spontaneous reactivation of somatic instability in inactive Mutator lines rarely occurs; however, reactivation can be induced with gamma irradiation. Reactivated plants regain both the spotted kernel phenotype indicative of element excision from the bz2-mu1 reporter allele and diagnostic restriction sites within the Mu elements indicative of a hypomethylated state. The reactivated plants transmit these characters to their progeny. These data support the hypothesis that genomic shock can elicit cryptic transposable element activities in maize. Possible mechanisms for inactivation and reactivation of the Mutator transposable element system are also discussed.     

Molecular analysis of the loss of somatic instability in the bz2::mu1 allele of maize

Summary Multiple genetic and epigenetic changes were detected within one plant generation at the bz2:: mu1 mutable allele in a population of 118 plants. Loss of somatic instability in bz2::mu1 was usually correlated with methylation of the Mu1 transposable element; in 6 plants, somatic instability was lost as a result of mutations in bz2::mu1. This is a surprisingly high frequency of mutation per allele (2.5%) for the Mutator family, for which germinal revertants occur at a frequency of about 10^–4 per gamete. One germinal excision event was found that contained an 8 by deletion, frameshift mutation in Bronze-2. The three other mutants described occurred as a result of abortive transposition, in which 75–77 by deletions were generated at the junction between Bronze-2 and Mu1. We discuss the possible mechanisms, and the role of host factors in abortive transposition in maize.     

Germinal and somatic products of Mu1 excision from the Bronze-1 gene of Zea mays

Summary Germinal and somatic excision products of Mu1 from the insertion allele bz::mu1 were selectively amplified from maize cob tissue. The sequence of these footprints often included deletions at the target site, suggesting that substantial exonucleolytic degradation occurs upon excision of the element. In addition to deletions of target site sequences, single base insertions were also found. The isolation of an excision product including a 4 by inverted duplication of the target site provides evidence that the double-stranded chromosomal break generated by Mu excision may be terminated by a covalently closed hairpin structure. The majority of excision products, however, do not include inverted duplications of target site sequences, suggesting that such structures are the result of occasional repair activities, rather than an essential step in the mechanism of Mu excision. The sequence of the Mu insertion sites of the bz::mu1 and bz::mu2 alleles is also presented.     

Developmental regulation of excision timing of Mutator transposons of maize: Comparison of standard lines and an early excision bzl::Mu1 line

Three characteristics of standard Mutator lines reflect developmental regulation: new mutants usually involve single gametes, somatic excision is restricted to terminal cell divisions during tissue development, and germinal excision is rare. By selection for earlier (larger) somatic sectors in the aleurone, a Mutator line was identified that exhibits a dramatic elevation in somatic excision frequency during the first three nuclear divisions of the endosperm and more than a 10-fold increase in germinal reversion from the bzl::Mul reporter gene. The programming of early sectoring is dominant in crosses with Mutator lines containing diverse reporter alleles. Germinal reversion is biased 5- to 10-fold for events through the pollen compared to the ear. The timing of germinal excision in the tassel is late because somatic excision sectors in the anthers are small; however, 98% of the germinal revertants are concordant. These observations indicate that in the early sectoring line Mu excision usually occurs before the mitotic divisions that separate gametic nuclei and may be restricted to the early stages of microsporogenesis. © 1992 Wiley-Liss, Inc.