Ac insertion site affects the frequency of transposon-induced homologous recombination at the maize p1 locus

The maize p1 gene regulates the production of a red pigment in the kernel pericarp, cob, and other maize floral tissues. Insertions of the transposable element Ac can induce recombination between two highly homologous 5.2-kb direct repeat sequences that flank the p1 gene-coding region. Here, we tested the effects of the Ac insertion site and orientation on the induction of recombination at the p1 locus. A collection of unique p1 gene alleles was used, which carry Ac insertions at different sites in and near the p1 locus, outside of the direct repeats, within the direct repeat sequences, and between the direct repeats, in both orientations. Recombination was scored by the numbers of colorless pericarp sectors (somatic frequency) and heritable mutations (germinal frequency). In both the somatic and germinal tests, the frequency of homologous recombination is significantly higher when Ac is inserted between the direct repeats than when Ac is inserted either within or outside the repeats. In contrast, Ac orientation had no significant effect on recombination frequency. We discuss these results in terms of the possible mechanisms of transposon-induced recombination.     

Tissue-specific patterns of a maize Myb transcription factor are epigenetically regulated

The maize p1 gene encodes a Myb-homologous regulator of red pigment biosynthesis. To investigate the tissue-specific regulation of the p1 gene, maize plants were transformed with constructs combining promoter and cDNA sequences of two alleles which differ in pigmentation patterns: P1-wr (white pericarp/red cob) and P1-rr (red pericarp/red cob). Surprisingly, all promoter/cDNA combinations produced transgenic plants with red pericarp and red cob (RR pattern), indicating that the P1-wr promoter and encoded protein can function in pericarp. Some of the RR patterned transgenic plants produced progeny plants with white pericarp and red cob (WR pattern), and this switch in tissue-specificity correlated with increased transgene methylation. A similar inverse correlation between pericarp pigmentation and DNA methylation was observed for certain natural p1 alleles, which have a gene structure characteristic of standard P1-wr alleles, but which confer red pericarp pigmentation and are consistently less methylated than standard P1-wr alleles. Although we cannot rule out the possible existence of tissue-specific regulatory elements within the p1 non-coding sequences or flanking regions, the data from transgenic and natural alleles suggest that the tissue-specific pigmentation pattern characteristic of the P1-wr phenotype is epigenetically controlled.     

Comparative Structural and Functional Characterization of Sorghum and Maize Duplications Containing Orthologous Myb Transcription Regulators of 3-Deoxyflavonoid Biosynthesis

Sequence characterization of the genomic region of sorghum yellow seed 1 shows the presence of two genes that are arranged in a head to tail orientation. The two duplicated gene copies, y1 and y2 are separated by a 9.084 kbp intergenic region, which is largely composed of highly repetitive sequences. The y1 is the functional copy, while the y2 may represent a pseudogene; there are several sequence indels and rearrangements within the putative coding region of y2. The y1 gene encodes a R2R3 type of Myb domain protein that regulates the expression of chalcone synthase, chalcone isomerase and dihydroflavonol reductase genes required for the biosynthesis of 3-deoxyflavonoids. Expression of y1 can be observed throughout the plant and it represents a combination of expression patterns produced by different alleles of the maize p1. Comparative sequence analysis within the coding regions and flanking sequences of y1, y2 and their maize and teosinte orthologs show local rearrangements and insertions that may have created modified regulatory regions. These micro-colinearity modifications possibly are responsible for differential patterns of expression in maize and sorghum floral and vegetative tissues. Phylogenetic analysis indicates that sorghum y1 and y2 sequences may have arisen by gene duplication mechanisms and represent an evolutionarily parallel event to the duplication of maize p2 and p1 genes.     

Transposon-induced inversions activate gene expression in the maize pericarp

Chromosomal inversions can have considerable biological and agronomic impacts including disrupted gene function, change in gene expression, and inhibited recombination. Here, we describe the molecular structure and functional impact of six inversions caused by Alternative Transpositions between p1 and p2 genes responsible for floral pigmentation in maize. In maize line p1-wwB54, the p1 gene is null and the p2 gene is expressed in anther and silk but not in pericarp, making the kernels white. By screening for kernels with red pericarp, we identified inversions in this region caused by transposition of Ac and fractured Ac (fAc) transposable elements. We hypothesize that these inversions place the p2 gene promoter near a p1 gene enhancer, thereby activating p2 expression in kernel pericarp. To our knowledge, this is the first report of multiple recurrent inversions that change the position of a gene promoter relative to an enhancer to induce ectopic expression in a eukaryote.     

Molecular evolution of FLORICAULA/LEAFY orthologs in the Andropogoneae (Poaceae)

Members of the grass family (Poaceae) exhibit a broad range of inflorescence structures and other morphologies, making the grasses an interesting model system for studying the evolution of development. Here we present an analysis of the molecular evolution of FLORICAULA/LEAFY-like genes, which are important developmental regulatory loci known to affect inflorescence development in a wide range of flowering plant species. We have focused on sequences from the Andropogoneae, a tribe within the grass family that includes maize (Zea mays ssp. mays) and Sorghum (Sorghum bicolor). The FLORICAULA/LEAFY gene phylogeny we generated largely agrees with previously published phylogenies for the Andropogoneae using other nuclear genes but is unique in that it includes both members of one of the many duplicate gene sets present in maize. The placement of these sequences in the phylogeny suggests that the duplication of the maize FLORICAULA/LEAFY orthologs, zfl1 and zfl2, is a consequence of a proposed tetraploidy event that occurred in the common ancestor of Zea and a closely related genus, Tripsacum. Our data are consistent with the hypothesis that the transcribed regions of the FLORICAULA/LEAFY-like genes in the Andropogoneae are functionally constrained at both nonsynonymous and synonymous sites and show no evidence of directional selection. We also examined conservation of short noncoding sequences in the first intron, which may play a role in gene regulation. Finally, we investigated the genetic diversity of one of the two maize FLORICAULA/LEAFY orthologs, zfl2, in maize and its wild ancestor, teosinte (Z. mays ssp. parviglumis), and found no evidence for selection pressure resulting from maize domestication within the zfl2-coding region.     

Pollination between maize and teosinte: an important determinant of gene flow in Mexico

Gene flow between maize [Zea mays (L.)] and its wild relatives does occur, but at very low frequencies. Experiments were undertaken in Tapachula, Nayarit, Mexico to investigate gene flow between a hybrid maize, landraces of maize and teosinte (Z. mays ssp. mexicana, races Chalco and Central Plateau). Hybridization, flowering synchrony, pollen size and longevity, silk elongation rates, silk and trichome lengths and tassel diameter and morphology were measured. Hybrid and open-pollinated maize ears produced a mean of 8 and 11 seeds per ear, respectively, when hand-pollinated with teosinte pollen, which is approximately 1–2% of the ovules normally produced on a hybrid maize ear. Teosinte ears produced a mean of 0.2–0.3 seeds per ear when pollinated with maize pollen, which is more than one-fold fewer seeds than produced on a maize ear pollinated with teosinte pollen. The pollination rate on a per plant basis was similar in the context of a maize plant with 400–500 seeds and a teosinte plant with 30–40 inflorescences and 9–12 fruitcases per inflorescence. A number of other factors also influenced gene-flow direction: (1) between 90% and 95% of the fruitcases produced on teosinte that was fertilized by maize pollen were sterile; (2) teosinte collections were made in an area where incompatibility systems that limit fertilization are present; (3) silk longevity was much shorter for teosinte than for maize (approx. 4 days vs. approx. 11 days); (4) teosinte produced more pollen on a per plant basis than the landraces and commercial hybrid maize; (5) teosinte frequently produced lateral branches with silks close to a terminal tassel producing pollen. Collectively these factors tend to favor crossing in the direction of teosinte to maize. Our results support the hypothesis that gene flow and the subsequent introgression of maize genes into teosinte populations most probably results from crosses where teosinte first pollinates maize. The resultant hybrids then backcross with teosinte to introgress the maize genes into the teosinte genome. This approach would slow introgression and may help explain why teosinte continues to co-exist as a separate entity even though it normally grows in the vicinity of much larger populations of maize.     

Evolution of Anthocyanin Biosynthesis in Maize Kernels: The Role of Regulatory and Enzymatic Loci

Understanding which genes contribute to evolutionary change and the nature of the alterations in them are fundamental challenges in evolution. We analyzed regulatory and enzymatic genes in the maize anthocyanin pathway as related to the evolution of anthocyanin-pigmented kernels in maize from colorless kernels of its progenitor, teosinte. Genetic tests indicate that teosinte possesses functional alleles at all enzymatic loci. At two regulatory loci, most teosintes possess alleles that encode functional proteins, but ones that are not expressed during kernel development and not capable of activating anthocyanin biosynthesis there. We investigated nucleotide polymorphism at one of the regulatory loci, c1. Several observations suggest that c1 has not evolved in a strictly neutral manner, including an exceptionally low level of polymorphism and a biased representation of haplotypes in maize. Curiously, sequence data show that most of our teosinte samples possess a promoter element necessary for the activation of the anthocyanin pathway during kernel development, although genetic tests indicate that teosinte c1 alleles are not active during kernel development. Our analyses suggest that the evolution of the purple kernels resulted from changes in cis regulatory elements at regulatory loci and not changes in either regulatory protein function nor the enzymatic loci.     

Cloning and characterization of a putative GS3 ortholog involved in maize kernel development

The GS3 gene was the first identified gene controlling the grain size in rice. It has been proven to be involved in the evolution of grain size during domestication. We isolated the maize ortholog, ZmGS3 and investigated its role in the evolution of maize grain size. ZmGS3 has five exons encoding a protein with 198 amino acids, and has domains in common with the rice GS3 protein. Compared with teosinte, maize has reduced nucleotide diversity at ZmGS3, and the reduction is comparable to that found in neutrally evolving maize genes. No positive selection was detected along the length of the gene using either the Hudson–Kreitman–Aguadé or Tajima’s D tests. Phylogenetic analysis reveals a distribution of maize sequences among two different clades, with one clade including related teosinte sequences. The nucleotide polymorphism analysis, selection test and phylogenetic analysis reveal that ZmGS3 has not been subjected to selection, and appears to be a neutrally evolving gene. In maize, ZmGS3 is primarily expressed in immature ears and kernels, implying a role in maize kernel development. Association mapping analysis revealed one polymorphism in the fifth exon that is significantly associated with kernel length in two environments. Also one polymorphism in the promoter region was found to affect hundred kernel weight in both environments. Collectively, these results imply that ZmGS3 is involved in maize kernel development but with different functional polymorphisms and thus, possibly different mechanisms from that of the rice GS3 gene.     

Insertional Mutagenesis of the Maize P Gene by Intragenic Transposition of Ac

The P-rr allele of the maize P gene regulates the synthesis of pigments derived from flavan-4-ol in the pericarp, cob glumes and other floral organs. We characterized 21 P alleles derived by intragenic transposition of Ac from three known positions. Ac transpositions can occur in either direction in the P gene, and with no apparent minimum distance: in one case Ac transposed just 6 bp from its original insertion site. However, the distribution of transposed Ac elements was markedly nonrandom: of 19 transposed Ac elements derived from a single Ac donor, 15 were inserted in a 1.1-kb region at the 5' end of P, while none had inserted in an adjacent 3.2-kb intronic region. All of the Ac insertions affect both pericarp and cob glume pigmentation, providing further evidence that the P-rr allele contains a single gene required for both pericarp and cob glume pigmentation. The distribution of the inserted Ac elements and the phenotype conditioned by each allele suggests a structure of P-rr which is similar to that previously determined molecularly. Possible explanations for the nonrandom distribution of transposed Ac elements are discussed.