The gene distribution of the maize genome.

Previous investigations from our laboratory showed that the genomes of plants, like those of vertebrates, are mosaics of isochores, i.e., of very long DNA segments that are compositionally homogeneous and that can be subdivided into a small number of families characterized by different GC levels (GC is the mole fraction of guanine+cytosine). Compositional DNA fractions corresponding to different isochore families were used to investigate, by hybridization with appropriate probes, the gene distribution in vertebrate genomes. Here we report such a study on the genome of a plant, maize. The gene distribution that we found is most striking, in that almost all genes are present in isochores covering an extremely narrow (1-2%) GC range and only representing 10-20% of the genome. This gene distribution, which seems to characterize other Gramineae as well, is remarkably different from the gene distribution previously found in vertebrate genomes.

Neighboring base composition and transversion/transition bias in a comparison of rice and maize chloroplast noncoding regions.

The correspondence between the transversion/transition ratio and the neighboring base composition in chloroplast DNA is examined. For 18 noncoding regions of the chloroplast genome, alignments between rice (Oryza sativa) and maize (Zea mays) were generated by two different methods. Difficulties of aligning noncoding DNA are discussed, and the alignments are analyzed in a manner that reduces alignment artifacts. Sequence divergence is < 10%, so multiple substitutions at a site are assumed to be rare. Observed substitutions were analyzed with respect to the A+T content of the two immediately flanking bases. It is shown that as this content increases, the proportion of transversions also increases. When both the 5'- and 3'-flanking nucleotides are G or C (A+T content of 0), only 25% of the observed substitutions are transversions. However, when both the 5'- and 3'-flanking nucleotides are A or T (A+T content of 2), 57% of the observed substitutions are transversions. Therefore, the influence of flanking base composition on substitutions, previously reported for a single noncoding region, is a general feature of the chloroplast genome.

Elongation factor 1 alpha concentration is highly correlated with the lysine content of maize endosperm.

Lysine is the most limiting essential amino acid in cereals, and for many years plant breeders have attempted to increase its concentration to improve the nutritional quality of these grains. The opaque2 mutation in maize doubles the lysine content in the endosperm, but the mechanism by which this occurs is unknown. We show that elongation factor 1 alpha (EF-1 alpha) is overexpressed in opaque2 endosperm compared with its normal counterpart and that there is a highly significant correlation between EF-1 alpha concentration and the total lysine content of the endosperm. This relationship is also true for two other cereals, sorghum and barley. It appears that genetic selection for genotypes with a high concentration of EF-1 alpha can significantly improve the nutritional quality of maize and other cereals.

A defective signal peptide in the maize high-lysine mutant floury 2.

The maize floury 2 (fl2) mutation enhances the lysine content of the grain, but the soft texture of the endosperm makes it unsuitable for commercial production. The mutant phenotype is linked with the appearance of a 24-kDa alpha-zein protein and increased synthesis of binding protein, both of which are associated with irregularly shaped protein bodies. We have cloned the gene encoding the 24-kDa protein and show that it is expressed as a 22-kDa alpha-zein with an uncleaved signal peptide. Comparison of the deduced N-terminal amino acid sequence of the 24-kDa alpha-zein protein with other alpha-zeins revealed an alanine to valine substitution at the C-terminal position of the signal peptide, a histidine insertion within the seventh alpha-helical repeat, and an alanine to threonine substitution with the same alpha-helical repeat of the protein. Structural defects associated with this alpha-zein explain many of the phenotypic effects of the fl2 mutation.

Dynamics of maize endosperm development and DNA endoreduplication.

Endosperm development in Zea mays is characterized by a period of intense mitotic activity followed by a period in which mitosis is essentially eliminated and the cell cycle becomes one of alternating S and G phases, leading to endoreduplication of the nuclear DNA. The endosperm represents a significant contribution to the grain yield of maize; thus, methods that facilitate the study of cellular kinetics may be useful in discerning cellular and molecular components of grain yield. Two mathematical models have been developed to describe the kinetics of endosperm growth. The first describes the kinetics of mitosis during endosperm development; the second describes the kinetics of DNA endoreduplication during endosperm development. The mitotic model is a modification of standard growth curves. The endoreduplication model is composed of six differential equations that represent the progression of nuclei from one DNA content to another during the endoreduplication process. Total nuclei number per endosperm and the number of 3C, 6C, 12C, 24C, 48C, and 96C nuclei per endosperm (C is the haploid DNA content per nucleus) for inbred W64A from 8 to 18 days after pollination were determined by flow cytometry. The results indicate that the change in number of nuclei expressed as a function of the number of days after pollination is the same from one yearly crop to another. These data were used in the model to determine the endosperm growth rate, the maximum nuclei number per endosperm, and transition rates from one C value to the next higher C value. The kinetics of endosperm development are reasonably well represented by the models. Thus, the models provide a means to quantify the complex pattern of endosperm development.

Developmentally related responses of maize catalase genes to salicylic acid.

The response of the maize catalase genes (Cat1, Cat2, and Cat3) to salicylic acid (SA) was examined at two distinct developmental stages: embryogenesis and germination. A unique, germination-related differential response of each maize catalase gene to various doses of SA was observed. During late embryogenesis, total catalase activity in scutella increased dramatically with 1 mM SA treatment. The accumulation of Cat2 transcript and CAT-2 isozyme protein provided the major contribution to the observed increase in total catalase activity. This increase was paralleled by the enhanced growth of germinated embryos at that stage. In a CAT-2 null mutant line, a full compensation of total catalase activity by the CAT-1 isozyme was observed in the presence of SA. This suggests that catalase is important for maintenance of normal cellular processes under stress conditions. SA at 1 mM, which enhances growth of precociously germinated embryos, appeared to inhibit seed germination at 1 day after inhibition. Furthermore, Cat2 transcript accumulation was inhibited at this stage. SA is probably not a direct signal for the induction of the catalase genes. Other signals, possibly germination-related regulator(s), might be responsible for the induction of the catalase genes. The effect of SA on the activity of purified catalase protein was also examined.

Auxins induce clustering of the auxin-binding protein at the surface of maize coleoptile protoplasts.

The predominant localization of the major auxin-binding protein (ABP1) of maize is within the lumen of the endoplasmic reticulum. Nevertheless, all the electrophysiological evidence supporting a receptor role for ABP1 implies that a functionally important fraction of the protein must reside at the outer face of the plasma membrane. Using methods of protoplast preparation designed to minimize proteolysis, we report the detection of ABP at the surface of maize coleoptile protoplasts by the technique of silver-enhanced immunogold viewed by epipolarization microscopy. We also show that ABP clusters following auxin treatment and that this response is temperature-dependent and auxin-specific.