Protoporphyrinogen oxidase inhibition by three peroxidizing herbicides: oxadiazon, LS 82-556 and M&B 39279

Three chemically unrelated peroxidizing molecules, namely oxadiazon [5-(t-butyl)-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2 -one], LS 82-556 [(S)3-N-(methylbenzyl)carbamoyl-5-propionyl-2,6-lutidine] and M&B 39279 [5-amino-4-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)pyrazol], are potent inhibitors of plant, yeast and mouse protoporphyrinogen oxidase.     

Fluorometric assays for coproporphyrinogen oxidase and protoporphyrinogen oxidase

We describe fluorometric assays for two enzymes of the heme pathway, coproporphyrinogen oxidase and protoporphyrinogen oxidase. Both assays are based on measurement of protoporphyrin IX fluorescence generated from coproporphyrinogen III by the two consecutive reactions catalyzed by coproporphyrinogen oxidase and protoporphyrinogen oxidase. Both enzymatic activities are measured by recording protoporphyrin IX fluorescence increase in air-saturated buffer in the presence of EDTA (to inhibit ferrochelatase that can further metabolize protoporphyrin IX) and in the presence of dithiothreitol (that prevents nonenzymatic oxidation of porphyrinogens to porphyrins). Coproporphyrinogen oxidase (limiting) activity is measured in the presence of a large excess of protoporphyrinogen oxidase provided by yeast mitochondrial membranes isolated from commercial baker's yeast. These membranes are easy to prepare and are stable for at least 1 year when kept at -80 degrees C. Moreover they ensure maximum fluorescence of the generated protoporphyrin (solubilization effect), avoiding use of a detergent in the incubation medium. The fluorometric protoporphyrinogen oxidase two-step assay is closely related to that already described (J.-M. Camadro, D. Urban-Grimal, and P. Labbe, 1982, Biochem. Biophys. Res. Commun. 106, 724-730). Protoporphyrinogen is enzymatically generated from coproporphyrinogen by partially purified yeast coproporphyrinogen oxidase. The protoporphyrinogen oxidase reaction is then initiated by addition of the membrane fraction to be tested. However, when very low amounts of membrane are used, low amounts of Tween 80 (less than 1 mg/ml) have to be added to the incubation mixture to solubilize protoporphyrin IX in order to ensure optimal fluorescence intensity. This detergent has no effect on the rate of the enzymatic reaction when used at concentrations less than 2 mg/ml. Activities ranging from 0.1 to 4-5 nmol protoporphyrin formed per hour per assay are easily and reproducibly measured in less than 30 min.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

The occurrence and frequency of 2n pollen in three diploid solanums from Northwest Argentina

Summary A group of wild, tuber-bearing species from Northwest Argentina, belonging to the series Tuberosa, Solarium spegazzini Bitt. (spg, 2n=2x=24), S. gourlayi Hawkes (grl, 2n=2x=24 and 2n=4x=48) and S. oplocense Hawkes (opl, 2n=6x=72), and Cuneolata, S. infundibuliforme Phil (ifd, 2n=2x=24), is being used to investigate the mode of origin of polyploids in the genus Solanum. 2n gametes have been detected in the diploid species ifd and spg and in a diploid race of grl, using cytological and breeding approaches. Twenty-two introductions of spg, 8 of grl and 26 of ifd have been tested for 2n pollen; 59%, 63% and 54% of them, respectively, had at least one 2n pollen producing plant. These introductions comprised 238, 76 and 235 plant respectively, of which 20, 16, and 32 plant produced 5% or more 2n pollen. The mechanism of 2n pollen formation was determined in several plant of 2x spg, 2x grl and 2x ifd. All of them were found to form diplandroids via parallel spindles. This mechanism, which gives meiotic products genetically equivalent to first division restitution gametes, is under control of the Mendelian recessive ps. The results suggest that the allele ps is widely distributed in natural populations of the three diploids, and that its frequency is very high. These species are seen as valuable material for population genetic studies, and for the eventual incorporation into a breeding scheme involving sexual polyploidization via 2n gametes.