Donald S. Robertson

Characterization of the maize gene sugary1, a determinant of starch composition in kernels

In maize kernels, mutations in the gene sugary1 (su1) result in (1) increased sucrose concentration; (2) decreased concentration of amylopectin, the branched component of starch; and (3) accumulation of the highly branched glucopolysaccharide phytoglycogen. To investigate further the mechanisms of storage carbohydrate synthesis in maize, part of the su1 gene locus and a cDNA copy of the su1 transcript were characterized. Five new su1 mutations were isolated in a Mutator background, and the mutant allele su1-R4582::Mu1 was isolated by transposon tagging. The identity of the cloned element as the su1 gene locus was confirmed by the cosegregation of restriction fragment length polymorphisms in the same or nearby genomic intervals with three additional, independent su1 mutations. Pedigree analysis was also used to confirm the identity of su1. A 2.8-kb mRNA that is homologous to the cloned gene was detected in maize kernels, and a 2.7-kb cDNA clone was isolated based on hybridization to the genomic DNA. Specific portions of the cDNA hybridized with multiple segments of the maize genome, suggesting that su1 is part of a multigene family. The cDNA sequence specified a polypeptide of at least 742 amino acids, which is highly similar in amino acid sequence to bacterial enzymes that hydrolyze alpha-(1-->6) glucosyl linkages of starch. Therefore, debranching of glucopolysaccharides is seemingly part of the normal process of starch biosynthesis, and the final degree of branch linkages in starch most likely arises from the combined actions of branching and debranching enzymes.

Characterization of a mutator system in maize

Abstract • A mutator system is described in maize that results in an approximately 30-fold increase in the seedling mutation rate. This rate is found to be stable for at least two generations. • The transmission of the mutator characteristic does not follow a simple Mendelian pattern. In outcrosses, for which a one-gene model would predict that 50 percent of the progeny should not possess the mutator, nearly 100 percent of the offspring showed mutability. Several hypotheses are considered as explanations for the observed transmission pattern. Data bearing on a couple of these hypotheses are considered. To date, the evidence would seem to rule out only one of the alternatives.

A possible technique for isolating genic DNA for quantitative traits in plants

Evidence is presented that qualitative and quantitative traits may be the result of different types of variation of genic DNA at the loci involved. At any given locus, variation of a minor nature may result in wild-type alleles responsible for gene products with different efficiencies (quantitative alleles) while major genic rearrangements or changes in the region of the gene essential for a normal functioning gene product may result in qualitative mutant alleles. If this relationship is valid, modern molecular genetic techniques, involving transposable DNA mutagenesis and other procedures available today, will permit the isolation of some of the quantitative genes for many traits.

Genetic Isolation, Cloning, and Analysis of a Mutator-Induced, Dominant Antimorph of the Maize amylose extender1 Locus.

We report the genetic identification, molecular cloning, and characterization of a dominant mutant at the amylose extender1 locus, Ae1-5180. The identities of our clones are corroborated by their ability to reveal DNA polymorphisms between seven wild-type revertants from Ae1-5180 relative to the Ae1-5180 mutant allele and between four of five independently derived, Mutator (Mu)-induced recessive ae1 alleles relative to their respective wild-type progenitor alleles. The Ae1-5180 mutation is associated with two Mu1 insertions flanked by complex rearrangements of ae1-related sequences. One of the Mu1 elements is flanked by inverted repeats of ae1-related DNA of at least 5.0 kb in length. This Mu1 element and at least some of this flanking inverted repeat DNA are absent or hypermethylated in six of seven wild-type revertants of Ae1-5180 that were analyzed. The second Mu1 element is flanked on one side by the 5.0-kb ae1-specific repeat and on the other side by a sequence that does not hybridize to the ae1-related repeat sequence. This second Mu1 element is present in revertants to the wild type and does not, therefore, appear to affect ae1 gene function. A 2.7-kb ae1 transcript can be detected in wild-type and homozygous ae1-Ref endosperms 20 days after pollination. This transcript is absent in endosperms containing one, two, or three doses of Ae1-5180. This result is consistent with a suppression model to explain the dominant gene action of Ae1-5180 and establishes Ae1-5180 as an antimorphic allele. Homozygous wild-type seedlings produce no detectable transcript, indicating some degree of tissue specificity for ae1 expression. Sequence analyses establish that ae1 encodes starch branching enzyme II.

Sequence Analysis of the Cloned glossy8 Gene of Maize Suggests That It May Code for a [beta]-Ketoacyl Reductase Required for the Biosynthesis of Cuticular Waxes

The gl8 locus of maize (Zea mays L.) was previously defined by a mutation that reduces the amount and alters the composition of seedling cuticular waxes. Sixty independently derived gl8 mutant alleles were isolated from stocks that carried the Mutator transposon system. A DNA fragment that contains a Mu8 transposon and that co-segregates with one of these alleles, gl8-Mu3142, was identified and cloned. DNA flanking the Mu8 transposon was shown via allelic cross-referencing experiments to represent the gl8 locus. The gl8 probe revealed a 1.4-kb transcript present in wild-type seedling leaves and, in lesser amounts, in other organs and at other developmental stages. The amino acid sequence deduced from an apparently full-length gl8 cDNA exhibits highly significant sequence similarity to a group of enzymes from plants, eubacteria, and mammals that catalyzes the reduction of ketones. This finding suggests that the GL8 protein probably functions as a reductase during fatty acid elongation in the cuticular wax biosynthetic pathway.

Cloning of the y1 Locus of Maize, a Gene Involved in the Biosynthesis of Carotenoids.

The y1 gene is one of the genes responsible for the production of [beta]-carotene in the endosperm and leaves of maize. We have cloned a Robertsons Mutator-tagged allele of the y1 gene (y1-mum) by using a Mu3 element as a hybridization probe. We substantiate that the cloned sequence is a portion of the y1 gene by molecular analyses of a revertant of a putative Mutator-induced y1 allele and the incidence of insertions within the cloned y1 sequence from several independently derived Mutator-induced y1 mutant stocks. The y1-mum sequence was used to isolate the standard Y1 allele, which conditions the presence of [beta]-carotene in the endosperm of the maize kernel.

The glossy1 locus of maize and an epidermis-specific cDNA from Kleinia odora define a class of receptor-like proteins required for the normal accumulation of cuticular waxes

Mutations at the glossy1 (gl1) locus of maize (Zea mays L.) quantitatively and qualitatively affect the deposition of cuticular waxes on the surface of seedling leaves. The gl1 locus has been molecularly cloned by transposon tagging with the Mutator transposon system. The epi23 cDNA was isolated by subtractive hybridization as an epidermis-specific mRNA from Senecio odora (Kleinia odora). The deduced amino acid sequence of the GL1 and EPI23 proteins are very similar to each other and to two other plant proteins in which the sequences were deduced from their respective mRNAs. These are the Arabidopsis CER1 protein, which is involved in cuticular wax deposition on siliques, stems, and leaves of that plant, and the protein coded by the rice expressed sequence tag RICS2751A. All four proteins are predicted to be localized in a membrane via a common NH2-terminal domain, which consists of either five or seven membrane-spanning helices. The COOH-terminal portion of each of these proteins, although less conserved, is predicted to be a water-soluble, globular domain. These sequence similarities indicate that these plant orthologs may belong to a superfamily of membrane-bound receptors that have been extensively characterized from animals, including the HIV co-receptor fusin (also termed CXCR4).

Chloroplast development in pigment deficient mutants of maize: I. Structural anomalies in plastids of allelic mutants at the w3 locus

A fine structure study of the plastids of normal and mutant seedlings was undertaken in an attempt to correlate what is known of the genetics and biochemistry of two allelic mutants to plastid structure and development. Seedlings of normal, albino (w3), pastel (pas8686), and the albino/pastel (F1) heterozygote were grown for 11–14 days in the dark, then exposed to light for 24 hours. Samples were taken in the dark and at intervals up to 24 hours, fixed, embedded, and sectioned for electron microscopy. Structural changes that occur in leaf mesophyll chloroplasts of the normal and mutant plants grown in dark and exposed to light are compared in four series of micrographs to show: structure and disorganization of the prolamellar body, lamellar formation, and subsequent breakdown or aberrant organization of internal structure. Probable relationships of chlorophyll, carotenoid, and carotenoid precursors to chloroplast structure are discussed.

Differential activity of the maize mutator Mu at different loci and in different cell lineages

SummaryMutator activity of the maize mutator (Mu) system varies for different loci. Mutation frequencies as high as 7.54x10−5 and as low as 4.0x10−6 are observed for 5 loci (i.e., y1,yg2, bz1, sh2, and wx). For the waxy locus, a higher mutation frequency is observed in Mu plants crossed as males than when Mu plants function as females. The frequency of unselected mutations also is found to be higher in Mu plants crossed as males than in the first-generation Mu plants crossed as females. The mutation frequency of the y1 locus, however, does not differ in the male or female crosses. Mu-induced mutation frequencies vary with respect to loci and, for some loci, may depend on other factors such as the sex of the Mu parent or the previous crossing history of the Mu parent. More limited data have been obtained for 4 additional loci(su1, c1, c2 and o2).

Mutator Activity in Maize: Timing of Its Activation in Ontogeny

Mutator activity in maize seems not to be active throughout the development of the plant, but to arise late in ontogeny, and to be developmentally rather than chronologically triggered.