Transposition of the En/Spm transposable element system in maize (Zea mays L.): reciprocal crosses of a1-m(Au) and a1-m(r) alleles uncover developmental patterns

Transposition studies of the transposon, En/Spm, have dealt with general aspects of the timing of the excision event with regard to DNA replication and plant development, but without describing details of the process. By following the excision events of an En transposon inserted at the a1 locus [a1-m(Au)], several features of this process can be elucidated. In progenies from reciprocal crosses between the a1-m(Au) allele containing an En insert, and a nonautonomous En allele, [a1-m(r) is a deficiency derivative of En], several features of the En at the a1-m(Au) allele can be observed taking place during ear development and during microsporogenesis. First, it has long been known that the distribution of mutant kernel phenotypes on an ear indicates that En transposes late in most of the events during ear development. Second, the phase change of En (presence and absence of activity) is observed during cob development. Third, discordant kernel phenotypes of two ears, reported herein, resulting from a reciprocal cross with the parental phenotype can be deduced to arise from the transposition of En during microsporogenesis and subsequent fertilization, leading to a discordant genotype between endosperm and embryo. The phase change and discordance lead us to conclude that these events can arise from transposition after host DNA replication. It can also be concluded that the activity of the En inserted in this a1-m(Au) allele is not limited to a specific stage or timing during plant development. Further, this study illustrates the power of genetic analysis in the determination of cellular events. Received: 26 May 1999 / Accepted: 11 November 1999     

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.     

Spm and I element changes with the a-m2 8004 allele in maize

Summary Among the mobile element systems in maize, the En (Spm) system (En — the regulatory element and I the receptive element — a nonfunctional En) has several interesting aspects of control of gene expression (En and Spm are homologous in structure and activity). One of the alleles arising from the Spm group included the a-m2 8004 allele that has a low spotting pattern and unique ringed areas. The interest in this allele is that Spm or En will induce it to co-express the A phenotype and mutability. Several exceptions of the allele were analyzed. Two are Spm changes and two are I changes. The analysis shows that the heritable changes include I changes that are co-expressed in various grades of color and different degrees of mutability. All these changes occur with I at the locus. The Spm changes also include changes in mutability patterns and a mottling pattern.Journal Paper No., J-11792 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 2381     

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.     

Genetic instability in Drosophila Melanogaster: Mutable miniature (mμ)

A new mutable gene, mutable miniature wing (m^μ), is described. This mutable gene mutates spontaneously at an inordinate rate both germinally and somatically. Two classes of reversions of m^μ have been found in approximately equal frequency: those to an allele with an intermediate phenotype (mⁱ) and those to a subliminal allele (m^s) equivalent to wild type in phenotype. Reversions appear to be mutationally stable. The chronology of genetic events leading to the discovery of m^μ implicates, but does not prove, the insertion of a “foreign” DNA segment as the basis of mutability.     

The KIT Gene Is Associated with the English Spotting Coat Color Locus and Congenital Megacolon in Checkered Giant Rabbits (Oryctolagus cuniculus)

The English spotting coat color locus in rabbits, also known as Dominant white spotting locus, is determined by an incompletely dominant allele (En). Rabbits homozygous for the recessive wild-type allele (en/en) are self-colored, heterozygous En/en rabbits are normally spotted, and homozygous En/En animals are almost completely white. Compared to vital en/en and En/en rabbits, En/En animals are subvital because of a dilated (“mega”) cecum and ascending colon. In this study, we investigated the role of the KIT gene as a candidate for the English spotting locus in Checkered Giant rabbits and characterized the abnormalities affecting enteric neurons and c-kit positive interstitial cells of Cajal (ICC) in the megacolon of En/En rabbits. Twenty-one litters were obtained by crossing three Checkered Giant bucks (En/en) with nine Checkered Giant (En/en) and two en/en does, producing a total of 138 F1 and backcrossed rabbits. Resequencing all coding exons and portions of non-coding regions of the KIT gene in 28 rabbits of different breeds identified 98 polymorphisms. A single nucleotide polymorphism genotyped in all F1 families showed complete cosegregation with the English spotting coat color phenotype (θ = 0.00 LOD  = 75.56). KIT gene expression in cecum and colon specimens of En/En (pathological) rabbits was 5–10% of that of en/en (control) rabbits. En/En rabbits showed reduced and altered c-kit immunolabelled ICC compared to en/en controls. Morphometric data on whole mounts of the ascending colon showed a significant decrease of HuC/D (P<0.05) and substance P (P<0.01) immunoreactive neurons in En/En vs. en/en. Electron microscopy analysis showed neuronal and ICC abnormalities in En/En tissues. The En/En rabbit model shows neuro-ICC changes reminiscent of the human non-aganglionic megacolon. This rabbit model may provide a better understanding of the molecular abnormalities underlying conditions associated with non-aganglionic megacolon.     

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     

Systemic infection of stalks and ears of corn hybrids by <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis>

This study was conducted to explore systemic infection by the Aspergillus flavus group into corn ears via the stalk. An A. parasiticus mutant which produces norsolorinic (NOR) acid (a visible orange intermediate of the aflatoxin biosynthetic pathway) was used in field studies to monitor systemic infection of corn stalk and ear tissues. Corn hybrids resistant and susceptible to aflatoxin contamination were grown in the field and inoculated prior to tasseling by inserting A. parasiticus infested toothpicks into stalks between the 5th and 6th node below the lowest ear shoot. Beginning 2 weeks after inoculation, systemic infection by the NOR mutant was assessed weekly by collecting ear shank tissue and stalk tissue from the nodes between the infection sites and the developing ears. Ears were collected at the end of the growing season to determine the level of kernel infection by the NOR mutant. In two separate studies, the A. parasiticus NOR mutant was isolated from stalk tissues at all of node positions and ear shank tissue from several susceptible corn hybrid plants at the first harvest date 2 weeks after inoculation. The NOR mutant was also isolated from stalk and ear tissue of a resistant hybrid. The NOR mutant was only isolated from kernels of susceptible hybrids in 2003 and 2004. Infection rates of kernels in infected ears were very low (<1%). In 2005, the fungus was found in only one kernel from an ear of the resistant hybrid. The NOR mutant was not isolated from stalks, ears, or kernels from control (uninoculated) plants grown in the plots with inoculated plants. Although infection levels of corn kernels were low, systemic movement of the A. parasiticus up the stalk appears to be another possible route to infection of developing corn ears.     

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     

The effect of sowing density and application of gibberellic acid on male sterility and ear emergence in ethephon-treated field-grown wheat

The induction of male sterility with ethephon and improvement in ear emergence with GA₃ (1-1 kg a.i./ha) applied 3 days later, was investigated in the winter wheat cv. Maris Nimrod sown in the field at 72 , 138 and 276 spaced plants/m². Increased sowing density resulted in fewer surviving tillers per plant, a larger number of tillers per unit area and consequently a narrower range of stage of tiller development. This permitted a more accurate assessment of the correct time for ethephon application and resulted in a larger proportion of tillers responding to GA₃ by having fully emerged ears. The most effective treatments for the production of emerged male sterile ears were 6-4 and 12-8 kg ethephon/ha applied to crops sown at the highest density.     

Controlling elements in a tribal maize from Colombia: fcu , a two-unit system

The genetic basis for a striking aleurone color variegation of a maize population from Colombia is described. The variegation is the result of the interaction of a particular r allele, designated r-cu , and a specific factor, Fcu (Factor Cuna), that collectively compose a controlling element system. In the absence of Fcu, a variable dilute (nonvariegated) pigmentation is expressed in the aleurone. The degree of variegation is determined by differences in the dosage of the Fcu element; differences in variegation between kernels with one r-cu vs. two r-cu's cannot be detected. Because of the specificity of the interaction, Fcu is different from the three other well-known controlling elements, Dt, Ac and En(=Spm), and therefore represents a fourth controlling element system in maize. In addition to Fcu, the Cuna tribal maize contains two other controlling elements, Dt and a very weak En. The prevalence of controlling elements in natural maize populations is discussed.     

Regulation of OsmiR156h through Alternative Polyadenylation Improves Grain Yield in Rice

Substantial increases in grain yield of cereal crops are required to feed a growing human population. Here we show that a natural variant of SEMIDWARF AND HIGH-TILLERING (SDT) increases harvest index and grain productivity in rice. Gain-of-function sdt mutation has a shortened polyadenylation tail on the OsmiR156h microRNA precursor, which cause the up-regulation of OsmiR156h. The plants carrying the semidominant sdt allele exhibit reduced plant height, enhanced lodging resistance, increased tiller numbers per plant, and resulting in an increased grain yield. We also show that combining the sdt allele with the OsSPL14^WFP allele can be effective in simultaneously improving tillering capacity and panicle branching, thereby leading to higher harvest index and grain yield. Most importantly, pyramiding of the sdt allele and the green revolution gene sd1 enhances grain yield by about 20% in hybrid rice breeding. Our results suggest that the manipulation of the polyadenylation status of OsmiR156 represents a novel strategy for improving the yield potential of rice over what is currently achievable.     

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.     

The TAG1 locus of Arabidopsis encodes for a diacylglycerol acyltransferase

Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) is a membrane enzyme that drives the final step in the formation of oils using diacylglycerol (DAG) and acyl-CoA to yield triacylglycerol (TAG). We identified a putative plant DGAT gene (TRIACYLGLYCEROL1: TAG1) and demonstrated its function by the cloning of two mutated alleles, designated AS11 (tag1-1) and ABX45 (tag1-2). One allele, AS11, has been previously characterised at the biochemical level. Mutant seeds contained less oil with a modified fatty acid profile and have reduced germination rates compared to wild-type controls. The TAG1 cDNA encodes for a 520-aa protein that possesses multiple putative transmembrane domains and shows 70 % similarity to a human DGAT cDNA.     

Reversion of the Maize Mutable Allele o2-hf in Plant Ontogeny

Reversions of the mutable allele o2-hfleading 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-hfand 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.     

Effect of foliar applications of urea on accelerated senescence of maize induced by ear removal

Field grown maize (Zea mays L. cv B73 × Mo17) plants, with and without ears, were sprayed with urea solutions to determine whether foliar application of N could prevent or delay the accelerated loss of reduced N from the leaf and leaf senescence induced by ear removal. Urea sprays were applied at 7, 14, and 21 days after anthesis in three separate and equal applications that provided a total of 67 kilograms N per hectare or 1 gram N per plant. Treatments were arranged in a 2 × 2 factorial in a randomized complete block with five replicates. Appropriate plant and leaf samplings and assays were made.

In response to spray treatments, net increases of reduced N were detected in the whole shoot and plant parts, especially the stalk of the earless plants and grain of the eared plants. There was no effect of urea spray treatment on the normal loss of N from the leaves or rate of senescence of the eared plants or on the accelerated loss of N from the leaves or rate of senescence induced by ear removal. Grain and stover yields were unaffected by the spray treatment.

Apparently the plants were unable to utilize the urea N applied to the vegetation (primarily leaves) after anthesis to enhance or extend the accumulation of dry weight by either eared or earless plants.

     

Pleiotropic effects of gibberellin-sensitive and giberrellin-insensitive dwarfing genes in bread wheat of the southern step region of the Black Sea

Investigation of the pleiotropic effects of GA-sensitive (Rht8) and GA-insensitive (Rht-B1 and Rht-D1) winter bread wheat dwarfing genes and the gene that determines the response of plants to photoperiod—Ppd-D1—were carried out for 3 years in the southern step region of the Black Sea bank on five different genetic backgrounds. It is shown that, in addition to direct effects on plant height, GA-sensitive and GA-insensitive dwarfing genes have pleiotropic effects on all studied traits except the number of fertile spikelets. Presence of the dwarfing genes in the genotype of tall forms led to the decrease of stem and ear length, and, at the same time, to the increase of ear density. The number of spikelets per spike decreased due to sterile spikelets, whereas the number of fertile spikelets did not change. There was a significant increase in the number of grains per ear as a result of increasing of spikelets in ears. The number and weight of grains did not decrease, even though the plants were characterized by a smaller number of productive tillers. The presence of Rht8x allele on genetic background of variety Stepnyak resulted in a significant decrease of plants productivity. However, in combination with Ppd-D1a allele, plants with Rht8x increased the potential productivity and surpassed the parental form (Rht8x Ppd-D1a). The presence of Rht-Ble allele resulted in reduction of weight of kernels from the main ear and 1000-kernels weight, increase of l/h, and left the number of seeds per spikelet stable in comparison with Rht8x.