The Fcu controlling-element system in maize

Summary The relationship between the Fcu controlling-element system and the spotted-dilute R system was investigated. The Fcu controlling-element system consists of the receptor element allele r-cu and the regulatory element Fcu. The equivalent components of the spotted-dilute R system are respectively R-r#2 (or R-r#2 Dil) and Spf. The R-r#2 allele of the latter system was shown to be responsive (mutable) to Fcu, provided that it has had an uninterrupted association with Dil or Dpf as evidenced by the color variegation of the aleurone tissue. The reverse test, in which the r-cu allele of the Fcu controlling element system was tested for its response to Spf, proved negative. This was surprising in view of the relationship and specificity between systems. The possibility was considered that maize controlling elements may have different sizes as is known for bacterial insertion sequences. —The variable dilute pigmenting capacity of the r-cu allele also was studied. A given level of r-cu-induced pigmentation, despite the wide range in pigmentation expression, was found to be generally non-heritable, as based on a test of correspondence between parent and progeny.Journal Paper No. J-9204 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 1884Now with Funk's Seeds, Casalmorano (Cremona), Italy     

The a2m(r-Pa-Pu) Allele of the En-Controlling Element System in Maize

The Enhancer (En) controlling element system in maize includes numerous alleles, one of which, a2m(r-pa-pu), was recovered as a derivative of the a2m(1 1511) allele. The behavior and composition of this allele is described. In the presence of the independently inherited regulatory element, En, the a2m(r-pa-pu) allele mutates and expresses in the aleurone purple, pale and colorless sectors within a colorless background. In the absence of En, the pigmentation is uniformly pale. Colored and colorless germinal mutations of the a2m(r-pa-pu) allele were recovered, although the derivative alleles tested did not respond to En. Uniformly colored derivatives, designated A2', arise only from states of a2m(r-pa-pu) that respond to an active En at relatively early stages in plant development, suggesting that somatic and germinal mutations may arise through similar events. Mutations to A2-expressing alleles appear to occur relatively late in plant development, that is, at or near meiosis, since recognizable mutant sectors do not appear on testcross ears. Kernels exhibiting such mutants are randomly distributed over the ear. The resulting A2' alleles express pigmentation ranging in a continual sequential series from dark pale to intense purple.     

Differential excision patterns of the <Emphasis Type="Italic">En</Emphasis>-transposable element at the <Emphasis Type="Italic">A2</Emphasis> locus in maize relate to the insertion site

Defined mutant alleles with resident transposons display characteristic patterns of germinal and somatic reversion, and heritable changes in the timing and frequency of reversions, which have been termed “change of state” by McClintock, constantly arise. Several mechanisms were proposed to account for these changes. They may be ascribed to the structure and composition of the elements themselves (composition hypothesis) or to their location (position hypothesis). In the current study, insertion positions were determined for three autonomous En-controlled mutable alleles of the A2 locus in maize that show different somatic reversion patterns. A relationship was observed between En insertion positions in the single coding region of the intronless A2 gene and anthocyanin variegation patterns in the aleurone. An insertion in the 5′ region of the coding sequence produced a very late somatic variegation pattern, whereas two early variegation patterns were caused by En insertions in the 3′ region of the coding sequence.     

Change in State following Transposition of a Regulatory Element of the Enhancer System in Maize

From an original A2 allele (colored aleurone), a mutable allele, a2-m-4 1629, that changes from a2 to A2 is described. Mutability is expressed as a very distinct pattern limited to the last cell division.—The mutability of a2-m-4 1629 is autonomously controlled by an En at the a2 locus. This En, inactive on standard a testers for En, is partially active on a2-m-1, an a2 tester for En, and expresses varied levels of activity from limited to nearly full suppression of the a2-m-1 color phenotype.—When the En of the a2-m-4 1629 allele transposes from the a2 locus, it behaves, at the new position, like a standard En in triggering a2-m-1, a-m-1 and a-m(r), which express colored spots on a colorless background. The activity of En is therefore different following the change in chromosome location. This finding supports the "position" hypothesis that has been proposed to explain diverse patterns observed among controlling elements. In this case mutation is related to the terminal cell state and not to tissue differences as shown with some phase-variation regulatory elements.     

Analysis of a case of somatic instability in a strain of maize from India

A case of somatic instability affecting aleurone colour in a strain of maize from India with flint background was analysed. The somatic instability is localized to theC ¹ (Inhibitor) allele ofC locus on the short arm of chromosome 9. Molecular tests indicated thatAc is not present in the Indian stock and the evidence is consistent with the involvement of theEn (Spm) transposable element in the instability. The presence of theEn (Spm)-like element in the stock would suggest that these elements have been present in the maize genome for a long time. A new allele ofshrunken (sh1) gene with a somewhat unorthodox breeding behaviour is also described.     

Genetic characterization of a mutable allele,R-marbled (R-mb), in maize (Zea mays L.)

TheR-marbled (R-mb) allele in maize confers a distinct pattern of anthocyanin pigmentation in the aleurone. We investigated the genetic mechanism involved in the formation and variability of this pattern. Wide variation was observed in the extent of anthocyanin pigmentation in marbled kernels. Progeny testing of different expressions resulted in distinct segregation profiles, indicating that the somatic variegation has a genetic basis. Drastic reduction in penetrance and expressivity was noticed whenR-mb was transmitted in a single dose through the pollen parent. Analysis of the colored kernels fromR-mb, including discordant endosperm-embryo phenotypes, showed that only germinal reversions fromR-mb toR-sc (self-colored) were transmissible. Unlike other pattern alleles at theR locus, viz.R-stippled (R-st) andR-Navajo (R-nj),R-mb reverts toR-sc at a higher frequency. No dominance-recessiveness relation was found among the three pattern alleles. Reciprocal-cross differences occurred whenR-mb was crossed withR-nj orR-st, but the interaction ofR-mb withR-st was not entirely similar to that withR-nj. The characteristic variegation pattern due toR-mb is attributed to the action of a transposable genetic element on the basis of somatic and germinal instability, occurrence of discordant endosperm-embryo phenotypes, and genetic analysis ofR-mb/R-st andR-mb/R-nj heterozygotes. The distinct genetic behaviour ofR-mb, in comparison withR-st andR-nj, indicates specificity of the controlling element operating at this allele.     

Basis for the diversity of states of controlling elements in maize

Summary In maize the insertion of a controlling element such as the regulatory element, En, (Enhancer) at a locus generates diverse derivatives. These derivatives include stable phenotypes that range from colorless to full color. Distinguishable patterns are determined by timing and frequency of mutation events. It has been hypothesized that diverse expressions are based either on position of the controlling element at the locus or composition differences among controlling elements. — Experiments with two target loci, A2 and C, indicate that the previous pattern of a controlling element does not influence the pattern generated at a new site. This finding supports the hypothesis that these diverse trans-effects of En are not caused by a change in the genetic information of En itself but rather by its position; diverse expressions are a manifestation of the effect of an element at a specific locus site. — Controlling elements are discussed in relation to chromosome structure, gene activation, gene programming and possible parallels in prokaryotes.Journal Paper No. J-8416 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa 50011. Project No. 1884. Support from NSF GB38328     

An altered state of a specific en regulatory element induced in a maize tiller

There are numerous states of the regulatory element, Enhancer (En). With specific receptor alleles, such as a2m(r-pa-pu) or a2m(r), specific mutability patterns are expressed. One specific derivative En allele, En-v (En-variable), was originally identified with a coarse pattern of mutability with the a2m(r-pa-pu) allele and giving progeny with varied En expression (standard to reduced within an ear progeny). Derivatives of En-v were subsequently found on numerous occasions to give only a very reduced expression (fewer mutant spots) with the a2m(r-pa-pu) allele in the ears derived from the main stalk of the corn plant. When a comparison is made of the effect of this changed En-v state between tiller ears and main stalk ears of the same plant, the tiller ears show an increased level of En-v expression (coarse pattern), while the main-stalk ears continue to show the very reduced level of En-v expression (low frequency of very late variegation). This increased level of mutability of the tiller ears is maintained when transmitted through the main-stalk ear in the subsequent generation. These results indicate that heritable alterations of controlling elements can be produced by endogenous environmental factors present during normal plant development.     

Variegation patterns caused by excision of the maize transposable element Dissociation (Ds) are autonomously regulated by allele-specific Activator (Ac) elements and are not due to trans-acting modifier genes

The Ac elements present in the unstable wxm7 and wx-m9 alleles of maize trigger different patterns of Ds excision in trans. To determine whether this differential regulation is a feature of the Ac alleles themselves or is mediated by genetically distinct factors, maize plants heterozygous for the wx-m7 and wx-m9 alleles were crossed to tester strains homozygous for Ds reporter alleles. Kernels showing the variegation pattern characteristic for the Ac elements carried in the wx-m7 and wx-m9 alleles were found to be present in the ratios expected from the genetic constitution of the strains. The aleurone variegation caused by excision of the Ds reporter element and the endosperm variegation caused by excision of Ac from the wx-m7 and wx-m9 alleles themselves segregated with the original wx-m alleles. In addition, stable Wx and wx derivatives of wx-m9 that have lost Ac no longer exert any trans effect on the wx-m7 allele (and vice versa). Therefore it is concluded that the observed variegation patterns are autonomously determined by specific trans effects of the particular Ac element.     

Genetic and molecular analysis of a three-component transposable-element system in maize

Summary Two different factors control the mutability of an unstable allele (c2-m8810581) of the C2 gene of maize. Both an autonomous En/Spm element and an unrelated independent factor, named Mediator, are coordinately required for the excision of the insert in c2-m881058Y. According to genetic analysis, Mediator does not have the suppressor (S) function or mutator (M) function of En/Spm. Mediator has no effect on the timing or frequency of excision of Enl, En-low, or various I/dSpm elements. Hence, Mediator only mediates a specific interaction between En and the insert at c2m881058Y. Molecular analysis of c2-m881058Y has revealed a 3.3 kb, complex, En-related receptor element inserted into the second exon of the C2 gene. The ends of this element are homologous to the ends of En/Spm, but an internal l.7 kb region shows no En/Spm homology. A great degree (11–14%) of nucleotide changes, relative to Enl, occur within and between the 12 bp TNPA binding motifs. Alterations of these critical cis-determinants may account for the need for a helper factor for excision. This element is named Irma, for Inhibitor that requires Mediator also, and represents a unique, low copy number class of receptor element.     

Variegation patterns caused by excision of the maize transposable element Dissociation (Ds) are autonomously regulated by allele-specific Activator (Ac) elements and are not due to trans-acting modifier genes

The Ac elements present in the unstable wxm7 and wx-m9 alleles of maize trigger different patterns of Ds excision in trans. To determine whether this differential regulation is a feature of the Ac alleles themselves or is mediated by genetically distinct factors, maize plants heterozygous for the wx-m7 and wx-m9 alleles were crossed to tester strains homozygous for Ds reporter alleles. Kernels showing the variegation pattern characteristic for the Ac elements carried in the wx-m7 and wx-m9 alleles were found to be present in the ratios expected from the genetic constitution of the strains. The aleurone variegation caused by excision of the Ds reporter element and the endosperm variegation caused by excision of Ac from the wx-m7 and wx-m9 alleles themselves segregated with the original wx-m alleles. In addition, stable Wx and wx derivatives of wx-m9 that have lost Ac no longer exert any trans effect on the wx-m7 allele (and vice versa). Therefore it is concluded that the observed variegation patterns are autonomously determined by specific trans effects of the particular Ac element.     

Genetic instability at the opaque-2 locus of maize

Summary A case of genetic variegation discovered at the opaque-2 locus of maize that includes a two-element system with a receptor and regulatory element is described. The somatic mutability depends on the existence of two genetic factors: a responsive allele (with receptor element), o2m(r), and a regulatory element, Bg, that induces mutability of o2m(r). In the absence of Bg, o2m(r) is indistiguishable from the recessive alleles of the O2 locus; in the presence of the regulatory element, o2m(r) mutates giving rise to sectors of flint-like endosperm in an opaque back-ground. The regulatory element Bg may be located independently or at the controlled locus. The genetic properties of the new system, somatic mutability, transposition, existence of different patterns of mutability, are apparently similar to those previously described in maize for the classical systems of controlling elements. In addition, the recovery of the o2 mutability from crosses between spontaneous o2 alleles suggests that transposable genetic elements may be involved in the origin of natural mutability.     

The inveterate wanderer: study of Enhancer wandering on chromosome 3 in maize

The transposition of the maize transposable element Enhancer (En) had been focused on one chromosome 3 for several generations. From the a1-m(Au) allele with an autonomous En, a new En reporter allele a1-m(r)3927-1, was isolated that undergoes very infrequent and late excision events, producing one or two small spots in the aleurone. This allele is seriously impaired in its capacity to excise. Coincident with the origin of this allele, an En was located at a site close to the a1 locus. From this initial insertion site, the movement of this En was followed for three to four generations in 974 families with a higher transposition rate of this En (50% of the testcross progeny) than that found in a previous study of En transposition. This is the first case reported where a particular En was followed for more than three generations. The higher rate of wanderings of this En along the same chromosome led to the term vagabond En (En ^vag ). Genetic evidence that En may transpose from a replicated donor site to an unreplicated site is provided. Speculative mechanisms on the origin of a1-m(r)3927-1 and En ^vag are discussed.Journal Paper No. J-15864 of Iowa Agricultural and Iowa Economical Experiment Station Project #3176     

Chromosome labeling with transposable elements in maize

Transposable elements randomly insert into a targeted locus at a frequency of 10^-6 to 10^-5. The En element has been shown in previous studies to transpose more frequently into closely linked sites. Thus, it is appropriate to place an En element onto each of the 20 chromosome arms in maize to maximize tagging efficiency. This is called chromosome labeling for tagging purposes with transposons. After a chromosome arm has been labeled with a transposon, genes residing in that arm will have a greater chance to be tagged by the transposon. To date, all of the maize chromosome arms have been labeled with at least one of five Encontaining alleles. The elements were linked to the arms using reciprocal translocations. The usage of these arm-labeled lines is discussed in the context of gene tagging.Journal Paper No. 15224 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa; Project No. 3176     

Controlling-element events at the shrunken locus in maize

We have examined insertions of the controlling element Ds at the Shrunken locus of maize. A cDNA probe complementary to a portion of the Shrunken locus mRNA was prepared. This probe recognizes a unique sequence in maize DNA. Using lines carrying derivatives of the same short arm of chromosome 9, we have detected modifications at the nucleic acid level caused by Ds. The changes appear to be large insertions, one of which may be more than 20 kilobase pairs in length. These observations provide a basis for the isolation and molecular characterization of one of the maize controlling elements.     

Enhancer transposable element induced changes at the A locus in maize: The a-m-1 6078 allele

Summary Transposable (controlling) elements in maize are highly variable in their versatility in inducing changes at any locus. With the Enhancer (En) (Suppressor-Mutator-Spm) controlling-element system,a multitude of changes result from the action of En on specific alleles. One such allele, McClintock's a-m-1 6078 allele, illustrates the diversity of events that can take place that involve three parameters; namely, spotting-timing, spotting-frequency, and background pigment formation with and without En. A series of derivative alleles has been isolated and described according to the three paramters. The multitude of changes that has been isolated illustrates the striking versatility of these transposing elements to initiate changes at the controlled locus.Journal Paper No. J-11135 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 2381     

Germinal derivatives of the En controlling-element system in maize: Characterization of colored,pale and colorless derivatives a2-m

Summary Several stable germinal derivatives, with varied phenotypic expression have been recovered from testcrosses of four different a2 mutable alleles (a2m-7 8018, a2m-6 8144, a2m-6 8140, and a2m-1 1511) under the control of the En controlling-element system. These are colored, pale, and colorless aleurone types; the former two represent mutations of a2-mA2 type. The phenotypic variation among colored and pale derivatives has been confirmed by quantitative determination of anthocyanin content in the aleurone tissue of the kernels.The data suggest that significant variation exists between phenotypically similar colored derivatives arising from different mutable alleles as well as from the same source allele. Every tested colored derivative is significantly different from the pale, colorless, and colored control (W22 color converted line). Genetic analysis of these colored derivatives suggests that all of them contain one or more En and, hence, they are characterized as permanent changes of a nonresponsive (nr) type. Their frequencies are variable among the testcrosses.Similarly, the pale derivatives, which are clearly different from colored and colorless, also show significant differences in terms of anthocyanin content. Significant differences exist between pales arising from the same allele as well as between pales arising from different alleles. All the tested pales are significantly different from colorless and colored derivatives. Thus, the pales, as well as colored derivatives, represent a differential impairment of anthocyanin synthesis in the aleurone tissue. The genetic analysis confirmed that all the pales, except the pales of a2m-1 1511, lack the capacity to respond to En. These pale derivatives can be with or without En.Qualitative differences among the colored, pale, and colorless types have been investigated by thin-layer chromatographic and spectroscopic techniques. The results suggest that there are no qualitative differences, in terms of anthocyanin pigments, between colored, pale, and colored control. All accumulate the same anthocyanins (namely, cyanidin-3-glucoside and pelargonidin-3-glucoside in appropriate proportions depending upon the Pr and pr constitution).Journal Paper No. J-8431 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011. Project No. 1884: Support from NSF GB38328.