PARAMUTATION is the fascinating ability of specific DNA sequences to communicate in trans to establish meiotically heritable expression states. Intriguingly, newly silenced sequences continue to issue instructions to naive alleles in subsequent generations. The term “paramutation” was first coined in the 1950s by Alexander Brink to describe this puzzling phenomenon at the r1 locus in maize (Brink 1956); an interaction between specific alleles in heterozygotes led to heritable decreases in gene expression of one allele. Not only was the reduced expression state stable through meiosis, but also the low-expressing allele could induce silencing of another high-expressing allele in subsequent generations. The frequency of the change was 100% and the stability of the change was lower than typical mutations; hence the term “paramutation.” A few years later, Ed Coe, Jr., described another maize example in which interaction between alleles at the b1 locus also led to heritable silencing (Coe 1959) and Rudolf Hagemann described interactions at the sulfurea locus in tomato (Hagemann 1969). Since that time other examples of paramutation have been identified in maize and in other species (reviewed in Chandler and Stam 2004; Stam and Mittelsten Scheid 2005; Chandler 2007), yet the two maize loci where paramutation was initially described, r1 and b1, remain the most extensively characterized and best understood. The r1 and b1 loci encode closely related, functionally equivalent transcription factors that activate the anthocyanin pigment biosynthetic pathway (Goff et al. 1990; Ludwig et al. 1990). They are likely related to each other through a duplication resulting from an ancient allotetraploidization event during maize evolution (Gaut and Doebley 1997). The two loci have multiple alleles with distinct expression patterns, which regulate the distribution of anthocyanin pigments during development (Styles et al. 1973; Coe 1979). Recent work demonstrates a key role for RNA in mediating both r1 and b1 paramutation, as the mop1 gene that encodes an RNA-dependent RNA polymerase (RDR; Alleman et al. 2006) is absolutely required for paramutation at both loci (Dorweiler et al. 2000). Yet, there are striking differences in the properties of r1 and b1 paramutation, which hint at distinct mechanisms. In this article, the most striking differences between r1 and b1 paramutation are described and potential mechanisms are discussed relative to our current understanding of the role of RNA interference (RNAi) in mediating transcriptional silencing.

How maize bends the rules Paramutation, first discovered in maize in the 1950s and since found in other plants, fungi, and even mice, is an inheritance pattern that breaks the rules. Most of the time Mendel's law holds sway, and gene pairs sort independently. Paramutation is an interaction in which one silent allele of a gene 'mutates' the actively expressed allele, so that it too is silenced. New work in maize now shows that paramutation is RNA-directed. Stability of the chromatin states associated with paramutation and transposon silencing requires the mop1 gene, which encodes an RNA-dependent RNA polymerase.

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.

The epigenetic phenomenon of genomic imprinting occurs among both plants and animals. In species where imprinting is observed, there are parent-of-origin effects on the expression of imprinted genes in offspring. This review focuses on imprinting in plants with examples from maize, where gene imprinting was first described, and Arabidopsis. Our current understanding of imprinting in plants is presented in the context of cytosine methylation and imprinting in mammals, where developmentally essential genes are imprinted. Important considerations include the structure and organization of imprinted genes and the role of regional, differential methylation. Imprinting in plants may be related to other epigenetic phenomena including paramutation and transgene silencing. Finally, we discuss the role of gene structure and evolutionary implications of imprinting in plants.

The R-sc gene of maize is a member of the R gene family of transcriptional activators that regulate anthocyanin biosynthesis. A derivative of R-sc, r-m9 conditions a reduced level of aleurone pigmentation due to the presence of a 2.1-kb Ds insertion near the 3' end of the coding region. Excision of Ds from r-m9 leaves a 7-bp insertion in the darker but still mutant v24 derivative. Both the 7-bp insertion in v24 and the 2.1-kb Ds in r-m9 are predicted to truncate their respective R proteins proximal to the carboxyl terminus, which was shown previously to contain one of three nuclear localization sequences. We find that the reduced expression of r-m9 and v24 are not due to mRNA or protein instability, but most likely reflect the inefficient localization of truncated R proteins to the nucleus. To our knowledge this is the first example of a transposable element insertion that alters gene expression by affecting nuclear localization. In addition, our data indicate that the carboxyl terminus of the R protein is far more important than previously suspected and illustrates the utility of natural mutations for defining functional domains in proteins.

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.

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.

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.

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.

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.

Forty unstable isolates of an R allele conferring strongly colored seed (R-sc:124) were established using a two-step procedure involving the Ac – Ds transposable element system. First, a series of full-color reversions to R-sc were isolated from two existing Ds mutable alleles, r-sc:m1 and r-sc:m3. Variegated kernels were then selected from large-scale testcrosses of the revenant strains (Ac still present) to establish new mutants. Four of the 9 R-sc revenants from r-sc:m3 gave no mutable alleles, whereas 5 produced a total of 40, in frequencies ranging from 0.8 × 10 −4 to 10.2 × 10 −4 . Upon removal of Ac, each of the 40 mutations was stabilized as colorless or pale, indicating insertion of a Ds element at the R locus. When placed in heterozygous combination with r-sc:m3 (Ac absent), all but possibly 1 of the 38 mutants tested gave R-sc recombinants, showing that insertion had occurred in sites different from that in r-sc:m3. Thirty-eight of the 40 new mutable alleles that were examined by Southern blotting contained a Ds insert of 2.1 kbp, the same size as that found for r-sc:m3. These findings are consistent with the excision of Ds from r-sc:m3, followed by its insertion into a linked site in the R-sc revertant, and the subsequent reinsertion of the element into R. The testcrosses of R-sc revenants obtained from r-sc:m3 also produced five stable mutations to colorless or pale. In contrast with the r-sc:m3 revertant series, no r-mutable or stable variant was obtained from 10 R-sc revenants of r-sc:m1. Either the Ds involved (~ 400 bp) does not transpose to linked sites preferentially or, when it does, it becomes relatively immobile.Key words: mutagenesis, transposable elements, Dissociation (Ds), Activator (Ac), maize.

The Mu transposon of maize exists in a highly mutagenic strain called Robertson's Mutator. Plants of this strain contain 10-50 copies of the Mu element, whereas most maize strains and other plants have none. When Mutator plants are crossed to plants of the inbred line 1S2P, which does not have copies of Mu, the progeny plants have approximately the same number of Mu sequences as did their Mutator parent. Approximately one-half of these copies have segregated from their parent and one-half have arisen by transposition and are integrated into new positions in the genome. This maintenance of copy number can be accounted for by an extremely high rate of transposition of the Mu elements (10-15 transpositions per gamete per generation). When Mutator plants are self-pollinated, the progeny double their Mu copy number in the first generation, but maintain a constant number of Mu sequences with subsequent self-pollinations. Transposition of Mu and the events that lead to copy number maintenance occur very late in the development of the germ cells but before fertilization. A larger version of the Mu element transposes but is not necessary for transposition of the Mu sequences. The progeny of crosses with a Mutator plant occasionally lack Mutator activity; these strains retain copies of the Mu element, but these elements no longer transpose.

AbstractThree new mutant alleles of maize alcohol dehydrogenase‐1 (Adh 1) were recovered following allyl alcohol selection of pollen. Each is altered in quantitative, organ‐specific, regulatory properties. All mutant sites act in cis to the structural gene component. One mutant arose spontaneously, one followed indirectly from irradiation with high Z accelerated particles, and one was induced by an autonomous mutator system. Each mutant is assessed in three organs by utilizing ADH allozyme ratios that were quantified at the level of ADH enzyme activity and either [3H]‐Leu incorporation into newly synthesized ADH 1 subunits or direct protein determinations. One mutation simultaneously raises Adh 1 expression in one organ and lowers it in another, another affects expression in one organ only, and another is extremely underexpressed in all organs but is unstable. This unstable allele has generated derivative mutant alleles that have less or zero ADH expression. We do not yet know whether or not coding sequences are involved in these mutants. We conclude that information for organ specificity and quantitative behavior resides near or within Adh 1 coding sequences.