Dinitroaniline herbicide-resistant transgenic tobacco plants generated by co-overexpression of a mutant alpha-tubulin and a beta-tubulin.

Dinitroaniline herbicides are used for the selective control of weeds in arable crops. Dinitroaniline herbicide resistance in the invasive weed goosegrass was previously shown to stem from a spontaneous mutation in an alpha-tubulin gene. We transformed and regenerated tobacco plants with an alpha/beta-tubulin double gene construct containing the mutant alpha-tubulin gene and showed that expression of this construct confers a stably inherited dinitroaniline-resistant phenotype in tobacco. In all transformed lines, the transgene alpha- and beta-tubulins increased the cytoplasmic pool of tubulin approximately 1.5-fold while repressing endogenous alpha- and beta-tubulin synthesis by up to 45% in some tissues. Transgene alpha- and beta-tubulin were overexpressed in every plant tissue analyzed and comprised approximately 66% of the total tubulin in these tissues. Immunolocalization studies revealed that transgene alpha- and beta-tubulins were incorporated into all four microtubule arrays, indicating that they are functional. The majority of the alpha/beta-tubulin pools are encoded by the transgenes, which implies that the mutant alpha-tubulin and the beta-tubulin can perform the majority, if not all, of the roles of microtubules in both juvenile and adult tobacco plants.

Herbicide resistance caused by spontaneous mutation of the cytoskeletal protein tubulin.

The dinitroaniline herbicides (such as trifluralin and oryzalin) have been developed for the selective control of weeds in arable crops. However, prolonged use of these chemicals has resulted in the selection of resistant biotypes of goosegrass, a major weed. These herbicides bind to the plant tubulin protein but not to mammalian tubulin. Here we show that the major alpha-tubulin gene of the resistant biotype has three base changes within the coding sequence. These base changes swap cytosine and thymine, most likely as the result of the spontaneous deamination of methylated cytosine. One of these base changes causes an amino-acid change in the protein: normal threonine at position 239 is changed to isoleucine. This position is close to the site of interaction between tubulin dimers in the microtubule protofilament. We show that the mutated gene is the cause of the herbicide resistance by using it to transform maize and confer resistance to dinitroaniline herbicides. Our results provide a molecular explanation for the resistance of goosegrass to dinitroanaline herbicides, a phenomenon that has arisen, and been selected for, as a result of repeated exposure to this class of herbicide.

Suppression of endogenous alpha and beta tubulin synthesis in transgenic maize calli overexpressing alpha and beta tubulins.

Maize Black Mexican Sweetcorn cells have been transformed with constructs containing alpha and beta tubulin coding sequences either singly or together. It is shown that recovery of stable maize transformants is dependent on the co-expression of transfected alpha and beta tubulin in the same lines, indicating that plant cells cannot tolerate an imbalance in the ratio of alpha tubulin to beta tubulin within the cytoplasm. The co-expression of transfected alpha and beta tubulin in maize cells results in an increase in the overall tubulin content (approximately threefold). The transfected alpha and beta tubulins are incorporated into cortical, spindle and phragmoplast microtubule arrays indicating that they are functional. Furthermore, the co-expression of the transfected alpha and beta tubulins results in the suppression of endogenous alpha and beta tubulin synthesis. This suppression increases both with the strength of the promoter in the constructs and with the number of copies of the transgenes inserted into the maize genome. The implications for the post-transcriptional and post-translational regulation of tubulin synthesis in plant cells are discussed.

Ambient UV-B radiation causes deformities in amphibian embryos.

There has been a great deal of recent attention on the suspected increase in amphibian deformities. However, most reports of amphibian deformities have been anecdotal, and no experiments in the field under natural conditions have been performed to investigate this phenomenon. Under laboratory conditions, a variety of agents can induce deformities in amphibians. We investigated one of these agents, UV-B radiation, in field experiments, as a cause for amphibian deformities. We monitored hatching success and development in long-toed salamanders under UV-B shields and in regimes that allowed UV-B radiation. Embryos under UV-B shields had a significantly higher hatching rate and fewer deformities, and developed more quickly than those exposed to UV-B. Deformities may contribute directly to embryo mortality, and they may affect an individual's subsequent survival after hatching.

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.

Cloning and sequence analysis of a flo/lfy homologue isolated from cauliflower (Brassica oleracea L. var. botrytis).

A cDNA library has been constructed from cauliflower curd in which floral development had been initiated. Two cDNAs (pBOFH3 and pBOFH8) have been isolated using the Antirrhinum flo gene as a heterologous probe. The two clones were sequenced and found to contain introns. Comparison of the deduced cDNA sequence of bofh with flo and the Arabidopsis homologue lfy reveals extensive homology. An mRNA transcript of 1.6 kb appears on northern RNA blots. This transcript can be detected, at low levels, before any obvious signs of floral differentiation, reflecting the role bofh plays in determining floral meristem identity.

Selenium in wetlands and waterfowl foods at Kesterson Reservoir, California, 1984.

Kesterson Reservoir (Kesterson) received subsurface agricultural drainwater containing high levels of salts and selenium from farmland in the San Joaquin Valley of California. The accumulation of selenium in wetlands and waterfowl foods at Kesterson was investigated during May, August, and December of 1984. High concentrations of selenium vegetation, and aquatic insects. Mean selenium concentrations in aquatic plants and insects ranged from 1.5 to 170 micrograms/g dry weight and were about 11 to 290 times those found at a nearby reference site. Concentrations in some waterfowl food plants and insects at Kesterson were up to 64 times those reported to be a health hazard to birds. Selenium concentrations were more seasonally variable in aquatic plants than in aquatic insects. Few differences in selenium accumulation were found among ponds. Deposition of selenium in plant parts was not uniform; rhizomes contained higher concentrations than seeds and leaves were intermediate. Most biota bioaccumulated maximum selenium concentrations that were 1,000 to nearly 5,000 times the concentration in the water.

Clinal genetic variation at enzyme loci in bald eagles (Haliaeetus leucocephalus) from the western United States.

Five polymorphic enzyme loci of about 50 sampled were discovered in blood extracts of bald eagles from Alaska, Washington, Oregon, and Arizona, representing the first biochemical genetic variation described for the species. All five loci exhibited trends of north-to-south clinal geographic variation in gene frequencies. Gene frequencies at three loci culminated in fixation in the Arizona population, which consists of 12 known breeding pairs. The Arizona birds were maximally heterozygous at the other two loci, suggesting the possibility of maintenance of some clines by natural selection. No significant discontinuities in gene frequencies were observed which correlated with earlier descriptions of two subspecies (northern and southern races) of bald eagles.

Occurrence of Escherichia coli in Wild Cottontail Rabbits.

Free-ranging cottontail rabbits (Sylvilagus floridanus) from two areas in central Pennsylvania were sampled over a 4-year period. Large numbers of coliforms were isolated from the intestinal tracts of these animals; in 136 of the 141 rabbits sampled, Escherichia coli was found to be a major component of the alimentary flora. Four serogroups (O7, O77, O73, and O103) were predominant among the isolates and were considered resistant coliflora of this species of cottontail rabbit.