Mu Element-Generated Gene Conversions in Maize Attenuate the Dominant Knotted Phenotype

The knotted1 gene was first defined by dominant mutations that affect leaf morphology. The original allele, Kn1-O, results from a 17-kb tandem duplication. Mutator (Mu) insertions near the junction of the two repeats suppress the leaf phenotype to different degrees depending on the position of the insertion. The Mu insertions also increase the frequency of recombination at Kn1-O to create derivative alleles in which the Mu element and one copy of the repeat are lost. These derivatives are normal in appearance. Here we describe two derivatives that retained the tandem duplication but gained insertions of 1.7 and 3 kb in length in place of the Mu element. In each case, the inserted DNA is a sequence that normally flanks the distal repeat unit. Thus, each derivative consists of a tandem duplication in which the repeat unit has been extended at its distal end by the length of the new insertion. The 1.7-kb insertion dampens the phenotype, as did the original Mu insertion, whereas the 3-kb insertion completely suppresses the knotted phenotype. We propose that gene conversion, stimulated by the double-strand break of the Mu excision, gave rise to these derivatives.     

A Tandem Duplication Causes the Kn1-O Allele of Knotted, a Dominant Morphological Mutant of Maize

Molecular and genetic techniques are used to define Kn1-O, a mutation which interferes with the normal differentiation of vascular tissue in leaves. Sequences associated with a previously cloned allele, Kn1-2F11, were used as hybridization probes in a Southern analysis of Kn1-O. By this analysis, Kn1-O lacks the Ds2 transposable element that causes Kn1-2F11 but instead is associated with a sequence duplication. Sequence and restriction analysis of genomic clones show that the duplication consists of a tandem array of two 17-kb repeats. Analysis of Kn1-O derivatives indicates that the duplication itself conditions the mutant phenotype; a severely knotted line, Kn1-Ox, has gained a repeat unit to form a triplication, whereas normal derivatives have either lost a repeat unit or sustained insertions that disrupt the tandem duplication. These insertions map near the central junction of the tandem duplication, suggesting that the mutant phenotype results from the novel juxtaposition of sequences. We discuss models that relate the tandem duplication of sequences to altered gene expression.     

Genetic analysis of mutations that alter cell fates in maize leaves: Dominant Liguleless mutations

The three major components of the maize leaf are the blade, the sheath, and at their junction, the ligular region. Each exhibits specific cell types and organization. Four dominant Liguleless (Lg) mutations (Lg3-O, Lg4-O, Lg*347, and Lg*9167) in at least three different genes cause a similar morphological phenotype in leaves, although each mutation affects a distinct domain of the blade. Mutant leaves display regions of altered cell fate in the blade, occompanied by elimination of ligule and auricle at their wild-type positions and development of ligule and auricle in the blade at the borders of the altered regions. The affected blade cells are transformed into sheath-like cells, as determined by morphological and genetic tests. Lg4-O expressivity is highly dependent on genetic background. For example, two different backgrounds may specify converse patterns of phenotypic expression. Lg4-O expressivity is also affected by the heterochronic mutation Teopod2 (Tp2). Gene dosage experiments indicate that Lg4-O is a neomorph. Interactions between recessive lg mutations (which eliminate ligular structures) and the dominant Lg mutations suggest that the lg⁺ genes act after the Lg mutations. Lg3-O and Lg4-O act semidominantly, and interact with each other and with other mutations in the Knotted1 (Kn1)-like family (a family in which dominant mutant alleles cause blade to sheath transformation phenotypes). These interactions suggest that the above Kn1-like mutations may function similarly in the leaf. We discuss the similarities between the Lg mutations and the other mutations of the Kn1-like family, which led us to postulate that lg3 and lg4 are members of a growing family of kn1-like (knox) homeobox genes that are identified by dominant mutant alleles causing leaf transformation phenotypes. We also propose that certain key characteristics of this family of dominant neomorphic mutations are important for generating meaningful morphological changes during evolution. © 1996 Wiley-Liss, Inc.     

Genetic Analysis of Rough Sheath1 Developmental Mutants of Maize

Maize Rough sheath1 (Rs1) mutants are dominant and cause a proliferation of sheath-like tissue at the base of the blade and throughout the ligular region. They also cause ligule displacement, a chaotic pattern of vasculature and abnormal cellular structure of vascular bundles. The affected region of Rs1-O leaves displays genetic and morphological attributes of both sheath and auricle, suggesting an overlap of these genetic programs. The rs1 locus maps approximately 26 map units distal to opaque2 (o2) on chromosome 7S, defining a new distal-most locus on the genetic map. Three mutant alleles, Rs1-O, Rs1-1025 and Rs1-Z, all display similar phenotypes. The mutations are completely dominant and the Rs1-O phenotype is not affected by dosage of the chromosome arm carrying the rs1(+) allele, indicating that these alleles are neomorphic. Analysis of genetic mosaics showed that the Rs1-O phenotype is non-cell-autonomous, suggesting that intercellular signals convey the phenotype. Rs1 mutant phenotypes are affected by modifiers present in particular genetic backgrounds. An enhancer of Rs1-O was identified; segregation data imply a single recessive gene, ers1. Rs1 mutants were also found to enhance the expression of unlinked rs2 and Rs4 mutants, suggesting that these mutations affect similar developmental processes. We discuss the phenotypic and genetic similarities between Rs1 and Knotted1 (Kn1) mutants that led to the identification of rs1 as a kn1-like homeobox gene (unpublished data).     

Mutant characters of knotted maize leaves are determined in the innermost tissue layers

Knotted (Kn1), a dominant mutation in maize, perturbs normal leaf development. Mutant leaves have localized regions of extra growth called knots and, in addition to the normal ligule, ectopic fringes of ligule are found on the leaf blade. Previous clonal analysis showed that the epidermal genotype was immaterial in knot formation. To establish which inner leaf layer was required for formation of knots and ectopic ligule we used a closely linked albino mutation to mark X-ray-induced clonal sectors of wild type (kn) tissue in Kn1 plants. The sectors examined frequently changed in composition of layers in the leaf both transversely and longitudinally. We present results that show that both mutant characters are determined in the middle mesophyll-bundle sheath (MMBS) layer. We show that a lateral vein can produce a knot when only half the MMBS layer around the lateral vein contains the mutant gene. We also show that the ectopic ligule in Kn1 has contributions from both the adaxial epidermal and adaxial mesophyll layer.     

The Knotted-1 mutants of maize: investigating the circuitry of leaf development

The maize homeobox gene, Knotted-1 (KN1), was first identified by dominant mutations conditioning aberrant leaf development. Thirteen mutant Kn1 alleles have facilitated a molecular and genetic dissection of the gene and some of its regulatory components. These studies illuminate areas of plant developmental biology that include a demonstration of how transposable elements can control the expression of genes specifying meristematic activity. Genetic approaches have been used to identify collaborating loci, while the Kn1 homeobox has permitted the identification and cloning of related plant homeobox genes. The functions of these genes are being addressed in the context of pattern formation and acquisition of cell fate. This review will focus upon the array of Kn1 mutations and how they have been utilized in genetics and molecular biology.     

Coordinate Suppression of Mutations Caused by Robertson''s Mutator Transposons in Maize

Transposable elements from the Robertson's Mutator family are highly active insertional mutagens in maize. However, mutations caused by the insertion of responder (non-autonomous) elements frequently depend on the presence of active regulator (autonomous) elements for their phenotypic effects. The hcf106::Mu1 mutation has been previously shown to depend on Mu activity in this way. The dominant Lesion-mimic 28 mutation also requires Mu activity for its phenotypic effects. We have used double mutants to show that the loss of Mu activity results in the coordinate suppression of both mutant phenotypes. This loss can occur somatically resulting in large clones of cells that have a wild-type phenotype. Autonomous and non-autonomous Mutator elements within these clones are insensitive to digestion with methylation-sensitive enzymes, suggesting extensive methylation of CG and non-CG cytosine residues. Our data are consistent with the sectors being caused by the cycling of MuDR regulatory elements between active and inactive phases. The pattern of sectors suggests that they are clonal and that they are derived from the apical cells of the vegetative shoot meristem. We propose that these cells are more likely to undergo epigenetic loss of Mu activity because of their longer cell division cycle during shoot growth. Coordinate suppression of unlinked mutations can be used to perform mosaic analysis in maize.     

Somatic excision of the Mu1 transposable element of maize.

The Mu transposons of the Robertsons's Mutator transposable element system in maize are unusual in many respects, when compared to the other known plant transposon systems. The excision of these elements occurs late in somatic tissues and very rarely in the germ line. Unlike the other plant transposons, there is no experimental evidence directly linking Mu element excision and integration. We have analyzed the excision products generated by a Mu1 transposon inserted into the bronze 1 locus of maize. We find that the excision products or 'footprints' left by the Mu1 element resemble those of the other plant transposable elements, rather than those of the animal transposable element systems. We also find some novel types of footprints resembling recombinational events. We suggest that the Mu1 element can promote intrachromosomal crossovers and conversions near its site of insertion, and that this may be another mechanism by which transposons can accelerate the evolution of genomes.     

Biomechanical analysis of the Rolled (RLD) leaf phenotype of maize

The pleiotropic effects of the Rld1-O/+ mutation of Zea mays (Poaceae) on leaf phenotype include a suppression of normal transverse unrolling, a reversed top/bottom epidermal polarity, and an apparently straighter longitudinal shape. According to engineering shell theory, there might be mechanical coupling between transverse and longitudinal habit, i.e., the leaf rolling itself might produce the longitudinal straightening. We tested this possibility with quantitative curvature measurements and mechanical uncoupling experiments. The contributions of elastic bending under self weight, mechanical coupling, and rest state of leaf parts to the longitudinal and transverse habit were assessed in Rld1-O/+ mutants and a population of sibling +/+ segregants. Elastic bending and curvature coupling are shown to be relatively unimportant. The Rld1-O/+ mutation is shown to alter not only the unrolling process, but also the developmental longitudinal curving in the growing leaf, leading to a straighter midrib and a rolled lamina. The Rld1-O/+ mutant is thus a suitable model to study the relation between tissue polarity and differential curvature development in the maize leaf. Since on the abaxial side of the leaf, more abundant sclerenchyma is found in +/+ than in Rld1-O/+, a gradient in sclerification may contribute to the development of midrib curvature.     

The establishment of axial patterning in the maize leaf

The maize leaf consists of four distinct tissues along its proximodistal axis: sheath, ligule, auricle and blade. liguleless1 (lg1) functions cell autonomously to specify ligule and auricle, and may propagate a signal that correctly positions the blade-sheath boundary. The dominant Wavy auricle in blade (Wab1) mutation disrupts both the mediolateral and proximodistal axes of the maize leaf. Wab1 leaf blades are narrow and ectopic auricle and sheath extend into the blade. The recessive lg1-R mutation exacerbates the Wab1 phenotype; in the double mutants, most of the proximal blade is deleted and sheath tissue extends along the residual blade. We show that lg1 is misexpressed in Wab1 leaves. Our results suggest that the Wab1 defect is partially compensated for by lg1 expression. A mosaic analysis of Wab1 was conducted in Lg1+ and lg1-R backgrounds to determine if Wab1 affects leaf development in a cell-autonomous manner. Normal tissue identity was restored in all wab1+/- sectors in a lg1-R mutant background, and in three quarters of sectors in a Lg1+ background. These results suggest that lg1 can influence the autonomy of Wab1. In both genotypes, leaf-halves with wab1+/- sectors were significantly wider than non-sectored leaf-halves, suggesting that Wab1 acts cell-autonomously to affect lateral growth. The mosaic analysis, lg1 expression data and comparison of mutant leaf shapes reveal previously unreported functions of lg1 in both normal leaf development and in the dominant Wab1 mutant.     

Cloning Knotted, the dominant morphological mutant in maize using Ds2 as a transposon tag

The Kn1-2F11 mutation causes protrusions or knots along the lateral veins of the first few leaves of the maize plant. The phenotype is visible when an unlinked gene, presumably Ac, is present in the genome. The mutation is closely linked to a genetically unstable Adh1 mutation that resulted from the insertion of a Ds2 element (Döring et al., 1984; Chen et al., 1986). Using a unique sequence from the Ds2 element as a hybridization probe, a genomic restriction fragment that cosegregated with the knotted phenotype was cloned. It carries the Kn1-2F11 locus by the following criteria. (i) Cosegregation of the fragment is tightly linked to the phenotype. (ii) Somatic and germinal excision produce a fragment which is the expected size of a revertant fragment; progeny containing the revertant size fragment are normal. (iii) The sequences that hybridize to this fragment are significantly altered in the chromosome containing the original knotted mutation, Kn1-O, (iv) The cloned fragment does not hybridize to a chromosome that contains a deletion of Kn1-O.     

Somatic chimerism, genetic inheritance, and mapping of the fleshless berry (flb) mutation in grapevine (Vitis vinifera L.).

The fleshless berry (flb) mutation of grapevine (Vitis vinifera L. 'Ugni Blanc') impairs the differentiation and division of inner mesocarp cells responsible for flesh in grapevine berries. In order to study the inheritance of the mutation and to map the flb locus, 5 segregating populations were created. Progeny plants were classified as mutant or wild type by scoring for the presence of an ovary phenotype associated with the Flb- phenotype at anthesis. Phenotypic segregation revealed the involvement of a single dominant allele that was heterozygous in the original mutant. Through bulk segregant analysis, microsatellite (simple sequence repeat (SSR)) markers linked to the mutation were identified, and the flb locus was assigned to linkage group 18. The locus position was then refined by analyzing individual progeny and the segregation of SSR markers in the target region with the closest marker 5.6 cM distant from the flb locus. All progeny with the Flb- ovary phenotype differed from the original fleshless berry mutant in that no berries formed after anthesis. Our data suggest that the original mutant plant was a chimera with the mutated allele present in only 1 cell layer (L2 layer) of the ovary and berry.     

The maize macrohairless1 locus specifically promotes leaf blade macrohair initiation and responds to factors regulating leaf identity

The leaf surfaces of almost all plant species possess specialized epidermal cell types that form hairs or trichomes. Maize leaves produce three distinct types of hairs, the most prominent being the macrohairs that serve as a marker for adult leaf identity and may contribute to insect resistance. This report describes the maize macrohairless1 (mhl1) locus, which promotes macrohair initiation specifically in the leaf blade. Each of seven recessive mhl1 mutant alleles significantly reduces or eliminates macrohairs in the leaf blade. The mhl1 mutations block macrohair initiation rather than interfering with macrohair morphogenesis. Genetic mapping placed mhl1 within bin 4 on chromosome 9. A second independently segregating locus was found to partially suppress the mhl1 mutant phenotype in certain genetic backgrounds. Macrohair density was observed to increase during early adult vegetative development and then progressively decline, suggesting macrohair initiation frequency is affected by factors that act throughout shoot development. Genetic analyses demonstrated that mhl1 acts in the same pathway but downstream of factors that either promote or repress adult leaf identity. Thus, mhl1 plays a key role in integrating developmental programs that regulate leaf identity during shoot development with those that specify macrohair initiation within the leaf blade.     

Molecular cloning of the a1 locus of Zea mays using the transposable elements En and Mu1

The a1 locus of Zea mays has been cloned using transposable elements as gene tags. The strategy was to make genomic libraries from maize stocks with a1 mutations induced either by En(Spm) or by Robertson's Mutator-system. These libraries were then screened with either Spm-I8 and En1, for the En-containing mutant, or with Mu1 for the Mu-induced mutation. There are many En and Mu1 hybridizing sequences present in the maize genome, however, by a process of cross-screening of the positives from the two libraries and by molecular analysis of the En-positive clones it was possible to identify clones in both libraries carrying all or part of the a1 gene.     

The Knotted leaf blade is a mosaic of blade,sheath, and auricle identities

The dominant Knotted-1 mutations in maize alter development of the leaf blade. Sporadic patches of localized growth, or knots, and fringes of ectopic ligule occur along lateral veins of mutant leaf blades. In addition, bundle sheaths do not completely encircle lateral veins on mutant leaf blades. We have compared mutant leaf blades with wild-type leaves to determine the precise nature of the perturbed regions. Our analysis includes characterization of epidermal cell shapes, localization of photosynthetic proteins and histology of the leaf. We show that mutant leaf blades are a mosaic of leaf organ components. Affected regions of mutant leaf blades resemble either sheath or auricle tissue in both external and internal features. This conversion of blade cells represents an acropetal shift of more basal parts of the leaf blade region and correlates with previously identified ectopic expression of the Knotted-1 protein in the leaf blade. We propose that inappropriate expression of Kn1 interferes with the process of establishment of cell identities, resulting in early termination of the normal blade development program or precocious expression of the sheath and auricle development programs. © 1994 Wiley-Liss, Inc.