Thermoregulation by Diabrotica undecimpunctata howardi and potential effects on overwintering biology

In North Carolina, southern corn rootworm (SCR), Diabrotica undecimpunctata howardi Barber, adults of both sexes (diapausing and nondiapausing) elevate body temperature (Tb) above ambient air temperature (Ta) by basking in direct sunlight on clear fall and winter days when Ta<13°C (Tb-Ta range: 0.8–13.3°C). On cloudy days, SCR adults did not exhibit basking behavior and Tb was more highly correlated with Ta, ground temperature (Tg), and substrate temperature (Ts) than on clear days. Ts was the best predictor of Tb regardless of Ta and the presence or absence of adult basking behavior (fall basking SCR, Ts vs. Tb: R²=0.94, Ta vs. Tb: R²=0.41; fall nonbasking SCR, Ts vs. Tb: R²=0.85, Ta vs. Tb: R²=0.55).These results suggest that SCR thermoregulation at low Ta is ectothermic regulation by microhabitat selection. Elevation of Tb by thermoregulation was often of sufficient magnitude to affect SCR behavior (flight, feeding, mating) and preovipositional development rate. This could explain why gravid SCR females have been found in North Carolina by 6 January when Ta's were too low for development. The relationship of Tb to Ta on cold sunny days would be important to include in any predictive day-degree model of SCR postdiapause development or SCR endoparasite development in the field.

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Résumé En Caroline du Nord, les adultes diapausants ou non des deux sexes de Diabrotica undecimpunctata howardi (SCR) élèvent la température de leurs corps (Tb) au-dessus de la température de l'air ambiant (Ta) en lézardant en plein soleil par jours clairs du printemps et de l'hiver, quand Ta<13°C (Tb-Ta de 0.8 à 13.3°C). Par temps nuageux, les adultes ne lézardent pas, et Tb dépend beaucoup plus de Ta, de la température du sol (Tg), et de la température du substrat (Ts), que par temps clair. Ts fournit la meilleure idée de Ta que les adultes lézardent ou non; quand SCR lézarde au printemps la corrélation de Ts avec Tb est R²=0.94, de Ta avec Tb:R²=0.41; au printemps, quand ils ne lézardent pas; Ts avec TbR²=0.85 et Ta avec Tb:R²=0.55. Ces résultats suggerent que la thermorégulation de SCR aux basses Ta est une régulation ectothermique par sélection du microhabitat. L'augmentation de Tb par thermorégulation est souvent suffisante pour modifier le comportement de SCR (vol, prise de nourriture, accouplement) et un développement préponte. Ceci pourrait expliquer pourquoi des femelles gravides ont été trouvées un 6 janvier en Caroline du Nord quand Ta était trop basse pour permettre le développement. II paraît important d'inclure la relation entre Tb et Ta par temps nuageux froid, dans tout modèle prédictif, basé sur la notion de degrés-jours, sur le développement de SCR après la diapause ou le dévelopment d'endoparasites de SCR dans la nature.

     

Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis

Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants. One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified. Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail). Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome. Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes. Chromosomal rearrangements may therefore be widespread following seed transformation. DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns. Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation. The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants. Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis.     

Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis

Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants. One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified. Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail). Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome. Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes. Chromosomal rearrangements may therefore be widespread following seed transformation. DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns. Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation. The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants. Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis.     

Positions of multiple insertions in SSU rDNA of lichen-forming fungi

Lichen-forming fungi, in symbiotic associations with algae, frequently have nuclear small subunit ribosomal DNA (SSU rDNA) longer than the 1,800 nucleotides typical for eukaryotes. The lichen-forming ascomycetous fungus Lecanora dispersa contains insertions at eight distinct positions of its SSU rDNA; the lichen-forming fungi Calicium tricolor and Porpidia crustulata each contain one insertion. Insertions are not limited to fungi that form lichens; the lichen ally Mycocalicium albonigrum also contains two insertions. Of the 11 insertion positions now reported for lichen-forming fungi and this ally, 6 positions are known only from lichen-forming fungi. Including the 4 newly reported in this study, insertions are now known from at least 17 positions among all reported SSU rDNA sequences. Insertions, most of which are Group I introns, are reported in fungal and protistan lineages and occur at corresponding positions in genomes as phylogenetically distant as the nuclei of fungi, green algae, and red algae. Many of these positions are exposed in the mature rRNA tertiary structure and may be subject to independent insertion of introns. Insertion of introns, accompanied by their sporadic loss, accounts for the scattered distribution of insertions observed within the SSU rDNA of these diverse organisms.      

Structural and functional relationships in two families of beta-1,4-glycanases.

CenA and Cex are beta-1,4-glycanases produced by the cellulolytic bacterium Cellulomonas fimi. Both enzymes are composed of two domains and contain six Cys residues. Two disulfide bonds were assigned in both enzymes by peptide analysis of the isolated catalytic domains. A further disulfide bond was deduced in both cellulose-binding domains from the absence of free thiols under denaturing conditions. Corresponding Cys residues are conserved in eight of nine other known C. fimi-type cellulose-binding domains. CenA and Cex belong to families B and F, respectively, in the classification of beta-1,4-glucanases and beta-1,4-xylanases based on similarities in catalytic domain primary structure. Disulfide bonds in the CenA catalytic domain correspond to the two disulfide bonds in the catalytic domain of Trichoderma reesei cellobiohydrolase II (family B) which stabilize loops forming the active-site tunnel. Sequence alignment indicates the probable occurrence of disulfides at equivalent positions in the two other family B enzymes. Partial resequencing of the gene encoding Streptomyces KSM-9 beta-1,4-glucanase CasA (family B) revealed five errors in the original nucleotide sequence analysis. The corrected amino acid sequence contains an Asp residue corresponding to the proposed proton donor in hydrolysis catalysed by cellobiohydrolase II. Cys residues which form disulfide bonds in the Cex catalytic domain are conserved in XynZ of Clostridium thermocellum and Xyn of Cryptococcus albidus but not in the other eight known family F enzymes. Like other members of its family, Cex catalyses xylan hydrolysis. The catalytic efficiency (kcat/Km) for hydrolysis of the heterosidic bond of p-nitrophenyl-beta-D-xylobioside is 14,385 min-1.mM-1 at 25 degrees C; the corresponding kcat/Km for p-nitrophenyl-beta-D-cellobioside hydrolysis is 296 min-1.mM-1.     

Unusual sequence organization in CenB, an inverting endoglucanase from Cellulomonas fimi.

The nucleotide sequence of the cenB gene was determined and used to deduce the amino acid sequence of endoglucanase B (CenB) of Cellulomonas fimi. CenB comprises 1,012 amino acids and has a molecular weight of 105,905. The polypeptide is divided by so-called linker sequences rich in proline and hydroxyamino acids into five domains: a catalytic domain of 607 amino acids at the N terminus, followed by three repeats of 98 amino acids each which are greater than 60% identical, and a C-terminal domain of 101 amino acids which is 50% identical to the cellulose-binding domains of C. fimi cellulases Cex and CenA. A deletion mutant of the cenB gene encodes a polypeptide lacking the C-terminal 333 amino acids of CenB. The truncated polypeptide is catalytically active and, like intact CenB, binds to cellulose, suggesting that CenB has a second cellulose-binding site. The sequence of amino acids 1 to 461 of CenB is 35% identical, with a further 15% similarity, to that of a cellulase from avocado, which places CenB in cellulase family E. CenB releases mostly cellobiose and cellotetraose from cellohexaose. Like CenA, CenB hydrolyzes the beta-1,4-glucosidic bond with inversion of the anomeric configuration. The pH optimum for CenB is 8.5, and that for CenA is 7.5.     

An embryo-lethal mutant of Arabidopsis thaliana is a biotin auxotroph

Lethal mutants have been used in a variety of animal systems to study the genetic control of morphogenesis and differentiation. Abnormal development has been shown in some cases to be caused by defects in basic cellular processes. We describe in this report an embryo-lethal mutant of Arabidopsis thaliana that can be rescued by the addition of biotin to arrested embryos cultured in vitro and to mutant plants grown in soil. Mutant plants rescued in culture produced phenotypically normal seeds when supplemented with biotin but became chlorotic and failed to produce fertile flowers in the absence of biotin. Arrested embryos were also rescued by desthiobiotin, the immediate precursor of biotin in bacteria. Langridge proposed 30 years ago (1958, Aust. J. Biol. Sci. 11, 58-68) that the scarcity of plant auxotrophs might be caused by lethality prior to germination. The bio1 mutant of Arabidopsis described in this report clearly demonstrates that some auxotrophs in higher plants are eliminated through embryonic lethality. Further analysis of this mutant should provide valuable information on the nature of plant auxotrophs, the biosynthesis and utilization of biotin in plants, and the underlying causes of developmental arrest in lethal mutants of Arabidopsis.     

Partial reconstruction of the microvillus core bundle: characterization of villin as a Ca(++)-dependent, actin-bundling/depolymerizing protein

The brush border, isolated from chicken intestine epithelial cells, contains the 95,000 relative molecular mass (M(r)) polypeptide, villin. This report describes the purification and characterization of villin as a Ca(++)-dependent, actin bundling/depolymerizing protein. Then 100,000 g supernatant from a Ca(++) extract of isolated brush borders is composed of three polypeptides of 95,000 (villin), 68,000 (fimbrin), and 42,000 M(r) (actin). Villin, following purification from this extract by differential ammonium sulfate precipitation and ion-exchange chromatography, was mixed with skeletal muscle F-actin. Electron microscopy of negatively stained preparations of these villin-actin mixtures showed that filament bundles were present. This viscosity, sedimentability, and ultrastructural morphology of filament bundles are dependent on the villin:actin molar ratio, the pH, and the free Ca(++) concentration in solution. At low free Ca(++) (less than 10(-6) M), the amount of protein in bundles, when measured by sedimentation, increased as the villin: actin molar ratio increased and reached a plateau at approximately a 4:10 ratio. This behavior correlates with the conversion of single actin filaments into filament bundles as detected in the electron microscope. At high free Ca(++) (more than 10(-6) M), there was a decrease in the apparent viscosity in the villin-actin mixtures to a level measured for the buffer. Furthermore, these Ca(++) effects were correlated with the loss of protein sedimented, the disappearance of filament bundles, and the appearance of short fragments of filaments. Bundle formation is also pH-sensitive, being favored at mildly acidic pH. A decrease in the pH from 7.6 to 6.6 results in an increase in sedimentable protein and also a transformation of loosly associated actin filaments into compact actin bundles. These results are consistent with the suggestions that villin is a bundling protein in the microvillus and is responsible for the Ca(++)-sensitive disassembly of the microvillar cytoskeleton. Thus villin may function in the cytoplasm as a major cytoskeletal element regulating microvillar shape.     

Expression of storage-protein genes during soybean seed development

Mature seeds of Glycine max (L.) Merr. contain two major storage proteins, a glycosylated 7S protein (conglycinin) and a non-glycosylated 11S protein (glycinin). Accumulation of these proteins and their mRNAs during seed development in cv. Provar was studied by SDS polyacrylamide gel electrophoresis and by Northern (DNA-RNA) hybridization. The 11S acidic and basic subunits and the 7S and subunits began to accumulate 18–20 d after pollination, shortly after the termination of cell division in developing cotyledons, whereas the 7S and 11S A-4 subunits were not detected until one to two weeks later, during the maturation phase of development. Messenger RNAs for 7S and 11S proteins were first detected 14–18 d after pollination, several days before the accumulation of storage proteins. Extracts from embryonic axes contained reduced levels of the 7S subunit, very little 11S protein, no detectable 7S or 11S A-4 subunits, and an additional 7S subunit not found in cotyledons. Soybean axes and cotyledons therefore differ in their synthesis of seed storage proteins.Abbreviations cDNA complimentary DNA - mRNA messenger RNA - SDS sodium dodecyl sulfate