Spatial configuration of transposable element Ac termini affects their ability to induce chromosomal breakage in maize

Abstract

Composite or closely linked maize (Zea mays) Ac/Ds transposable elements can induce chromosome breakage, but the precise configurations of Ac/Ds elements that can lead to chromosome breakage are not completely defined. Here, we determined the structures and chromosome breakage properties of 15 maize p1 alleles: each allele contains a fixed fractured Ac (fAc) element and a closely linked full-length Ac at various flanking sites. Our results show that pairs of Ac/fAc elements in which the termini of different elements are in direct or reverse orientation can induce chromosome breakage. By contrast, no chromosome breakage is observed with alleles containing pairs of Ac/fAc elements in which the external termini of the paired elements can function as a macrotransposon. Among the structures that can lead to chromosome breaks, breakage frequency is inversely correlated with the distance between the interacting Ac/Ds termini. These results provide new insight into the mechanism of transposition-induced chromosome breakage, which is one outcome of the chromosome-restructuring ability of alternative transposition events.

Figure 1.

Ac Transposition Generates Multiple p1 Alleles. (A) Kernel phenotypes of representative p1 alleles and their molecular structures. The solid black boxes indicate p1 gene exons 1, 2, and 3 (left to right). The thick red lines represent Ac (two arrowheads) or fAc (one arrowhead). The closed and open red arrowheads represent the 5′ and 3′ termini of Ac/fAc, respectively. (B) Locations of Ac and fAc insertions in p1 alleles used in this study. The solid black boxes indicate p1 gene exons 1, 2, and 3 (left to right). Triangles with allele numbers represent Ac/fAc insertion sites in each allele. Open triangles indicate Ac elements in the same transcriptional orientation as the p1 gene (5′ to 3′, left to right); closed triangles indicate Ac insertions in the opposite orientation. The P1-rr904 allele is marked with an asterisk because it contains an intact Ac insertion and an additional 685 bp fAc insertion in the same position as that of the Ac element in p1-vv9D9A.

Figure 2.

Five Standard Ears for Evaluating the Frequency of Chromosome Breakage. The ears are from dek1/+ tester plants crossed with pollen from plants homozygous for various Ac-containing p1 alleles. Kernels containing a functional Dek1 gene develop solid purple aleurone color; chromosome breakage at the proximal p1 locus results in loss of Dek1 as evidenced by colorless aleurone sectors. The ears were borne on heterozygous dek1/+ tester plants; hence, only half of the kernels can show Dek1 loss. The photographs show Grade 0 (no Dek1-loss sectors) to Grade 4 (highest frequency Dek1-loss sectors) standard ears produced by crossing with pollen from plants homozygous for the following alleles: Grade 0, P1-rr458; Grade 1, P1-rr459; Grade 2, P1-rr910; Grade 3, p1-vv9D9A; Grade 4, P1-rr904. [See online article for color version of this figure.]

Figure 3.

Detection of Somatic Macrotransposon Excision by PCR. (A) Molecular structures of p1 alleles containing possible macrotransposons. Five alleles (P1-ovov455, P1-rr905, P1-rr908, P1-rr458, and P1-rr460) have the Ac situated downstream of the fAc element, such that the 5′ end of Ac and the 3′ end of fAc could function together as a macrotransposon. The large closed and open arrowheads represent the 5′ and 3′ termini of Ac/fAc. The positions of oligonucleotide primers used for PCR are indicated by the small arrows. The figure is drawn to scale, except that an 8.5-kb segment (indicated by / /), including the p1 5′ region, exon 1, intron 1, exon 2, and part of intron 2, is not shown. (B) Results of PCR analysis of somatic macrotransposon excision. Primers used are shown at top in the two rows above the brackets. Alleles tested are shown above each lane: Pvv, p1-vv; 455, P1-ovov455; 11, P1-rr11; 908, P1-rr908; 458, P1-rr458; 905, P1-rr905; and 460, P1-rr460. Total genomic DNA from young leaves of plants homozygous for the indicated alleles was used as template. Bands corresponding to expected macrotransposition excision products are indicated by arrows and apparent sizes. For each pair of primers, P1-rr11 is used as a negative control and to identify probable nonspecific or background amplification products, for example, the ∼0.33-kb band observed in lanes 1, 2, and 3; the ∼0.3-kb band in lane 3; the ∼0.65-kb band in lane 8; and the 0.70-kb band in lanes 9 and 10. In addition, the standard p1-vv allele, which contains a single Ac element between the binding sites of primers PA-A13 and PP1’ (lanes 1 to 3), was used as a positive control. PCR of this allele produced a 0.80-kb band (lane 1), which is the size expected from Ac excision.

Figure 4.

Bridges and Fragments at Anaphase I and Telophase I of Meiosis in Microsporocytes of P1-rr11 Plants. (A) Bridge and fragment at anaphase I of meiosis. (B) Bridge and fragment at telophase I of meiosis.

Figure 5.

Chromosome Breakage Models. (A) Model for chromosome breakage by directly oriented Ac/Ds termini (sister chromatid transposition). The two lines indicate sister chromatids joined at the centromere (oval). The closed and open triangles represent the 5′ and 3′ termini of Ac/fAc. The black X indicates the footprint generated by transposition. (1) Ac transposase (open circles) recognizes the 3′ and 5′ termini of Ac/fAc on different sister chromatids. (2) Cleavage by transposase occurs at Ac or fAc termini. (3) The cleavage results in excision of the entire chromosome arm distal to the p1 gene. (4) A chromatid bridge is formed. The bridge will break in the subsequent anaphase. An acentric chromosome fragment is also produced. (B) Model for chromosome breakage by reverse oriented Ac/Ds structure. (1) Ac transposase recognizes the reverse oriented 3′ and 5′ termini of Ac/fAc on the same chromatid. (2) Cleavage by transposase occurs at the Ac or fAc terminus. (3) Excised 5′ and 3′ termini of Ac/fAc insert into a target site in the sister chromatid (black arrow). (4) A chromatid bridge is formed. The bridge will break in the subsequent anaphase. An acentric chromosome fragment is also formed. [See online article for color version of this figure.]