A Ds insertion alters the nuclear localization of the maize transcriptional activator R.

The R-sc gene of maize is a member of the R gene family of transcriptional activators that regulate anthocyanin biosynthesis. A derivative of R-sc, r-m9 conditions a reduced level of aleurone pigmentation due to the presence of a 2.1-kb Ds insertion near the 3' end of the coding region. Excision of Ds from r-m9 leaves a 7-bp insertion in the darker but still mutant v24 derivative. Both the 7-bp insertion in v24 and the 2.1-kb Ds in r-m9 are predicted to truncate their respective R proteins proximal to the carboxyl terminus, which was shown previously to contain one of three nuclear localization sequences. We find that the reduced expression of r-m9 and v24 are not due to mRNA or protein instability, but most likely reflect the inefficient localization of truncated R proteins to the nucleus. To our knowledge this is the first example of a transposable element insertion that alters gene expression by affecting nuclear localization. In addition, our data indicate that the carboxyl terminus of the R protein is far more important than previously suspected and illustrates the utility of natural mutations for defining functional domains in proteins.

Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins.

In pollen development, a dramatic reorganization of the actin cytoskeleton takes place during the passage of the pollen grain into dormancy and on activation of pollen tube growth. A role for actin-binding proteins is implicated and we report here the identification of a small gene family in maize that encodes actin depolymerizing factor (ADF)-like proteins. The ADF group of proteins are believed to control actin polymerization and depolymerization in response to both intracellular and extracellular signals. Two of the maize genes ZmABP1 and ZmABP2 are expressed specifically in pollen and germinating pollen suggesting that the protein products may be involved in pollen actin reorganization. A third gene, ZmABP3, encodes a protein only 56% and 58% identical to ZmABP1 and ZmABP2, respectively, and its expression is suppressed in pollen and germinated pollen. The fundamental biochemical characteristics of the ZmABP proteins has been elucidated using bacterially expressed ZmABP3 protein. This has the ability to bind monomeric actin (G-actin) and filamentous actin (F-actin). Moreover, it decreases the viscosity of polymerized actin solutions consistent with an ability to depolymerize filaments. These biochemical characteristics, taken together with the sequence comparisons, support the inclusion of the ZmABP proteins in the ADF group.

A single mutation that increases maize seed weight.

The maize endosperm-specific gene shrunken2 (Sh2) encodes the large subunit of the heterotetrameric starch synthetic enzyme adenosine diphosphoglucose pyrophosphorylase (AGP; EC 2.7.7.27). Here we exploit an in vivo, site-specific mutagenesis system to create short insertion mutations in a region of the gene known to be involved in the allosteric regulation of AGP. The site-specific mutagen is the transposable element dissociation (Ds). Approximately one-third (8 of 23) of the germinal revertants sequenced restored the wild-type sequence, whereas the remaining revertants contained insertions of 3 or 6 bp. All revertants retained the original reading frame 3' to the insertion site and involved the addition of tyrosine and/or serine. Each insertion revertant reduced total AGP activity and the amount of the SH2 protein. The revertant containing additional tyrosine and serine residues increased seed weight 11-18% without increasing or decreasing the percentage of starch. Other insertion revertants lacking an additional serine reduced seed weight. Reduced sensitivity to phosphate, a long-known inhibitor of AGP, was found in the high seed-weight revertant. This alteration is likely universally important since insertion of tyrosine and serine in the potato large subunit of AGP at the comparable position and expression in Escherichia coli also led to a phosphate-insensitive enzyme. These results show that single gene mutations giving rise to increased seed weight, and therefore perhaps yield, are clearly possible in a plant with a long history of intensive and successful breeding efforts.

Single-amino acid substitutions eliminate lysine inhibition of maize dihydrodipicolinate synthase.

Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) catalyzes the first step in biosynthesis of lysine in plants and bacteria. DHPS in plants is highly sensitive to end-product inhibition by lysine and, therefore, has an important role in regulating metabolite flux into lysine. To better understand the feedback inhibition properties of the plant enzyme, we transformed a maize cDNA for lysine-sensitive DHPS into an Escherichia coli strain lacking DHPS activity. Cells were mutagenized with ethylmethanesulfonate, and potential DHPS mutants were selected by growth on minimal medium containing the inhibitory lysine analogue S-2-aminoethyl-L-cysteine. DHPS assays identified surviving colonies expressing lysine-insensitive DHPS activity. Ten single-base-pair mutations were identified in the maize DHPS cDNA sequence; these mutations were specific to one of three amino acid residues (amino acids 157, 162, and 166) localized within a short region of the polypeptide. No other mutations were present in the remaining DHPS cDNA sequence, indicating that altering only one of the three residues suffices to eliminate lysine inhibition of maize DHPS. Identification of these specific mutations that change the highly sensitive maize DHPS to a lysine-insensitive isoform will help resolve the lysine-binding mechanism and the resultant conformational changes involved in inhibition of DHPS activity. The plant-derived mutant DHPS genes may also be used to improve nutritional quality of maize or other cereal grains that have inadequate lysine content when fed to animals such as poultry, swine, or humans.

Red/far-red and blue light-responsive regions of maize rbcS-m3 are active in bundle sheath and mesophyll cells, respectively.

Leaves of the C4 plant maize have two major types of photosynthetic cells: a ring of five large bundle sheath cells (BSC) surrounds each vascular bundle and smaller mesophyll cells (MC) lie between the cylinders of bundle sheath cells. The enzyme ribulose bisphosphate carboxylase/oxygenase is encoded by nuclear rbcS and chloroplast rbcL genes. It is not present in MC but is abundant in adjacent BSC of green leaves. As reported previously, the separate regions of rbcS-m3, which are required for stimulating transcription of the gene in BSC and for suppressing expression of reporter genes in MC, were identified by an in situ expression assay; expression was not suppressed in MC until after leaves of dark-grown seedlings had been illuminated for 24 h. Now we have found that transient expression of rbcS-m3 reporter genes is stimulated in BSC via a red/far-red reversible phytochrome photoperception and signal transduction system but that blue light is required for suppressing rbcS-m3 reporter gene expression in MC. Blue light is also required for the suppression system to develop in MC. Thus, the maize gene rbcS-m3 contains certain sequences that are responsive to a phytochrome photoperception and signal transduction system and other regions that respond to a UVA/blue light photoperception and signal transduction system. Various models of "coaction" of plant photoreceptors have been advanced; these observations show the basis for one type of coaction.

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.

Interactions of maize bushy stunt phytoplasma with the leafhopper vector, Dalbulus maidis (Delong and Wolcott) (Hemiptera: Cicadellidae) and associated microbiota

Phytoplasmas are bacteria with a persistent propagative transmission by insect vectors that generates direct and indirect interactions among them. In order to understand these interactions for maize bushy stunt phytoplasma (MBSP) and the leafhopper vector Dalbulus maidis (Hemiptera: Cicadellidae), two research lines were addressed. The first one aimed to determine the indirect effects of maize infection by MBSP on some biological and behavioral parameters of the vector, whereas a second line investigated direct interactions of the phytoplasma with D. maidis during its movement through the vector body following acquisition from plants, and associated microbiota. Indirect effects were investigated in choice experiments in which alighting and oviposition preferences by D. maidis were compared on healthy vs. MBSP-infected plants with variable incubation time (diseased plants with early and advanced symptoms, or still asymptomatic). Likewise, indirect effect of MBSP on the D. maidis biology was determined in two life table experiments in which the vector was reared on healthy vs. MBSP-infected plants expressing advanced disease symptoms or still asymptomatic. Choice experiments showed that alighting and oviposition preferences of D. maidis on MBSP-infected plants compared to healthy plants depend on the pathogen incubation period in the plant. The leafhopper preferred MBSP-infected plants over healthy ones during the asymptomatic phase of the disease, but rejected infected plants with advanced symptoms. The vector was able to acquire MBSP from asymptomatic infected plants shortly (3 days) after inoculation, but transmission efficiency increased when acquisition occurred at later stages of the pathogen incubation period (≥14 days) in the source plants and the test plants showed disease symptoms faster. These results suggest that MBSP modulates D. maidis preference for asymptomatic infected plants in the early stages of the crop, allowing rapid spread of this pathogen. Maize infection by the phytoplasma had a neutral effect on most life table parameters of D. maidis; a lower net reproductivity rate (Ro) was observed in the cohort reared on MBSP-infected plants with advanced symptoms, which was compensated to some extent by a higher sexual ratio. MBSP acquisition by all vector nymphal stadia was confirmed by PCR, and the pathogen as detected in both male and female reproductive organs. Concerning direct MBSP-vector interactions, transmission electron microscopy analyses showed phytoplasma-like cells in the midgut lumen, microvilli and epithelial cells, suggesting that MBSP enters the epithelium midgut through the microvilli wall. Within the epithelial cells, mitochondria and bacteria-like cells (possibly endosymbionts) were observed together with masses of phythoplasma-like cells. In the hemocoel, phytoplasma-like cells grouped into a matrix were also observed in association with bacteria-like cells similar to those observed in the midgut epithelium. Similar associations were found in the salivary gland. Interestingly, in-situ hybridization (FISH) technique revealed a variation in diversity and abundance of the microbiota in intestine and salivary glands of D. maidis adults over time after MBSP acquisition from plants. Sulcia sp., Cardinium sp. and eubacteria increased their abundance over time, whereas Rickettsia sp. decreased. The frequent association of the vector microbiota with the phytoplasma in some tissues of D. maidis suggests that endosymbiotic bacteria may play some role in MBSP-vector interactions.