Reversion of the maize mutable allele o2-hf in plant ontogeny

Reversions of the mutable allele o2-hf leading to formation of the phenotypically normal kernels or whole endosperm revertants (WER) are studied in the plant ontogeny. The pattern of WER kernel distribution on the ear maps and analysis of their progeny showed that the reversion of the mutable allele o2-hf occurs at the late premeiotic stages of the ear development. Most of whole endosperm revertants on the ears homozygous for both the mutable allele o2-hf and regulatory element Bg-hf are grouped into clusters. The WER kernels are mostly formed during the period from the gamete fusion to the first division of the primary endosperm nucleus and are not embryo revertants. This clustering of revertant kernels seems to be caused by the joint effect of two factors on the early stages of endosperm development. These factors are (1) diffusion of an additional amount of transposase into the nearby Kernels from the developing endosperm, where the level of this enzyme is sufficient to induce excision of the receptor element and (2) the high proportion of the developing kernels with supra- and subthreshold levels of the Bg-hf-encoded transposase.     

Control of the Frequency of Reversion of the Mutable Alleles at the opaque2 Locus by the Bg-rbg System of Transposable Elements in Maize

Maize lines differing in the frequency of reversion of the opaque2 (o2) mutable alleles controlled by the Bg-rbg system of transposable elements were studied. In the presence of the Bg regulatory element, these alleles can revert to normal. When reversion occurs prior to the first division of the primary endosperm nucleus, phenotypically normal kernels or whole endosperm revertants (WER) develop. It was shown that the low frequency of formation of whole endosperm revertant may be produced by different genetic mechanisms. The frequency of WER formation was shown to nonlinearly depend on the dose of the Bg-hf regulatory element. A dose increase from two to three failed to cause an essential increase in the number of revertants. The regulatory elements Bg-lf andBg-hfdiffered in their ability to induce excision of the receptor element at the same dose. The frequency of reversion of the receptive alleles was shown to be regulated by epigenetic mechanisms so that high frequency of reversion of receptive alleles requires preliminary premeiotic association between the regulatory and receptor elements. The inheritance of the maize alleles o2-hfand o2-lf proved to be similar to that of an3 mutable alleles in petunia.     

The frequency of reversion of the opaque2 locus mutable alleles controlled by the Bg-rbg system of mobile elements in maize

Maize lines differing in the frequency of reversion of the opaque2 (o2) mutable alleles controlled by the system of Bg-rbg transportable elements were studied. In the presence of the Bg regulatory element, these alleles can revert to normal. When reversion occurs prior to the first division of the primary endosperm nucleus, either phenotypically normal kernels or whole endosperm revertants (WER) develop. Low frequency of whole endosperm revertant formation may be produced by different genetic mechanisms. The frequency of WER formation was shown to nonlinearly depend on the dose of the Bg-hf regulatory element. A dose increase from two to three failed to cause an essential increase in the number of revertants. The regulatory elements Bg-lf and Bg-hf differed in ability to induce excision of the receptor element at the same dose. The frequency of reversion of the receptive alleles was shown to be regulated by epigenetic mechanisms so that high frequency of reversion of receptive alleles requires preliminary premeiotic association between the regulatory and receptor elements. The inheritance of the maize alleles o2-hf and o2-lf proved to be similar to that an3 mutable alleles in petunia.     

Novel, developmentally specific control of Ds transposition in maize

Plants form their gametes late in somatic development and, as a result, often pass somatic mutations on to their progeny. Classic examples of this process are the germinal revertants of unstable, Ac/Ds transposon-induced kernel mutations in maize: frequent and early reversion events during somatic development are generally correlated with a high frequency of revertant gametes. We have characterized a Ds allele of the maize waxy(wx) gene, wx-m5:CS7, for which the correlation between somatic and germinal reversion frequencies no longer holds. The ability of wx-m5:CS7 (CS7) to produce revertant gametes is suppressed ∼100-fold in comparison with a second Ds allele, wx-m5:CS8 (CS8), which has an identical insertion at Wx and the same frequent and early somatic reversion pattern in endosperm. The excision of Ds from wx is not reduced 100-fold in the somatic tissues of CS7 plants as compared with CS8 plants. Suppressed formation of CS7 revertant gametes is independent of the Ac transposase source and is heritably passed to the embryos of progeny kernels; however, frequent and early somatic reversion is observed again in endosperms of these progeny kernels. This suppression appears to be caused by a dominant mutation in a trans-acting product that can suppress the germinal reversion of other Ds-induced alleles as well; the mutation is tightly linked to Wx but is not in the CS7 Ds itself. Taken together, the data suggest a novel mode of developmental control of Ac/Ds elements by the host plant, suppressing element excision in the shoot meristem. Received: 16 December 1996 / Accepted: 4 March 1997     

Studies on the Mx transposable element system in maize recovered from X-irradiated stocks

Summary The unstable mutant bz-x3m arose in a plant subjected to X-irradiation. The element at the bronze locus is non-autonomous and recombination data indicate that an autonomous element is tightly linked. The autonomous element has been designated Mx (mobile element induced by X-rays) and the non-autonomous element, rMx (responder to Mx). Linkage data indicate that a second Mx lies near the end of the short arm of chromosome 9; in one plant, an Mx that is unlinked was detected. Distinguishing characteristics of bz-x3m are a large window of time in endosperm development during which somatic reversions can arise and a wide range in the frequency at which they occur; these features are heritable. With increasing doses of bz-x3m and Mx, the window expands and the frequency range increases. In kernels containing the bz-x3m allele and the tightly linked Mx, breakage occurs in chromosome 9 distal to the C locus, resulting in breakage-fusion-bridge patterns for endosperm markers that lie proximal to the break. The frequency of breaks and the developmental time at which they occur exhibit the same dosage effect as the somatic reversions of the bz-x3m allele. These observations suggest that an rMx (designated rMxBr) that causes chromosome breakage is positioned distal to the C locus. At the molecular level, the bz-x3m allele is associated with a 0.5 kb increase in fragment size in DNA samples digested with BglII, EcoRI, HindIII and PstI; in germinal revertants, the fragment size returns to that of the progenitor.     

Alfalfa transposable elements and variegation

Alfalfa with unstable anthocyanin pigmentation has been independently discovered on six occasions since 1958. Genetic studies showed that each of the six unstable stocks was due to an allele mutable at the basic anthocyanin locus C2 in alfalfa. The alleles are designated c2-m1 through c2-m6. Variegated phenotypes of m1, m2, and m3 are similar and express reversion from the recessive to the dominant state. This reversion produces streaks and sectors of pigment in flower petals and seeds that are otherwise white. Reversion occurs at various times in development and may result in periclinal chimeras. The c2-m4 allele is unique in that it arose during tissue culture, whereas the other mutables were discovered in plant populations. Interestingly, m4 is very stable in planta and only rarely produces a sectored flower, but is very unstable in vitro as measured by about 23% revertant plants regenerated from tissue cultures. Most m4 reversion occurs relatively early in development and results in completely pigmented in vitro revertants, and in large sectors on in planta revertants. Alleles m5 and m6 are phenotypically and genetically similar. Their flowers are basic purple with white streaks thus representing mutation from dominant purple to recessive white. White progeny of m5 and m6 are very stable both in planta and in vitro; reversion of white to purple was never observed. Thus, the loss of function of the dominant allele results in a stable recessive or a deficiency. The absolute stability of m5 white derivatives favors the deficiency model, because transposable element mutations might show reversion. Finally, several mutations are described that reoccur in the mutable populations. It is speculated that they are recent mutations due to transposition of transposable elements.     

Ds-Induced Alleles at the OPAQUE-2 Locus of Maize

Transposon mutagenesis has been used to isolate mutable alleles at the Opaque-2 (O2) locus of maize. Plants with the Activator-Dissociation (Ac-Ds) system of transposable elements and O2 were crossed as males to a stable o2 tester line. Among a population of 200,000 kernels, 198 exceptional kernels with somatic instability were recovered. In four cases, designated O2-m1, o2-m2, O2-m3 and O2-m4, variegated phenotypes appeared in F₂ and subsequent generations. Genetic analyses indicated that the presence of Ds near or within the O2 gene was responsible for the observed somatic instability at the O2 locus. The phenotypes of the newly induced alleles were of two types. Alleles O2-m1, O2-m3 and O2-m4, in the absence of Ac, were characterized by kernel phenotypes indistinguishable from the wild type; in the presence of Ac they generated kernels with opaque sectors interspersed within a vitreous background. In contrast, the mutable allele o2-m2, in the absence of Ac, was characterized by kernels with a recessive phenotype similar to o2 recessive mutants. In the presence of Ac, it reverted somatically to wild-type-producing kernels with vitreous spots in an o2 background. The association of the Ds element with the O2 locus may prove a valuable tool directed to the isolation of DNA fragments bearing the O2 gene.     

Molecular analysis of the loss of somatic instability in the bz2::mu1 allele of maize

Summary Multiple genetic and epigenetic changes were detected within one plant generation at the bz2:: mu1 mutable allele in a population of 118 plants. Loss of somatic instability in bz2::mu1 was usually correlated with methylation of the Mu1 transposable element; in 6 plants, somatic instability was lost as a result of mutations in bz2::mu1. This is a surprisingly high frequency of mutation per allele (2.5%) for the Mutator family, for which germinal revertants occur at a frequency of about 10^–4 per gamete. One germinal excision event was found that contained an 8 by deletion, frameshift mutation in Bronze-2. The three other mutants described occurred as a result of abortive transposition, in which 75–77 by deletions were generated at the junction between Bronze-2 and Mu1. We discuss the possible mechanisms, and the role of host factors in abortive transposition in maize.     

Molecular cloning of the o2-m5 allele of Zea mays using transposon marking

Summary The deposition of zein protein in maize endosperm is under the control of several regulatory loci. The isolation of DNA sequences corresponding to Opaque-2 (O2), one of such loci, is described in this paper. The mutable allele, o2-m5 was first induced moving the Ac transposable element present at the wx-m7 allele to the O2 locus. Genetic data suggest that a functional Ac element is responsible for the observed somatic mutability of o2-m5. The isolation of genomic clones containing flanking sequences corresponding to the O2 gene was possible by screening an o2-m5 genomic libary with a probe corresponding to internal Ac sequences usually absent in the defective element Ds. Out of 27 clones isolated with homology to the central part of Ac element, only clones 6IP and 21IP generated a 2.5 kb internal fragment size of an active Ac element when digested with PvuII restriction enzyme. A sequence representing a XhoI fragment of 0.9 kb lying, in the 6IP clone, adjacent to the Ac elements, was subcloned and utilized to prove that it corresponded to a part of the O2 gene. To obtain this information we made use of: (1) DNAs from several reversions originating from the unstable (o2mk-(r) allele, which, when digested with SstI, showed a correct 3.4 kb fragment typical of non-inserted alleles of the O2 locus; and (2) recessive alleles of the O2 locus which were devoid of a 2.0 kb mRNA, present on the contrary in the wild type and in other zein regulating mutants different from O2.This paper is dedicated to the memory of R. Marotta, who actively participated in the realization of this work     

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.     

Regulation of programmed cell death in maize endosperm by abscisic acid

Cereal endosperm undergoes programmed cell death (PCD) during its development, a process that is controlled, in part, by ethylene. Whether other hormones influence endosperm PCD has not been investigated. Abscisic acid (ABA) plays an essential role during late seed development that enables an embryo to survive desiccation. To examine whether ABA is also involved in regulating the onset of PCD during endosperm development, we have used genetic and biochemical means to disrupt ABA biosynthesis or perception during maize kernel development. The onset and progression of cell death, as determined by viability staining and the appearance of internucleosomal DNA fragmentation, was accelerated in developing endosperm of ABA-insensitive vp1 and ABA-deficient vp9 mutants. Ethylene was synthesized in vp1 and vp9 mutant kernels at levels that were 2–4-fold higher than in wild-type kernels. Moreover, the increase and timing of ethylene production correlated with the premature onset and accelerated progression of internucleosomal fragmentation in these mutants. Treatment of developing wild-type endosperm with fluridone, an inhibitor of ABA biosynthesis, recapitulated the increase in ethylene production and accelerated execution of the PCD program that was observed in the ABA mutant kernels. These data suggest that a balance between ABA and ethylene establishes the appropriate onset and progression of programmed cell death during maize endosperm development.     

w4-Mutable line in soybean

An unstable mutation for anthocyanin pigmentation in soybean (Giycine max [L.] Merr.) was identified in 1983. The mutability is conditioned by an allele at the w4 locus that is recessive to wild type. The population containing the mutable allele is known as the w4-mutable line. Most plants in the line have chimeric flowers with purple sectors on a near-white background. The mutable allele yields germinal revertants at a rate that varies from 5 to 10% per generation, and the revertant alleles are stable. Approximately 1% of the progenies derived from germinal revertant plants contain mutations at other loci These features, as well as the occurrence of pale flower phenotypes and changes of state, suggest that a transposable element system is producing the unstable phenotype. Several new mutants were isolated in an experiment designed to tag loci. The first three chlorophyll-deficient mutants found (CD-1, CD-2, and CD-3) are inherited as single-gene recessives. Each of the mutants lacks the same two mitochondrial malate dehydrogenase (MDH) bands. No recombination has been detected between the MDH phenotype and the chlorophyll-deficient phenotype. Genetic data indicate that the three mutants are allelic, and additional evidence suggests that each of the CD mutants is the result of a deletion. In the CD-1, CD-2, and CD-3 mutants, the deletions result in the silencing of an MDH locus, atypical chloroplast development, and an altered chlorophyll composition. Additional mutants for root necrosis, partial and near sterility, chlorophyll deficiency, and flower color isolated from the transposon tagging study have provided material for future research.     

Effects of floury-2 locus on zein accumulation and RNA metabolism during maize endosperm development

Zein accumulation patterns during mutant and normal maize endosperm development were determined. Accompanying an increase in the number of floury-2 alleles present in the endosperm was a well-defined stepwise depression in zein accumulation. Analysis of the zein accumulated in endosperms containing zero, one, two, and three doses of the floury-2 allele by sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed a proportionate reduction in the two major zein components, Z1 and Z2. In contrast, the relative proportions of the minor zein bands were altered. Membrane-bound polysomes isolated from kernels of floury-2 and normal maize were predominantly large size classes. The presence of increasing numbers of the floury-2 allele in the endosperm decreased recovery of membrane-bound polysomal material in a stepwise fashion. However, major alterations in polysome size-class distributions were not observed. The reduction in membrane-bound polysome material correlated linearly with reductions in in vitro zein synthesis and in vivo zein accumulation.     

In vivo aggregation of maize Activator (Ac) transposase in nuclei of maize endosperm and Petunia protoplasts

The transposase (TPase) of the maize transposon Activator (Ac) accumulates in the nuclei of maize endosperm and transfected Petunia protoplasts, where it aggregates into rod-like structures about 2 μm in length. In petunia protoplasts the amount of TPase aggregates increases with the strength of the promoter fused to the Ac-coding region. The excision frequency of a Ds element, however, does not increase proportionally. The data suggest that the aggregated TPase is not responsible for the mobilization of the Ds element, but rather is a transpositionally inactive form of the protein. In contrast to the full-length TPase, a functional, N-terminally truncated TPase derivative is inefficiently transported into the nucleus at high expression levels and aggregates predominantly in the cytoplasm. Accordingly, the N-terminus of the TPase is involved in nuclear localization and/or aggregation.     

Novel, developmentally specific control of Ds transposition in maize

Plants form their gametes late in somatic development and, as a result, often pass somatic mutations on to their progeny. Classic examples of this process are the germinal revertants of unstable, Ac/Ds transposon-induced kernel mutations in maize: frequent and early reversion events during somatic development are generally correlated with a high frequency of revertant gametes. We have characterized a Ds allele of the maize waxy(wx) gene, wx-m5:CS7, for which the correlation between somatic and germinal reversion frequencies no longer holds. The ability of wx-m5:CS7 (CS7) to produce revertant gametes is suppressed ～100-fold in comparison with a second Ds allele, wx-m5:CS8 (CS8), which has an identical insertion at Wx and the same frequent and early somatic reversion pattern in endosperm. The excision of Ds from wx is not reduced 100-fold in the somatic tissues of CS7 plants as compared with CS8 plants. Suppressed formation of CS7 revertant gametes is independent of the Ac transposase source and is heritably passed to the embryos of progeny kernels; however, frequent and early somatic reversion is observed again in endosperms of these progeny kernels. This suppression appears to be caused by a dominant mutation in a trans-acting product that can suppress the germinal reversion of other Ds-induced alleles as well; the mutation is tightly linked to Wx but is not in the CS7 Ds itself. Taken together, the data suggest a novel mode of developmental control of Ac/Ds elements by the host plant, suppressing element excision in the shoot meristem.