Biomedical subjects

M Freeling

Publications and source records attributed to M Freeling.

At least 19 recordsLinked to original sources

PubMed1992

Maize mutants and variants altering developmental time and their heterochronic interactions.

It is useful to envision two fundamentally different ways by which the timing of plant development is regulated: developmental stage-transition mechanisms and time-to-flowering mechanisms. The existence of both mechanisms is indicated by the behavior of various mutants. Shoot stage transitions are defined by dominant mutants representing at least four different genes; each mutant retards transitions from juvenile shoot stages to more adult shoot stages. In addition, dominant leaf stage-transition mutants in at least seven different genes have similar phenotypes, but the leaf rather than the shoot is the focus (and at least two of these genes encode homeodomain proteins.) One mutant, Hairy sheath frayed 1-O (Hsf1-O) simultaneously affects shoot and leaf; this mutant's behavior initiated our interest in plant heterochronism. The second type of timekeeping involves time-to-flowering. As with most plant but not animal species, cultivars of the maize species vary greatly for the time-to-flowering quantitative trait: between 6 and 14 weeks is common. It is via the 'slipping time frames' interaction that takes place between stage-transition mutants and time-to-flowering genetic backgrounds that unexpected and radical phenotypes occur. We see a reservoir of previously unsuspected morphological possibilities among the few heterochronic genotypes we have constructed, possibilities that may mimic the sort of variation needed to fuel macroevolution without having to posit (as done by Goldschmidt) any special macromutational mechanisms.

Biological Evolution↗

PubMed1992

The TATA box promoter region of maize Adh1 affects its organ-specific expression.

We have isolated two lineage-related Mutator (Mu3) transposon-induced Adh1 promoter mutants in maize: Adh1-3F1124 carries a duplicated TATA box and its revertant, Adh1-3F1124r17, bears a deleted TATA box. Both alterations lead to unique patterns of organ-specific ADH1 enzyme expression. Enzyme activity in Adh1-3F1124 sporophytic organs (scutellum and roots) is greatly reduced, while activity levels remain normal in the male gametophyte (pollen). Conversely, enzyme activity in Adh1-3F1124r17 roots and scutellum is partially restored, but is concomitantly reduced in pollen. Transcript analysis suggests (i) that the TATA box region of the Adh1 gene influences post-transcriptional processes in the male gametophyte but not in roots and (ii) that organ-specific transcription signals in the promoter are distinct from the previously identified anaerobic environment-specific cis-acting transcription signals. Different organs appear to provide surrogate TATA function in different ways, leading to organ-specific differences in the length of the Adh1 message 5' leader.

Alcohol Dehydrogenase↗

PubMed1992

Expression and distribution of cytosolic 6-phosphogluconate dehydrogenase isozymes in maize.

Maize (Zea mays L.) cytosolic 6-phosphogluconate dehydrogenase isozymes (EC 1.1.1.44; 6-PGD) are encoded by unlinked loci Pgd1 and Pgd2. Two families from a Robertson's Mutator line were isolated which have no detectable expression of Pgd2. These Pgd2-null mutants and a Pgd1-null line were used to generate plants homozygous for null alleles at both cytosolic 6-PGD loci. The specific activity of 6-PGD in the double-null mutant was between 20 and 30% of wild-type levels in root extracts. The double-null mutant was reproductively viable in a moderate environment, suggesting that wild-type levels of cytosolic 6-PGD activity are not essential for growth. Isozyme dimer ratios in roots, leaves, and scutellum were binomial and reflected the wild-type gene copy number. 6-PGD isozymes showed tissue- and cell type-specific expression.

Cytosol↗

PubMed1992

A conceptual framework for maize leaf development.

What is and is not known about the maize leaf is reviewed. Analysis of genetic mosaics and direct observation with the SEM have broken leaf development into three distinct phases: recruitment of cells within the meristem, cell division into the 0.6-mm tall primordium, and postprimordial division and differentiation into the mature leaf. New data are presented that imply that cell division rates in the leaf are coordinated by inductive signals from the internal cells. Leaf cells that tend to divide more are held in check by slower growing neighbors; this complicates the search for developmental compartments. Experiments with recessive mutants that remove the ligule and auricle have been important in identifying an inducer signal with the specific meaning "make ligule-auricle." We have studied many dominant mutant alleles at seven different genes. Each mutant alters the position of the ligule boundary. We conclude the following. First, the mutants act in particular domains of the primordium. Second, the dominant mutants all move the ligule boundary in the same direction. Third, the mutants all retard developmental stage transitions. Fourth, three and probably four of the seven genes for which dominant mutants have been studied specify homeodomain proteins in the wrong place. The concept of "maturation schedule" is used to explain these data. All of the dominant mutant phenotypes are seen as consequences of immature cells being in the wrong place when inductive signals pass through the leaf. Several specific questions of leaf development and especially questions as to source of inductive signals or homologies among juvenile and adult organ parts are recast in light of this "maturation schedule" hypothesis.

Embryonic Induction↗

PubMed1992

Cell interactions in plants.

Plant cells interact during development through diverse mechanisms that range from genetically encoded signals to physical stresses. Pollen self-incompatibility is the best understood cell interaction in plants. Analysis of genes that appear to be involved in specific developmental signals, such as liguleless1 from maize and GLABROUS1 from Arabidopsis, will provide clues as to the nature of cell interactions in plant development. Recent data suggest that intercellular connections may be more similar in plants and animals than previously thought.

Cell Communication↗

PubMed1992

The role of initial cells in maize anther morphogenesis.

The near absence of cell movement in plants makes clonal analysis a particularly informative method for reconstructing the early events of organ formation. We traced the patterns of cell division during maize anther development by inducing sector boundaries that preceded the earliest events of anther initiation. In doing this, we were able to estimate the smallest number of cells that are fated to form an anther, characteristic cell division patterns that occur during anther morphogenesis, and the relationship between the pre-existing symmetry of the initial cells and the final symmetry of the mature anther. Four general conclusions are made: (1) anthers are initiated from small groups of 12 or fewer cells in each of two floral meristematic layers; (2) the early growth of the anther is more like a shoot than a glume or leaf; (3) cell ancestry does not dictate basic structure and (4) the orientation of initial cells predicts the orientation of the four pollen-containing microsporangia, which define the axes of symmetry on the mature anther. The final point is discussed with other data, and an explanation involving a 'structural template' is invoked. The idea is that the orientation of initial cells within the floral meristem establishes an architectural pattern into which anther cells are recruited without regard to their cellular lineages. The structural template hypothesis may prove to be generally applicable to problems of pattern formation in plants.

Gametogenesis↗

PubMed1991

Identification of a regulatory transposon that controls the Mutator transposable element system in maize.

The Mutator system of maize consists of more than eight different classes of transposable elements each of which can be found in multiple copies. All Mu elements share the approximately 220-bp terminal inverted repeats, whereas each distinct element class is defined by its unique internal sequences. The regulation of instability of this system has been difficult to elucidate due to its multigenic inheritance. Here we present genetic experiments which demonstrate that there is a single locus, MuR1, which can regulate the transposition of Mu1 elements. We describe the cloning of members of a novel class of Mu elements, MuR, and demonstrate that a member of the class is the regulator of Mutator activity, MuR1. This conclusion is based on several criteria: MuR1 activity and a MuR-homologous restriction fragment cosegregate; when MuR1 undergoes a duplicative transposition, an additional MuR restriction fragment is observed, and MuR1 activity and the cosegregating MuR fragment are simultaneously lost within clonal somatic sectors. In addition, the MuR element hybridizes to transcripts in plants with Mutator activity. Our genetic experiments demonstrate that the MuR1 transposon is necessary to specify Mutator activity in our lines.

Blotting, Northern↗

PubMed1991

Sectors of liguleless-1 tissue interrupt an inductive signal during maize leaf development.

The ligule and auricles separate the blade and sheath of normal maize leaves and are absent in liguleless-1 (lg1) mutant leaves. We induced chromosome breakage using X-rays to create plants genetically mosaic for lg1. In genetically mosaic leaves, when an lg1 mutant sector interrupts the normal ligule, the ligule is often displaced basipetally on the marginal side of the sector. Therefore, lg1 mutant sectors not only fail to induce ligule and auricle, but are also disrupting some form of intercellular communication that is necessary for the normally coordinated development of the ligular region. Our data are consistent with a model in which an inductive signal originates near the midvein, cannot traverse the lg1 mutant sector, and reinitiates in the wild-type tissue across the sector toward the leaf margin. The lg1 gene product, therefore, appears to be required for the transmission of this signal and could be involved with reception.

Cell Communication↗

PubMed1990

The liguleless-1 gene acts tissue specifically in maize leaf development.

The liguleless-1 (lg1) gene affects maize leaf development. In a normal maize leaf, a ligule and auricles separate the blade and sheath. The recessive lg1 mutation prevents formation of ligules and auricles during leaf development. To determine the timing and site of lg1 gene action, we compared development of wild-type and lg1 mutant leaves, and analyzed genetic mosaics composed of wild-type and lg1 mutant cells. In wild-type leaves the first sign of differentiation of the ligular region is a series of specialized anticlinal divisions in the adaxial epidermis. This establishes a distinct band of cells, from which the ligule arises via periclinal divisions. The anticlinal divisions preceding ligule formation are altered in the mutant; therefore, the gene acts early in development, before the periclinal divisions, and possibly during basipetal vascularization. Genetic mosaic analysis indicates that the lg1 gene has at least two functions with different tissue specificities: The Lg1+ wild-type allele acts autonomously in the adaxial epidermis for normal ligule development, and in internal tissues for auricle formation. Wild-type internal tissue in direct contact with lg1 epidermis appears able to induce the mutant epidermis to form a rudimentary ligule. The results indicate that the lg1 gene acts tissue specifically in an early step of ligule and auricle initiation.

In Vitro Techniques↗

PubMed1990

Clonal analysis of the cell lineages in the male flower of maize.

The cell lineages in the male flower of maize were characterized using X-rays and transposable elements to produce clonal sectors differing in anthocyanin pigmentation. Less than 50% of the somatic tassel mutations (caused by reversion of unstable color mutations) that were visible on the anther wall were sexually transmitted by the male gametes, unless the sectors were larger than half the tassel circumference. This result is explained by showing that: (a) both the outer (LI) and inner (LII) lineages of the shoot apical meristem form a cell layer in the bilayered anther wall, and that anther pigmentation can be derived from either cell layer; and that (b) the male germ cells are derived almost exclusively from the LII. Therefore, while reversion events in either the LI or LII are visible on the anther, only the LII events are heritable. Reversion events that occur prior to the organization of the shoot apical meristem however, produce large (usually more than one-half tassel) sectors that include both the outer and inner lineages. In contrast to the high level of cell layer invasion previously reported during leaf development, during anther development less than 10(-3) cells in the LI invade the LII to form male gametes. The strong correlation between cell lineage and cell fate in the maize anther has implications for studies on plant evolution and the genetic improvement of cereals by DNA transformation.

Anthocyanins↗

PubMed1990

Somatically heritable switches in the DNA modification of Mu transposable elements monitored with a suppressible mutant in maize.

Many transposable elements in maize alternate between active and inactive phases associated with the modification of their DNA. Elements in an inactive phase lose their ability to transpose, their ability to excise from reporter alleles and, in some cases, their ability to enhance or suppress mutant phenotypes caused by their insertion. The maize mutant hcf106 is a recessive pale green seedling lethal caused by the insertion of the transposable element Mu1. We show that the hcf106 mutant phenotype is suppressed in lines that have lost Mu activity. That is, homozygous hcf106 seedlings are dark green and viable when transposable elements belonging to the Robertson's Mutator family are modified in their terminal inverted repeats, a diagnostic feature of inactive lines. This property of the mutant phenotype has been used to follow clonal leaf sectors containing modified Mu elements that arise from single somatic cells during plant development. The distribution of these sectors indicates that epigenetic switches involving Mu DNA modification occur progressively as the meristem ages.

Blotting, Southern↗

PubMed1990

Division and differentiation during normal and liguleless-1 maize leaf development.

The maize leaf is composed of a blade and a sheath, which are separated at the ligular region by a ligule and an auricle. Mutants homozygous for the recessive liguleless-1 (lg1) allele exhibit loss of normal ligule and auricle. The cellular events associated with development of these structures in both normal and liguleless plants are investigated with respect to the timing of cell division and differentiation. A new method is used to assess orientation of anticlinal division planes during development and to determine a division index based on recent epidermal cross-wall deposition. A normal leaf follows three stages of development: first is a preligule stage, in which the primordium is undifferentiated and dividing throughout its length. This stage ends when a row of cells in the preligule region divides more rapidly in both transverse and longitudinal anticlinal planes. During the second stage, ligule and auricle form, blade grows more rapidly than sheath, divisions in the blade become exclusively transverse in orientation, and differentiation begins. The third stage is marked by rapid increase in sheath length. The leaf does not have a distinct basal meristem. Instead, cell divisions are gradually restricted to the base of the leaf with localized sites of increased division at the preligule region. Divisions are not localized to the base of the sheath until near the end of development. The liguleless-1 homozygote shows no alteration in this overall pattern of growth, but does show distinct alteration in the anticlinal division pattern in the preligule region. Two abnormal patterns are observed: either the increase in division rate at the preligule site is absent or it exhibits loss of all longitudinal divisions so that only transverse (or cell-file producing) divisions are present. This pattern is particularly apparent in developing adult leaves on older lg1 plants, in which sporadic ligule vestiges form. From these and results previously published (Becraft et al. (1990) Devl Biol. 14), we conclude that the information carried by the Lg1+ gene product acts earlier in development than formation of the ligule proper. We hypothesize that Lg1+ may be effective at the stage when the blade-sheath boundary is first determined.

Cell Differentiation↗

PubMed1989

Molecular cloning of a maize gene involved in photosynthetic membrane organization that is regulated by Robertson's Mutator.

The maize photosynthetic mutant hcf106 has a distinctive and unusual thylakoid membrane organization, and fails to accumulate three of the five thylakoid membrane protein complexes. This mutant arose in a Robertson's Mutator background, and shows somatic instability typical of a transposon-induced allele. In addition, hcf106 is suppressed when Mu1 elements are inactive and modified in their terminal inverted repeats. Thus plants homozygous for the mutant allele adopt a mutant phenotype only when Mu1 elements are active and unmodified. DNA from the mutant allele has been cloned by 'transposon-tagging' using the transposon Mu1, and the identity of the clone confirmed by observing somatic excision of the transposon in a revertant sector. A 1.2 kb transcript homologous to the cloned DNA is found in wild-type and suppressed seedlings, but is not found in mutant seedlings, suggesting that suppression is mediated at the level of transcript accumulation.

Alleles↗

PubMed1989

Cell lineage analysis of maize bundle sheath and mesophyll cells.

Maize leaves are divided into repeated longitudinal units consisting of vascular tissue, bundle sheath (BS), and mesophyll (M) cells. We have carried out a cell lineage analysis of these cell types using six spontaneous striping mutants of maize. We show that certain cell division patterns are preferentially utilized, but not required, to form the characteristic arrangement of cell types. Our data suggest that early in development a central cell layer is formed, most frequently by periclinal divisions in the adaxial subepidermal layer of the leaf primordium. Lateral and intermediate veins are initiated in this central layer, most often by divisions which contribute daughter cells to both the procambium and the ground meristem. These divisions generate "half vein" units which comprise half of the bundle sheath cells around a vein and a single adjacent M cell. We show that intermediate veins are multiclonal both in this transverse direction and along their lengths. BS cells are more closely related to M cells in the middle layer of the leaf than to those in the upper and lower subepidermal layers. An examination of sector boundaries has shown that photosynthetic differentiation in M cells is affected by the phenotype of neighboring BS cells.

Cell Differentiation↗

PubMed1987

An extrachromosomal form of the Mu transposons of maize.

Maize lines known as Robertson's Mutator (Mu) lines generate unstable recessive mutations at high frequencies. These lines carry actively transposing copies of the transposons (Tn) Mu1 and Mu1.7. TnMu1 and TnMu1.7 are approximately 1400 and 1700 base pairs long, respectively, and they have 210-base-pair terminal inverted repeats. We report here extrachromosomal forms of TnMu1 and TnMu1.7. The extrachromosomal Mu1 and Mu1.7 molecules are resistant to alkaline denaturation and to proteinase treatment and have circular restriction maps; therefore, they are probably covalently closed circular DNA. Further, we show that their occurrence is correlated with Mu activity, so they are probably generated during Mu transposition as transposition intermediates or as products of Mu excision. When the total extrachromosomal supercoiled DNA from immature male flowers of a Mu line was examined by electron microscopy, the Mu transposons appeared to constitute a significant fraction of the extrachromosomal DNA circles in Mu lines.

Base Sequence↗

PubMed1987

Organ-specific expression of maize Adh1 is altered after a Mu transposon insertion.

A new, unstable, organ-specific Adh1 mutant was isolated from a Robertson's mutator line by germinating kernels under partial anaerobic conditions. Families of kernels which showed segregation of a conditional anaerobic lethal phenotype were identified. One mutant, Adh1-3F1124, was shown to express approximately 6% normal levels of ADH1 in seed and anaerobically treated seedlings but expresses normal levels of ADH1 in pollen, the male gametophyte. The ADH1 polypeptide encoded by the mutant allele was found to be indistinguishable from that encoded by the Adh1-3F progenitor but its message levels were lower in seed and seedlings. Robertson's mutator lines are known to carry Mu transposons that cause increased mutation rates. Genomic Southern analysis of Adh1-3F1124 and Adh1-3F showed the presence of a 1.85 kbp insertion at the 5' region of Adh1. Comparison of the DNA sequences revealed that a Mu 1-like element was inserted 31 bp 5' from the transcriptional start site of Adh1-3F1124 gene. The insertion of the Mu element creates an additional TATA box by duplicating the 9 bp genomic sequence--ATATAAATC--at the site of insertion. Consequently, there are two potentially functional TATA sequences, separated by the 1.85 kbp Mu element, 5' to the transcriptional start site. It is not yet understood how such an arrangement alters the organ-specific expression of Adh1.

Alcohol Dehydrogenase↗

PubMed1986

The Shrunken gene on chromosome 9 of Zea mays L is expressed in various plant tissues and encodes an anaerobic protein.

The Shrunken gene, located on the short arm of chromosome 9 of Zea mays, encodes the enzyme sucrose synthase (EC 2.4.1.13). The gene is known to be expressed in the endosperm of the developing maize kernel and seems to be involved in sucrose breakdown prior to starch synthesis. We have analyzed different tissues of the maize plant for transcripts of the Shrunken gene and have found rather high transcription rates in the etiolated shoot and the primary root of the germinating kernel. If the etiolated seedlings are illuminated, the transcript level drops by about 95% in the greening plant parts (1st and 2nd leaves) which are active in photosynthesis. A very low transcript level is found in mature green leaves where sucrose is formed from products of photosynthesis via a separate pathway. Upon anaerobic stress of the young seedling, the level of Shrunken transcripts increases 10 and 20 times in shoot and root tissue respectively. Apparently anaerobic induction supersedes the negative control that is observed after illumination in the 1st and 2nd leaves. From the experiments outlined here we conclude that the anaerobic protein 87 (ANP87, Hake et al. 1985) is encoded by the Shrunken locus. While the expression of the Shrunken gene varies in different tissues and in response to external stimuli, transcription of the second sucrose synthase (B) gene seems to be irresponsive to anaerobic stress and to be expressed at a similar low level in all of the tissues examined.

Alcohol Dehydrogenase↗

PubMed1986

The Mu transposable elements of maize: evidence for transposition and copy number regulation during development.

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.

Base Sequence↗