Maize P1 Research Articles

Transcribed enhancer sequences are required for maize p1 paramutation.

Paramutation is a transfer of heritable silencing states between interacting endogenous alleles or between endogenous alleles and homologous transgenes. Prior results demonstrated that paramutation occurs at the P1-rr (red pericarp and red cob) allele of the maize p1 (pericarp color 1) gene when exposed to a transgene containing a 1.2-kb enhancer fragment (P1.2) of P1-rr. The paramutable P1-rr allele undergoes transcriptional silencing resulting in a paramutant light-pigmented P1-rr' state. To define more precisely the sequences required to elicit paramutation, the P1.2 fragment was further subdivided, and the fragments transformed into maize plants and crossed with P1-rr. Analysis of the progeny plants showed that the sequences required for paramutation are located within a ∼600-bp segment of P1.2 and that this segment overlaps with a previously identified enhancer that is present in 4 direct repeats in P1-rr. The paramutagenic segment is transcribed in both the expressed P1-rr and the silenced P1-rr'. Transcription is sensitive to α-amanitin, indicating that RNA polymerase II mediates most of the transcription of this sequence. Although transcription within the paramutagenic sequence was similar in all tested genotypes, small RNAs were more abundant in the silenced P1-rr' epiallele relative to the expressed P1-rr allele. In agreement with prior results indicating the association of RNA-mediated DNA methylation in p1 paramutation, DNA blot analyses detected increased cytosine methylation of the paramutant P1-rr' sequences homologous to the transgenic P1.2 subfragments. Together these results demonstrate that the P1-rr enhancer repeats mediate p1 paramutation.

Excision and reinsertion of Ac macrotransposons in maize.

Eukaryotic Macrotransposons (MTns) can be formed by 2 nearby elements flanking a segment of host DNA. The maize Ac transposon can form Ac::MTns, but little is known about Ac::MTn transposition activities. Here, we studied 3 Ac::MTns at the maize p1 locus, each of which is composed of a segment of maize p1 genomic DNA (up to 15 kb) bounded by a fractured Ac element (fAc, 2039 bp), and a full-length Ac element in direct orientation. The resulting Ac::MTns are of 16, 16.5, and 22 kb total length. From these 3 Ac::MTns, we identified 10 independent cases of macrotransposition, and observed similar features of transposition between Ac::MTn and standard Ac/Ds, including characteristic excision footprints and insertion target site duplications. Nine out of the 10 Ac::MTn reinsertion targets were genetically linked to the donor sites, another similarity with Ac/Ds standard transposition. We also identified a MTn-like structure in the maize B73 reference genome and 5 NAM founder lines. The MTn in diverse lines is flanked by target site duplications, confirming the historic occurrence of MTn transposition during genome evolution. Our results show that Ac::MTns are capable of mobilizing segments of DNA long enough to include a typical full-length plant gene and in theory could erode gene colinearity in syntenic regions during plant genome evolution.

Alternative Transposition Generates New Chimeric Genes and Segmental Duplications at the Maize p1 Locus.

The maize Ac/Ds transposon family was the first transposable element system identified and characterized by Barbara McClintock. Ac/Ds transposons belong to the hAT family of class II DNA transposons. We and others have shown that Ac/Ds elements can undergo a process of alternative transposition in which the Ac/Ds transposase acts on the termini of two separate, nearby transposons. Because these termini are present in different elements, alternative transposition can generate a variety of genome alterations such as inversions, duplications, deletions, and translocations. Moreover, Ac/Ds elements transpose preferentially into genic regions, suggesting that structural changes arising from alternative transposition may potentially generate chimeric genes at the rearrangement breakpoints. Here we identified and characterized 11 independent cases of gene fusion induced by Ac alternative transposition. In each case, a functional chimeric gene was created by fusion of two linked, paralogous genes; moreover, each event was associated with duplication of the ∼70-kb segment located between the two paralogs. An extant gene in the maize B73 genome that contains an internal duplication apparently generated by an alternative transposition event was also identified. Our study demonstrates that alternative transposition-induced duplications may be a source for spontaneous creation of diverse genome structures and novel genes in maize.

Analysis of the Transcriptome of Erigeron breviscapus Uncovers Putative Scutellarin and Chlorogenic Acids Biosynthetic Genes and Genetic Markers

Background Erigeron breviscapus (Vant.) Hand-Mazz. is a famous medicinal plant. Scutellarin and chlorogenic acids are the primary active components in this herb. However, the mechanisms of biosynthesis and regulation for scutellarin and chlorogenic acids in E. breviscapus are considerably unknown. In addition, genomic information of this herb is also unavailable.Principal FindingsUsing Illumina sequencing on GAIIx platform, a total of 64,605,972 raw sequencing reads were generated and assembled into 73,092 non-redundant unigenes. Among them, 44,855 unigenes (61.37%) were annotated in the public databases Nr, Swiss-Prot, KEGG, and COG. The transcripts encoding the known enzymes involved in flavonoids and in chlorogenic acids biosynthesis were discovered in the Illumina dataset. Three candidate cytochrome P450 genes were discovered which might encode flavone 6-hydroase converting apigenin to scutellarein. Furthermore, 4 unigenes encoding the homologues of maize P1 (R2R3-MYB transcription factors) were defined, which might regulate the biosynthesis of scutellarin. Additionally, a total of 11,077 simple sequence repeat (SSR) were identified from 9,255 unigenes. Of SSRs, tri-nucleotide motifs were the most abundant motif. Thirty-six primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism. The result revealed that 34 (94.40%) primer pairs were successfully amplified and 19 (52.78%) primer pairs exhibited polymorphisms.ConclusionUsing next generation sequencing (NGS) technology, this study firstly provides abundant genomic data for E. breviscapus. The candidate genes involved in the biosynthesis and transcriptional regulation of scutellarin and chlorogenic acids were obtained in this study. Additionally, a plenty of genetic makers were generated by identification of SSRs, which is a powerful tool for molecular breeding and genetics applications in this herb.

Genome Rearrangements in Maize Induced by Alternative Transposition of ReversedAc/DsTermini

Alternative transposition can induce genome rearrangements, including deletions, inverted duplications, inversions, and translocations. To investigate the types and frequency of the rearrangements elicited by a pair of reversed Ac/Ds termini, we isolated and analyzed 100 new mutant alleles derived from two parental alleles that both contain an intact Ac and a fractured Ac (fAc) structure at the maize p1 locus. Mutants were characterized by PCR and sequencing; the results show that nearly 90% (89/100) of the mutant alleles represent structural rearrangements including deletions, inversions, translocations, or rearrangement of the intertransposon sequence (ITS). Among 37 deletions obtained, 20 extend into the external flanking sequences, while 17 delete portions of the intertransposon sequence. Interestingly, one deletion allele that contains only a single nucleotide between the retained Ac and fAc termini is not competent for further alternative transposition events. We propose a new model for the formation of intertransposon deletions through insertion of reversed transposon termini into sister-chromatid sequences. These results document the types and frequencies of genome rearrangements induced by alternative transposition of reversed Ac/Ds termini in maize.

Spatial Configuration of Transposable ElementAcTermini Affects Their Ability to Induce Chromosomal Breakage in Maize

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.

Gene Structure Induced Epigenetic Modifications of pericarp color1 Alleles of Maize Result in Tissue-Specific Mosaicism

BackgroundThe pericarp color1 (p1) gene encodes for a myb-homologous protein that regulates the biosynthesis of brick-red flavonoid pigments called phlobahpenes. The pattern of pigmentation on the pericarp and cob glumes depends upon the allelic constitution at the p1 locus. p1 alleles have unique gene structure and copy number which have been proposed to influence the epigenetic regulation of tissue-specific gene expression. For example, the presence of tandem-repeats has been correlated with the suppression of pericarp pigmentation though a mechanism associated with increased DNA methylation.Methodology/Principal FindingsHerein, we extensively characterize a p1 allele called P1-mosaic (P1-mm) that has mosaic pericarp and light pink or colorless cob glumes pigmentation. Relative to the P1-wr (white pericarp and red cob glumes), we show that the tandem repeats of P1-mm have a modified gene structure containing a reduced number of repeats. The P1-mm has reduced DNA methylation at a distal enhancer and elevated DNA methylation downstream of the transcription start site.Conclusions/SignificanceMosaic gene expression occurs in many eukaryotes. Herein we use maize p1 gene as model system to provide further insight about the mechanisms that govern expression mosaicism. We suggest that the gene structure of P1-mm is modified in some of its tandem gene repeats. It is known that repeated genes are susceptible to chromatin-mediated regulation of gene expression. We discuss how the modification to the tandem repeats of P1-mm may have disrupted the epigenetic mechanisms that stably confer tissue-specific expression.

RNA-Dependent RNA Polymerase Is Required for Enhancer-Mediated Transcriptional Silencing Associated With Paramutation at the Maizep1Gene

Paramutation is the ability of an endogenous gene or a transgene to heritably silence another closely related allele or gene. At the maize p1 (pericarp color1) gene, paramutation is associated with decreases in transcript levels and reduced pigmentation of the endogenous allele that normally specifies red seed coat (pericarp) and cob pigmentation. Herein we demonstrate that this silencing occurs at the transcriptional level and that a specific enhancer fragment from p1 is sufficient to induce all aspects of paramutation. Further, we demonstrate that a mutation in the mop1 gene (mediator of paramutation1), which encodes a RNA-dependent RNA polymerase, is absolutely required for establishing the silencing associated with p1 paramutation. In contrast to its effects on other paramutation loci, the mop1 mutation does not immediately reactivate a previously silenced allele; several generations in the presence of the mop1 mutation are required. In addition, the mop1 mutation was also able to release tissue-specific silencing of another p1 allele that does not participate in paramutation, but does contain a tandem repeated structure and is likely regulated through epigenetic mechanisms. These results demonstrate that RNA-mediated gene-silencing mechanisms play key roles in p1 paramutation and the spectrum of roles for MOP1 is broadened to include tissue-specific expression patterns.

Expression of a Maize Myb Transcription Factor Driven by a Putative Silk-Specific Promoter Significantly Enhances Resistance to Helicoverpa zea in Transgenic Maize

Hi II maize (Zea mays) plants were engineered to express maize p1 cDNA, a Myb transcription factor, controlled by a putative silk specific promoter, for secondary metabolite production and corn earworm resistance. Transgene expression did not enhance silk color, but about half of the transformed plant silks displayed browning when cut, which indicated the presence of p1-produced secondary metabolites. Levels of maysin, a secondary metabolite with insect toxicity, were highest in newly emerged browning silks. The insect resistance of transgenic silks was also highest at emergence, regardless of maysin levels, which suggests that other unidentified p1-induced molecules likely contributed to larval mortality. Mean survivor weights of corn earworm larvae fed mature browning transgenic silks were significantly lower than weights of those fed mature nonbrowning transgenic silks. Some transgenic pericarps browned with drying and contained similar molecules found in pericarps expressing a dominant p1 allele, suggesting that the promoter may not be silk-specific.

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.

A Segmental Deletion Series Generated by Sister-Chromatid Transposition of Ac Transposable Elements in Maize

Certain configurations of maize Ac/Ds transposon termini can undergo alternative transposition reactions leading to chromosome breakage and various types of stable chromosome rearrangements. Here, we show that a particular allele of the maize p1 gene containing an intact Ac element and a nearby terminally deleted Ac element (fAc) can undergo sister-chromatid transposition (SCT) reactions that generate large flanking deletions. Among 35 deletions characterized, all begin at the Ac termini in the p1 gene and extend to various flanking sites proximal to p1. The deletions range in size from the smallest of 12,567 bp to the largest of >4.6 cM; >80% of the deletions removed the p2 gene, a paralog of p1 located approximately 60 kb from p1 in the p1-vv allele and its derivatives. Sequencing of representative cases shows that the deletions have precise junctions between the transposon termini and the flanking genomic sequences. These results show that SCT events can efficiently generate interstitial deletions that are useful for in vivo dissection of local genome regions and for the rapid correlation of genetic and physical maps. Finally, we discuss evidence suggesting that deletions induced by alternative transposition reactions can occur at other genomic loci, indicating that this mechanism may have had a significant impact on genome evolution.

Transformation of maize with the p1 transcription factor directs production of silk maysin, a corn earworm resistance factor, in concordance with a hierarchy of floral organ pigmentation

The maize p1 gene encodes an R2R3-MYB transcription factor that controls the biosynthesis of red flavonoid pigments in floral tissues of the maize plant. Genetic and quantitative trait locus analyses have also associated the p1 gene with the synthesis of maysin, a flavone glycoside from maize silks that confers natural resistance to corn earworm. Here, we show directly that the p1 gene induces maysin accumulation in silk tissues. Transformation of maize plants that had low or no silk maysin with p1 transgenes elevated silk maysin concentrations to levels sufficient for corn earworm abiosis. The p1 transgenes also conferred red pigment to pericarp, cob, husk and tassel tissues, as expected; however, different subsets of these tissues were pigmented within individual transgenic plants. Statistical analysis shows that the pigmentation patterns observed amongst the p1 transgenic plants conform to a hierarchy that is similar to the temporal ordering of floral organ initiation. We propose that the observed hierarchy of pigmentation patterns is conferred by variation due to epigenetic control of the p1 transgenes. The production of plants with improved traits through genetic engineering can depend in large part on the achievement of tight organ-specific expression of the introduced transgenes. Our results demonstrate that the production of transgenic plants using a promoter with well-defined tissue specificity, such as the p1 promoter, can result in unexpected variation in tissue specificity amongst the resulting transgenic plants.

Two Cysteines in Plant R2R3 MYB Domains Participate in REDOX-dependent DNA Binding

Plant R2R3 MYB domain proteins comprise one of the largest known families of transcription factors. Discrete evolutionary steps have shaped the plant-specific R2R3 MYB family from the broadly distributed R1R2R3 MYB proteins. R1R2R3 MYB domains have a single Cys residue (Cys-130) that needs to be reduced for DNA binding and transcriptional activity. In contrast, most R2R3 MYB domains contain two cysteines, Cys-49 and Cys-53, with Cys-53 at the equivalent position as Cys-130 in R1R2R3 MYB. Using the maize P1 regulator of flavonoid biosynthesis as a typical R2R3 MYB-domain protein, we investigated here the in vitro REDOX requirement for DNA binding by P1. We show that the C53S mutation requires reducing conditions for DNA-binding, whereas C53A binds DNA under oxidizing and reducing conditions. Neither mutation impairs the in vivo regulatory activity of P1. The C49S and C49A mutants bind DNA in vitro irrespective of the REDOX conditions. A C49I mutant, which simulates the MYB domain of c-MYB, binds DNA only under reducing conditions, and its binding is significantly affected by the C53S replacement. It is interesting that under non-reducing conditions, Cys-49 and Cys-53 form a disulfide bond that prevents the R2R3 MYB domain from binding DNA. Together, our results suggest that the evolutionary origin of Cys-49 within the plants has provided R2R3 MYB domains with a regulatory feature not present in animal MYB domains, highlighting fundamental structural and functional differences between similar DNA-binding domains from plants and animals.

Transposition of reversed Ac element ends generates chromosome rearrangements in maize.

In classical "cut-and-paste" transposition, transposons are excised from donor sites and inserted at new locations. We have identified an alternative pathway in which transposition involves the 5' end of an intact Ac element and the 3' end of a nearby terminally deleted fAc (fractured Ac). The Ac and fAc elements are inserted at the maize p1 locus on chromosome 1s in the same orientation; the adjacent ends of the separate elements are thus in reversed orientation with respect to each other and are separated by a distance of approximately 13 kb. Transposition involving the two ends in reversed orientation generates inversions, deletions, and a novel type of local rearrangement. The rearrangement breakpoints are bounded by the characteristic footprint or target site duplications typical of Ac transposition reactions. These results demonstrate a new intramolecular transposition mechanism by which transposons can greatly impact genome evolution.

A maize QTL for silk maysin levels contains duplicated Myb-homologous genes which jointly regulate flavone biosynthesis.

The maize p1 locus coincides with a major QTL (quantitative trait locus) determining levels of maysin, a C-glycosyl flavone that deters feeding by corn ear-worm. The p1 gene is tightly linked with a second gene, p2, and both genes encode similar Myb-domain proteins. We show here that maize cell cultures transformed with either the p1 or p2 genes expressed under a constitutive promoter accumulate transcripts for flavonoid biosynthetic genes, and synthesize phenylpropanoids and C-glycosyl flavones related to maysin. Additionally, maize plants that are deleted for the p1 gene have reduced maysin levels and moderate silk-browning reaction, whereas plants with a deletion of both p1 and p2 have non-detectable silk maysin and non-browning silks. We conclude that both p1 and p2 induce maysin biosynthesis in silk, although the two genes differ in their expression and pigmentation effects in other tissues. These results show that a QTL for flavone biosynthesis actually comprises two tightly linked genes with related functions.

Functional analysis of two matrix attachment region (MAR) elements in transgenic maize plants.

Matrix attachment regions (MARs) are binding sites for nuclear scaffold proteins in vitro, and are proposed to mediate the attachment of chromatin to the nuclear scaffold in vivo. Previous reports suggest that MAR elements may stabilize transgene expression. Here, we tested the effects of two maize MAR elements (P-MAR from the P1-rr gene, and Adh1-MAR from the adh1 gene) on the expression of a gusA reporter gene driven by three different promoters: the maize p1 gene promoter, a wheat peroxidase (WP) gene promoter, or a synthetic promoter (Rsyn7). The inclusion of P-MAR or Adh1-MAR on P::GUS transgene constructs did not reduce variation in the levels of GUS activity among independent transformation events, nor among the progeny derived from each event. The Adh1-MAR element did not affect GUS expression driven by the WP promoter, but did modify the spatial pattern of expression of the Rsyn7::GUS transgene. These results indicate that, in transgenic maize plants, the effects of MAR elements can vary significantly depending upon the promoter used to drive the transgene.

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.

Transgene-Induced Silencing Identifies Sequences Involved in the Establishment of Paramutation of the Maize p1 Gene

Transgene-Induced Silencing Identifies Sequences Involved in the Establishment of Paramutation of the Maize p1 Gene

A segmental gene duplication generated differentially expressed myb-homologous genes in maize.

The myb-homologous p1 gene regulates the synthesis of flavonoid pigments in maize kernel pericarp and cob; these floral organs are greatly modified in size and shape compared with their counterparts in teosinte, the progenitor of maize. To elucidate the molecular evolution of the p1 gene in relation to its expression and possible functions in maize and teosinte, we have isolated a second maize gene (p2) that is highly homologous with the p1 gene, and a related gene (p2-t) from Zea mays subsp parviglumis. We present evidence that the maize p1 and p2 genes were generated by duplication of an ancestral p gene (p(pre)) and its downstream sequences; the duplicated 3' flanking sequences were inserted upstream of the p(pre) gene, thereby changing its transcription pattern. This model accounts for the structural organization and the observed differential expression of the p1 and p2 genes: p1 transcripts accumulate in kernel pericarp, cob, tassel glumes, and silk, whereas p2 transcripts are found in developing anther and silk. The duplication is estimated to have occurred 2.75 million years ago; subsequently, multiple retroelements have been inserted between the p1 and p2 genes. Our results demonstrate the evolution of a single gene into a compound locus containing two component genes with different tissue specificities. Expression of the p1 gene in the kernel pericarp may have provided a selective advantage during the evolution of maize kernel morphology.

Genome rearrangements by nonlinear transposons in maize.

Transposable elements have long been considered as potential agents of large-scale genome reorganization by virtue of their ability to induce chromosomal rearrangements such as deletions, duplications, inversions, and reciprocal translocations. Previous researchers have shown that particular configurations of transposon termini can induce chromosome rearrangements at high frequencies. Here, we have analyzed chromosomal rearrangements derived from an unstable allele of the maize P1 (pericarp color) gene. The progenitor allele contains both a full-length Ac (Activator) transposable element and an Ac terminal fragment termed fAc (fractured Ac) inserted in the second intron of the P1-rr gene. Two rearranged alleles were derived from a classical maize ear twinned sector and were found to contain a large inverted duplication and a corresponding deficiency. The sequences at the junctions of the rearrangement breakpoints indicate that the duplication and deletion structures were produced by a single transposition event involving Ac and fAc termini located on sister chromatids. Because the transposition process we describe involves transposon ends located on different DNA molecules, it is termed nonlinear transposition (NLT). NLT can rapidly break and rejoin chromosomes and thus could have played an important role in generating structural heterogeneity during genome evolution.