Interaction between maternal effect and zygotic effect mutations during maize seed development.

Double fertilization of the embryo sac by the two sperm cells of a pollen grain initiates seed development. Proper development of the seed depends not only on the action of genes from the resulting embryo and endosperm, but also on maternal genes acting at two stages. Mutations with both sporophytic maternal effects and gametophytic maternal effects have been identified. A new maternal effect mutation in maize, maternal effect lethal1 (mel1), causes the production of defective seed from mutant female gametophytes. It shows reduced pollen transmission, suggesting a requirement in the male gametophyte, but has no paternal effect on seed development. Interestingly, the defective kernel phenotype of mel1 is conditioned only in seeds that inherit mel1 maternally and are homozygous for the recessive allele (endogenous to the W22 inbred line) of either of two genes, sporophyte enhancer of mel1 (snm1) or snm2, suggesting redundancy between maternally and zygotically required genes. Both mel1 and snm1 map to the short arm of chromosome 2, and snm2 maps to the long arm of chromosome 10. The mode of action of mel1 and the relationship between mel1 and snm1 and snm2 are discussed.

Gene conversion within regulatory sequences generates maize r alleles with altered gene expression.

The maize r locus encodes a transcription factor that regulates the developmental expression of the plant pigment anthocyanin. In an unusual example of gene regulatory diversity, the R-sc (Sc, strong seed color) and the R-p (P, plant color) alleles of r have nonoverlapping tissue specificity and nonhomologous 5' flanking sequences. Heterozygotes between wild-type P and Sc mutants with Ds6 transposable element inserts (r-sc:m::Ds6 or sc:m) produce colored seed derivatives (Sc+) during meiotic recombination. The sc:m alleles with Ds6 insertion in 3' regions of r produce crossover Sc+ derivatives. sc:m alleles with Ds6 elements inserted in 5' regions produce rare Sc+ derivatives borne on nonrecombinant chromosomes. Among 52 such noncrossover Sc+ derivatives, 18 are indistinguishable from the Sc progenitor in phenotype and DNA sequence [Scp(+) alleles]. The remaining 34 derivatives have strong Sc+ expression, including darkly pigmented aleurone, scutellum, coleoptile, and scutellar node [Scp(e) alleles]. The coleoptile and scutellar node phenotypes are unique from either progenitor but are similar to those of some naturally occurring r alleles. Both classes of Sc+ derivatives are explained by gene conversion between the promoter region of Sc:124 and a homologous region located proximal to P. The recombinational intermediate formed between sc:m alleles and P results in deletion of the Ds6 element alone or both Ds6 and a nearby unrelated transposable element-like sequence.

mediator of paramutation1 is required for establishment and maintenance of paramutation at multiple maize loci.

Paramutation is the directed, heritable alteration of the expression of one allele when heterozygous with another allele. Here, the isolation and characterization of a mutation affecting paramutation, mediator of paramutation1-1 (mop1-1), are described. Experiments demonstrate that the wild-type gene Mop1 is required for establishment and maintenance of the paramutant state. The mop1-1 mutation affects paramutation at the multiple loci tested but has no effect on alleles that do not participate in paramutation. The mutation does not alter the amounts of actin and ubiquitin transcripts, which suggests that the mop1 gene does not encode a global repressor. Maize plants homozygous for mop1-1 can have pleiotropic developmental defects, suggesting that mop1-1 may affect more genes than just the known paramutant ones. The mop1-1 mutation does not alter the extent of DNA methylation in rDNA and centromeric repeats. The observation that mop1 affects paramutation at multiple loci, despite major differences between these loci in their gene structure, correlations with DNA methylation, and stability of the paramutant state, suggests that a common mechanism underlies paramutation. A protein-based epigenetic model for paramutation is discussed.

Molecular consequences of Ds insertion into and excision from the helix-loop-helix domain of the maize R gene.

The R and B proteins of maize are required to activate the transcription of several genes in the anthocyanin biosynthetic pathway. To determine the structural requirements for R function in vivo, we are exploiting its sensitive mutant phenotype to identify transposon (Ds) insertions that disrupt critical domains. Here we report that the ability of the r-m1 allele to activate transcription of at least three structural genes is reduced to only 2% of wild-type activity because of a 396-bp Ds element in helix 2 of the basic helix-loop-helix (bHLH) motif. Residual activity likely results from the synthesis of a mutant protein that contains seven additional amino acids in helix 2. This protein is encoded by a transcript where most of the Ds sequence has been spliced from pre-mRNA. Two phenotypic classes of stable derivative alleles, very pale and extremely pale, condition <1% of wild-type activity as a result of the presence of two- and three-amino-acid insertions, respectively, at the site of Ds excision. Localization of these mutant proteins to the nucleus indicates a requirement for an intact bHLH domain after nuclear import. The fact that deletion of the entire bHLH domain has only a minor effect on R protein activity while these small insertions virtually abolish activity suggests that deletion of the bHLH domain may bypass a requirement for bHLH-mediated protein-protein interactions in the activation of the structural genes in the anthocyanin biosynthetic pathway.

Insertions of a novel class of transposable elements with a strong target site preference at the r locus of maize.

The r locus of maize regulates anthocyanin synthesis in various tissues of maize through the production of helix-loop-helix DNA binding proteins capable of inducing expression of structural genes in the anthocyanin biosynthetic pathway. The complex r variant, R-r: standard (R.r), undergoes frequent mutation through a variety of mechanisms including displaced synapsis and crossing over, and intrachromosomal recombination. Here we report a new mechanism for mutation at the R-r complex: insertion of a novel family of transposable elements. Because the elements were first identified in the R-p gene of the R-r complex, they have been named P instability Factor (PIF). Two different PIF elements were cloned and found to have identical sequences at their termini but divergent internal sequences. In addition, the PIF elements showed a marked specificity of insertion sites. Six out of seven PIF-containing derivatives examined had an element inserted at an identical location. Two different members of the PIF element family were identified at this position. The seventh PIF-containing derivative examined had the element inserted at a distinct position within r. Even at this location, however, the element inserted into a conserved target sequence. The timing of PIF excision is unusual. Germinal excision rates can range up to several percent of progeny. Yet somatic sectors are rare, even in lines exhibiting high germinal reversion rates.

Organization of paramutagenicity in R-stippled maize.

In heterozygotes, R-stippled (R-st) reduces the pigmenting potential of sensitive r alleles heritably (paramutation). R-st is comprised of four r genes arranged in direct orientation. Unequal crossing over within R-st generates deletion products retaining from one to three r genes. Paramutagenic strength decreased in parallel with copy number, both among internal and distal deletions. Single-gene R-st derivatives were nonparamutagenic. This was so whether or not the single gene retained the transposable element (I-R) responsible for seed spotting. Adding back r genes by intragenic recombination increased paramutagenicity in proportion to total gene number. Each member of a set of overlapping deletions retained moderately strong activity, showing that no single r gene or intragenic region is required for paramutagenicity. Proximal and distal loss R-st derivatives, each retaining two r genes, were less paramutagenic in trans than the corresponding four copy cis combination, indicating R-st's paramutagenic determinants function as a cis-interdependent unit in bringing about modification of a sensitive allele.

Molecular organization and germinal instability of R-stippled maize.

The spotted seed allele R-stippled (R-st) is comprised of the following genetic components: strong seed color (Sc), inhibitor-of-R (I-R) and near-colorless seed (Nc). I-R is a mobile element that represses (Sc) expression irregularly. Germinal I-R losses produce progeny with fully colored seed. Southern blot analysis revealed four r-hybridizing segments in R-st and three, two or one in two sets of unequal crossover deletion products. By comparison to published reports of r gene structure, we maintain that each segment contains at least one r gene. The proximal r gene, Sc, confers strong seed color; the three distal r genes together produce near-colorless seed. R-st's seed spotting phenotype is correlated with the presence of a 3.3-kb insert in Sc identified as I-R. The level of the near-colorless phenotype is inversely correlated with the number of r genes present, suggesting involvement of a multiple copy silencing mechanism in their regulation. Phenotypic changes in R-st occurred primarily by unequal exchange between r genes. The locations of exchange positions showed a strong polarity, nearly all occurring in the 3' portions of the identified r genes.

Transposon-mediated chromosomal rearrangements and gene duplications in the formation of the maize R-r complex.

R-r controls the production of anthocyanin pigment in plant parts and the aleurone layer of seeds through the production of a family of related transcriptional activating proteins of the helix-loop-helix type. The R-r complex comprises a series of repeated, homologous components arranged in both direct and inverted orientations. These include the P component, a simple R gene that confers pigmentation of plant parts, and the S subcomplex that consists of a truncated inactive R gene called q, and two functional R genes, S1 and S2, that pigment the aleurone. The S genes are arranged in an unusual inverted head-to-head orientation. The identity of each functional component was confirmed by microprojectile bombardment of intact maize tissues with cloned genomic DNA and by analysis of in vivo mRNA populations. Sequence analysis suggests that the S subcomplex was derived through the rearrangement of a simple P-like progenitor element. At the rearrangement breakpoints, features typical of the CACTA family of transposable elements were found. The location and arrangement of these CACTA element sequences implies that this element may have mediated the chromosomal rearrangements that led to the formation of the R-r complex. The unusual structure of R-r explains much of the meiotic instability of the complex.

Teosinte glume architecture 1: A Genetic Locus Controlling a Key Step in Maize Evolution.

Teosinte, the probable progenitor of maize, has kernels that are encased in hardened fruitcases, which interfere with the use of the kernels as food. Although the components of the fruitcase are present in maize, their development is disrupted so that the kernels are not encased as in teosinte but exposed on the ear. The change from encased to exposed kernels represents a key step in maize evolution. The locus that largely controls this morphological difference between maize and teosinte, teosinte glume architecture 1, is described and genetically mapped.

Somatic variegation and germinal mutability reflect the position of transposable element Dissociation within the maize R gene.

The R gene regulates the timing and tissue-specificity of anthocyanin deposition during maize development. The Ac/Ds system of transposable elements was used to induce insertional mutants of the R-sc:124 allele during two cycles of mutagenesis. Of 43 unstable, spotted-aleurone mutants generated, 42 contain inserts of the Ds6 transposable element differing only in the position and orientation of the element. The remaining mutant, r-sc:m1, contained an insert of a Ds element of the approximate size of the Ds1 transposable element. The patterns of somatic variegation of these mutants, resulting from excision of Ds, define a spectrum of phenotypes ranging from sparse to dense variegation. The sparsely variegated mutants produce few germinal revertants but relatively many stable null derivative alleles; densely variegated mutants produce many germinal revertants and few stable null derivatives. Molecular analysis shows that the sparsely variegated alleles are caused by Ds6 insertions in protein coding regions of R-sc:124 whereas the densely variegated mutants result from insertions in introns or in flanking regions of the gene. The excision rate of Ds6 from R, estimated as the proportion of R genomic DNA restriction fragments lacking the element, was uniform regardless of position, orientation or whether the element was inserted in R-sc:124 or another R allele. The excision rate was greater, however, for the mutable alleles involving the Ds element from r-sc:m1. These data indicate that, although the excision rates are uniform for a given Ds element, the somatic and germinal mutability patterns of alleles associated with that element vary widely and depend primarily on the position of the transposable element within coding or noncoding regions of the gene.

Meiotic instability of the R-r complex arising from displaced intragenic exchange and intrachromosomal rearrangement.

The R complex of Zea mays encodes a tissue-specific transcriptional activator of the anthocyanin pigment biosynthetic pathway. Certain R alleles comprise two genetically distinct components that confer the plant (P) and seed (S) aspects of the pigmentation pattern. These alleles are meiotically unstable, losing (P) or (S) function, often accompanied by exchange of flanking markers. We show that the (P) component consists of a single gene within the R-r complex, whereas the (S) component is part of a more complex arrangement of multiple R genes or gene subfragments. A third, cryptic region of the complex, termed (Q), consists of a truncated R sequence. The analysis of R-r crossover derivative alleles shows they arise from unequal exchange between the (P) gene and one of several distinct regions of the R-r complex. Restriction site polymorphisms were used to show that most of these unequal exchanges are intragenic. The frequency of displaced intragenic recombination is comparable to previous estimates for intragenic recombination in maize involving genes that are not duplicated. These exchange events have been used to determine the arrangement of components within the complex and their orientation in the chromosome. We also show that localized rearrangements in the (P) or (S) components are responsible for noncrossover derivative alleles. The organization of R-r has implications for these noncrossover derivatives and models for their origin are discussed.

Gametic imprinting in maize in relation to the angiosperm life cycle.

Differences in the activity of maternally and paternally derived genomes in maize endosperm have been observed at three levels of genetic manipulation. When the balance of entire chromosome sets departs from the standard ratio of two of maternal origin to one of paternal origin, development is impaired, often leading to seed failure. At the level of individual chromosomes, absence of a paternal representative for 8 of the 19 chromosome arms tested causes a marked reduction in kernel size. Replacement of the missing arms by ones of maternal origin does not complement this defect. At the gene level, some alleles of R confer solid coloration on the aleurone layer when transmitted maternally but patchy coloration (mottled) when transmitted via pollen. In contrast with the endosperm, no effect of parentage on R phenotype has been detected in embryonic and seedling tissues. Furthermore, gynogenetic and androgenetic haploid plants are viable in maize and are similar in appearance. The detection of parental effects in the endosperm, but not the embryo, points to the few cell divisions of the gametophytes as a critical stage in imprinting. Chromosomally based epigenetic variation originating at this stage would be reflected as imprinting effects. A separate fertilization establishes a line of genetic descent in the embryo that appears to be relatively free of imprinted genes.

The Transposable Element Ds Affects the Pattern of Intragenic Recombination at the bz and R Loci in Maize.

Insertion of the transposable element Ds into either the bz or R locus affects intragenic recombination in various ways. We have examined here one aspect of this problem; namely, the distribution of flanking markers among intragenic recombinations produced by different types of heterozygotes carrying Ds insertion mutations. Heteroallelic combinations of a Ds insertion mutation and a mutation borne on a structurally normal chromosome generate a majority of intragenic recombinants of a crossover type. In contrast to this, most intragenic recombinants obtained from heterozygotes between two different Ds insertion mutations have a parental arrangement of outside markers. Therefore, the resolution of the recombination intermediate would appear to depend on the nature of the mutations in the heterozygote.

R-stippled maize as a transposable element system.

The I-R element at the R locus destabilizes kernel pigmentation giving the variegated pattern known as stippled ( R-st). In trans linkage phase with R-st the element was shown to act as a modifier of stippled, intensifying seed spotting in parallel with effects of the dominant linked modifier M-st. Presence of I-R in the genome was, therefore, shown to be detectable as a modifier of R-st. When this test was used, new modifiers resembling M-st were often detected following mutations of R-st to the stable allele R-sc. Such mutations evidently occurred by transposition of I-R away from the R locus to a site where it was identifiable as a modifier. M-st may be such a transposed I-R. Analysis of mutations to R-sc during the second (sperm-forming) mitosis in pollen grains showed that some of the transposed I-R elements were linked with R, whereas others assorted independently. Their strengths varied from barely discernible to a level equal to M-st. Overreplication frequently accompanied transposition at the sperm-forming mitosis, leading to transposed I-R elements in both the mutant and nonmutant sperm.

Recombination between Components of a Mutable Gene System in Maize.

An unstable component (I-R) of the R-stippled allele interacts with a linked modifier which enhances stippled's expression ( M-st) to delete the intervening segment. The precision of deletion formation suggests a recombinational basis, specifically unequal crossing over between I-R and M-st. Four deletions were selected as losses of R function from plants homozygous for R-st and M-st. Also lost are stippled's near-colorless seed phenotype, its paramutagenicity and a closely linked gene, Inhibitor of striate. The deletion chromosomes, missing a 6-cM segment, are transmitted normally by ovules but in reduced frequency by pollen. Homozygous and heteroallelic combinations of the deletions confer defective seed lethality. The four did not differ detectably in transmissibility or breakpoint termini. The recurrence of deletions that have the same termini is explained by recombination between I-R and M-st. The homology between M-st and I-R, and their presence in the same chromosome arm, favors the view that M-st originated by I-R transposition.

Probing the component structure of a maize gene with transposable elements.

Three instances of R gene instability were found in maize stocks carrying the controlling elements Dissociation (Ds) and Modulator (Mp). In each, Ds or a Ds-like element had transposed to R, inhibiting kernel pigmentation irregularly. When Mp was removed from the genome, R expression stabilized at lowt to intermediate levels. Strong pigmenting action was restored through recombination in heterozygotes of the three new forms with an R allele that specifies only plant pigmentation. The sites of Ds insertion mapped distal to the region that specifies seed versus plant expression. The evidence suggests that an R functional unit consists of one component that both governs tissue-specific expression and another that is common to alleles of different tissue-specific activities.

Organelle DNA variation and systematic relationships in the genus Zea: Teosinte.

Chloroplast and mitochondrial DNAs from six races of annual teosinte (Guatemala, Huehuetenango, Balsas, Central Plateau, Chalco, and Nobogame), perennial teosinte, and maize were compared and grouped by restriction endonuclease fragment analyses. Three groups of chloroplast DNAs were detected: (i) perennial teosinte and Guatemala; (ii) Balsas and Huehuetenango; and (iii) all other teosintes. Four groups of mitochondrial DNAs were separated: (i) perennial teosinte; (ii) Guatemala; (iii) Nobogame; and (iv) all other teosintes. Separation of the teosinte and maize organelle DNAs into five groups (Guatemala; perennial teosinte; Balsas and Huehuetenango; Central Plateau and Chalco; Nobogame and maize) approximated the biosystematic relationships of the taxa. It was suggested that the evolutions of the chloroplast and mitochondrial DNAs may be independent of each other, that variation of organelle DNA within a species complex of an organism may be the common condition, and that the DNAs of the organelle and nuclear systems evolve in reasonable harmony.

Displaced and tandem duplications in the long arm of chromosome 10 in maize.

Lc, an anthocyanin pigmenting factor mapping somewhat more than one unit distal to R, is borne on a chromosomal segment which is homologous with part of the R-r:standard duplicated segment. Deficiencies and tandem duplications of the R to Lc region arise from exchanges within these obliquely paired homologous segments. The deficiencies are transmitted with a high, although reduced, frequency by the male gametophyte and are homozygous viable. Yet, the R to Lc region is not duplicated either proximal to R or distal to Lc. Thus the Lc-marked segment and either the P- or the S-marked segment of R-r constitute a displaced duplication. Such an arrangement can initiate a tandem and displaced duplication cycle.---No evidence was obtained for fractionation of the compound phenotype conditioned by Lc.

Reconstitution of the R compound allele in maize.

The R(r):standard allele in maize, which conditions anthocyanin pigmentation in plant and seed tissues in the presence of appropriate complementary factors, is associated with a tandem duplication. The proximal member of the duplication carries P, the plant pigmenting determiner and the distal member member carries S, the seed pigmenting determiner. Derivatives from R(r) that have lost S function are designated r(r). They represent either losses of the distal member of the duplication (P derivatives) or mutations of S to s (P s). Derivatives that have lost P function are designated R(g), and represent either losses of the proximal member of the duplication (S derivatives) or mutations of P to p (p S).-All four possible types of r(r)/R(g) heterozygotes were tested for their capacity to yield R(r) reconstitution by crossing over. No R(r) derivatives were obtained from P/S heterozygotes, a result consistent with the view that P and S occupy corresponding positions in homologous chromosome segments. R(r) reconstitution was detected in both tandem duplication heterozygotes P s/S and P/p S, and was found to be about ten times more frequent in the latter. The ratio of R(r) reconstitution in the two heterozygotes is a function of position of the anthocyanin marker within the duplicated segment. The data from these heterozygotes allow one to measure the distance between P and S, that is to say, the genetic length of the duplicated segment. This distance was found to be 0.16 map units. The highest frequency of R(r) reconstitution was obtained from P s/p S heterozygotes, since direct pairing (see PDF) as well as the p//s type of displaced pairing have the potential to produce R(r) derivatives. One of the R(g) derivatives used in this study, R(g) (6), was found to back-mutate in some sublines to R(r). The basis for this instability remains unknown.